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Urinary proteomics and the role of orosomucoid

(ORM) in vascularization of bladder cancer

Dissertation Inaugural-

Zur Erlangung des

aften hDoktorgrads der Naturwissensc

(Dr. rer. nat.)

am Fachbereich Chemie

ersität Duisburg-Essenan der Univ

vorgelegt von

Ster Irmak

aus Mardin

Essen 2007

Die der vorliegenden Arbei

t zugrunde liegenden Experiment

e wurden am Institut

-Essen und des Universitätsklinikum Duisburgfür Anatomie der Universität

Hamburg-Eppendorf durchgeführt.

Gutachter: Prof. Dr. R. Sustmann 1.

Gutachter: Prof. Dr. Dr2. t o. H. de Gro

Gutachter: Prof. Dr. S. Ergün 3.

r chönbuchehusses: Prof. Dr. A. SVorsitzender des Prüfungsaussc

hen PrTag der mündlicüfung: 03.07.2007


To my Family and

nephew Yusuf Heja

Ever tried, ever failed. Try again, fail again. Fail better Samuel Beckett



tween March 2003 - August 2006 at the The present study has been carried out be

spital Hamburg-Eppendorf, Germany and Department of Anatomy, University Ho

Anatomy, University 2007 at the Department ofbetween August 2006  March

Hospital Essen, Germany.

ted andto everyone who aided, suppor my sincere gratitude I wish to express

, in one way to another, throughout this study. einspired m

thank my supervisor Professor Dr. Süleyman I would especially like to First of all,

ting field of research, for his constant Ergün for introducing me to work in an exci

e study and for the friendly atmosphere interest and support in the progress of th

within the department.

Hamburg-Eppendorf, I would ogy, University Hospital From the Department of Urol

support and the opportunity to work in the like to thank Professor Dr. Huland for his

e to thank PD Dr. Martin y likiallogical Department. I would speclaboratory of the Urol

Friedrich for his encouragement, optimism and for giving the clinicians point of view. I

laboratory of the Urologyformer and present, at the thank all the lab members,

Hospital Hamburg -Eppendorf. Department at the University

ra-e to thank Dr. Leticia OliveiI would likerrer for her help and scientific and F

in the first months of my life in sometimes non-scientific support especially

uschild for her support, always having a ssica HaeGermany. I would like to thank J

breakfasts and coffe breaks. smile and the happy times, daily

I thank Dr. Nerbil Kilic, Dr. Ergin Kilic and Dr. Derya Tilki for their warm support and

fellowship. I would like to thank Kiersten Miete, Elvin Zengin, Ege Erenler, Biranda

ly atmosphere in lab.doviding a frienKocaoglu, Mehmet Varol, Deniz Tilki for pr

al Essen, I would firstly like to omy, University HospitWithin the Department of Anat

-Yasar for her help anthank Dr. Ferya Banazd support during the writing of my thesis.

Prof. Dr. Ergün. Among them, I speciallyI thank all members of the research group of

thank Dorothee Schünke for her help.

for his help in editing of figures. I would like to thank Oguzhan A. Polat

support encouragement and their continuousFinally, I want to thank my parents for

during all the years. Without them this work would not have been possible. I would

en for her deep friendship and motivation. like to thank my best friend Elmas Yurtsev



Index 1 Introduction.......................................................................................................1
1.2 1.1 ProteomicsTechnology of proteomics: 2-Dim...................................................................................................ensional Gelelectrophoresis (2-DE).........3 2
1.2.2 Protein 1.2.1 Sample extractionpreparation.................................................................................................................................................................4 4 De1.2.2.1 Chaottergentsropic agent........................................................................................................................................................................5 5
6 ................................................................................gentsing a1.2.2.3 Reduc1.2.3 1.2.4 Second First Dimension: Isoeldimension: SDectric focuS-PAGEsing (IEF)......................................................................................................10 7
1.2.5 Equi1.2.6 Identificatiolibrationn of proteins..............................................................................................................................................................11 11
1.3.1 1.3 Urinary proteomicsFormation of urine........................................................................................12 12
1.4 Bladder1.3.2 Sources cancerof urinary proteins............................................................................................................................................................15 12
1.4.2 Cellular 1.4.1 Staging of classifibladder cationcancer..............................................................................................................................................16 15
1.5 Clinical 1.6 Urinary proteombiomarkersics and biomarkers.................................................................................................................................................18 18
19 ...................................................................................isangiogenes1.7 Tumor 1.8 1.9 The One of urinplasminogen ary proteiactivatins: Orosomon systemucoid (ORM).......................................................................................................2220

2 The aim of study..............................................................................................26
3 Material and Methods......................................................................................27
3.1 Mate3.1.1 Chemical rialsand Consumables....................................................................................................................................................................27 27
28 ......................................................................................................Kits3.1.2 3.1.3 Stock Solutions and buffers................................................................29
3.1.5 Anti3.1.4 Equipment bodiesand applications...........................................................................................................................................................31 32
3.1.6 Cell lines and medium for cultivation of cell lines................................32
3.1.8 Primers3.1.7 Bacterial strains...................................................................................................................................................................................33 32
34 .....................................................................................................thods3.2 Me3.2.1 Protei3.2.1.1 Determinatn analysesion of total protein..........................................................................................................................................34 34 Preparation of Two dimensional polyacrylurine Samples for 2-DEamide gel electrophoresis (2D-PAGE)...........................................35 35 Rehydration First dimensional separation: Isoelectric ...................................................................................focusing (IEF)................36 36 E Second dimquilibrationensional separation........................................................................................................................................37 37 Silv Proter staiein detecningtion............................................................................................................................................................38 38 Coomassie staining.......................................................................39



39 ...............................................................analysisage and data Im3.2.1.13 SDS-polyacry3.2.1.12 Mass spectrometrylamide gelele and bioictrophornformaticsesis.................................................................................41 39 We3.2.2 Molecularbiostern bllogical ottingmethods...........................................................................................................................................42 41
42 ..................................................E. coli Cultivation and storage of Bacter Preparatiial transfoon of comprmaettionent cells.....................................................................................................................43 43 Miniprepar Purification of ation of plDNA from soluasmid-DNAtion and gel bands.............................................................................44 44 Max Restriictpreparion digesation of plt of DNAasmid-DNA................................................................................................................45 44 S Agarose equencing ofgel electr DNAophoresis...............................................................................................................................45 45 Determination Construction of pcDNA3.of DNA conc1(-)/ORMentration expression vector..............................................................46 46 Ligation Polymerase of Pchain reactiCR product into pCRon (PCR)® 2.1-TOPO®................................................ Vector................46 47 siRNA Subcloning construcinto pcDNtionA3.1(-)..............................................................................................................................48 48 3.2.3 Cellbiolog Culturing ofical me cellsthods..................................................................................................................................................49 49 Freezi Determination of ng and thawing cell numof ceberlls.............................................................................................................50 49 Cell extraction..............................................................................50 Overex Transfection pression/ of HDMECs via gene silencing of NucleofeORM in ctorHDMECs....................................................51 51
3.2.5 Histologi3.2.4 Endothelial tube cal methodsformation assay...................................................................................................................................53 52 I Fixammunohistochemtion and HE staiistryning for tissue samples an.................................................................d cells.................53 53 Immunocytochemistry..................................................................54

4 4.1 Profiling Resultsof urinary proteins by 2D gel electr.............................................................................................................ophoresis...............................5555
preparation method for 2D gel Optimization of the sample 4.1.1 4.1.2 electrophoresis Determination of urinary protein patteon human urinern in rela..........................................................tion to bladder cancer by 55
56 .................................................................................................... 2-DE4.1.3 Protein pattern of normal urine versus urine of patients with pTa.......57
4.1.4 4.1.5 Protein pattern of normProtein pattern of normaal urine l urine versus urine of patients with pT2versus urine of patients with pT1..............60 58
of follow-up versus urine samples of Protein pattern of urine samples 4.1.6 4.1.7 bladder cancProtein pattern of urine samples er pateints.......................................................................of patients on follow-up versus that of 61
patients of tumor stage pTa.................................................................64
4.1.8 patients of tumor sProtein pattern of urine samples tage pT1.................................................................of patients on follow-up versus that of 64
of patients on follow-up versus that of Protein pattern of urine samples 4.1.9 patients of bladder cancer stage pT2..................................................64



cancer4.1.10 Identification of protein spots in urine samp.................................................................................................les of patients with bladder 66
Human serum albumin in normal urine versus in urine samples of 4.1.11 4.1.12 patients with bladder Mass spectrums and matched peptcancer and follow-upides for identified proteins.....................................................70 68
4.2 Idendification of two urinary proteins using Western blot analyses............75
4.2.1 patients with bladder cancerDetection of orosomucoid (ORM) in and patients on follow-up urine samples of healthy persons, .......................75
urine samples of noprotein (ZAG) iDetection of zinc-alpha-2-glyc4.2.2 healthy persons, patients with bladder cancer and patients on ..............
4.3 follow-upExpression pattern of ORM and ZAG bladder cancer versus normal 76
4.4 bladder tissueLocalization of ORM on endothelial cells (HDMECs) by .............................................................................................76
79 .................................................................................hemistry immunocytoc4.5 ORM overexpression versus ORM gene silencing in HDMECs.................82
4.6 Mechanistic studies via in-vitro angiogenesis assays.................................83
ng ORM-overexpressing versus endothelial tube formation usiIn-vitro4.6.1 4.6.2 ORMsilencThe interaction between ORed HDMECsM and PAI-1 in endot.....................................................................helial tube .................83
84 ............................................................................................. formation4.7 Immunolocalization of ORM in in-vitro induced endothelial tubes..............87

5 5.1 DiscussionThe impact of 2-DE for characterization of ur......................................................................................................inary proteins.......................9494
5.2 Optimized powerful tool for identificseparation ofation of uri urinary proteins for ne proteinsperforming of 2-DE as a ..........................................95
the identification of proteins related to bladder Urine proteomics enables 5.3 5.4 cancerNovel protein spots in urine samples ........................................................................................................of patients with bladder cancer using 96
5.5 2-DE may be of diagnostic relevanceORM is increased in urine samples of patients with bladder cancer...................................................................97 97
5.6 Endothelial ORM support the VEoverexpressiGF-inducedon of ORM as well as endothelia endothelial tube formationl stimulation by ...................100
5.7 formationORM interaction with PAI-1 influen..................................................................................................ces the VEGF-induced endothelial tube 101
5.8 bladder cancer particularlZinc-alpha-2-glycoprotein is increasy in the invasive stagesed in urine samples.................................... of patients with 103

6 Conclusion.....................................................................................................105
7 Literature........................................................................................................106
8 8.1 AppendiMap of 3.x1(-) vector..........................................................................................................................................................................................117117
118 .......................................................................................m Vitae8.2 Curriculu

Figure Index

Figure Index

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

Figure 10:

Figure 11:

Figure 12:

Figure 13:

Figure 14:

Figure 15:

Figure 16:

Figure 17:

Figure 18:

Figure 19:

Figure 20:

Figure 21:

Figure 22:

Figure 23:

Figure 24:


formation to protein products2 The flow of genomic in

Proteomics and .2 genomics integration

5 Urea and ThioureafoStructure

Structure of detergents used in solubilisation of proteins.6

dithiothreitol (DTT)6 fStructure o

.7 tributylphosphine (TBP) a reducing agentfoStructure

s, isoelectric point of protein as amphoteric moleculeStructures of


yacrylamide gel matrix9 polfStructure o

ome formation and excretion..14 os of urinary exMechanism

bladder cancer stages.16 ation of Represent

..20 and initiation of angiogenesisVascular destabilization

for the inhibition ofHypothesis leukocyte extravasation by ORM.22

Plasminogen activation system..23

ns of plasminogen, idomaentation of the protein Graphical repres

tPA and uPA..24

teracting partners..25 entation of uPA and its inSchematic pres

Schematical representation of two dimensional separation..35

Experimental flow diagram..40

..48 of gene silencing via siRNAMechanism

le of a patient with bladder cancer Protein pattern in urine samp

of the sage pTa...............56 t

in normal urine (A) and in urine of Comparison of protein pattern

e stage pTa,GI (B).58 ancer of thpatients with bladder c

Comparison of protein pattern in normal urine (A) and in urine of

e stage pT1, GIII (B).59 thancer of patients with bladder c

in normal urine (A) and in urine of Comparison of protein pattern

patients with bladder c (B).61 ancer of the stage pT2, GIII

in urine of patients on follow-up (A) Comparison of protein pattern

of the stage pTa, GI (B)62 s with bladder cancer and patient

s on follow-up (A) in urine of patientComparison of protein pattern

pT1, GIII (B)..63 cancer of the stage s with bladder and patient

Figure Index

s on follow-up (A) in urine of patientComparison of protein pattern Figure 25:


pT2, GIII (B)..65 cancer of the stage s with bladderand patient

ne of a patient with DE image of uriProtein identification from 2-Figure 26:

...66 bladder cancer of the stage pTa/GI

pots indicating HSA in all urine Comparison of protein sFigure 27:

sample groups...69

..70 ion of UromodulinMS identificatFigure 28:

ing glycoprotein and Ribozomal Protein71 MS identification of DNA bindFigure 29:

..72 MS identification of HSAFigure 30:

on of Orosomucoid 1...73 MS identificatiFigure 31:

glycoprotein and Zinc-alpha-2-MS identification ofFigure 32:

Complex forming glycoprotein74

Detection of ORM and ZAG in urine samples..75 Figure 33:

adder tissue..77 normal and tumor blLocalization of ORM inFigure 34:

M in bladder tumor cells..78 Localization of ORFigure 35:

bladder tumor tissueG inLocalization of ZAFigure 36: .79

ation of ORM and ZAG ng for localizImmunocytochemical stainiFigure 37:

HDMECs80 in

ation of ORM and ZAG in r localizImmnucytochemical staining foFigure 38:

bladder cancer cell line81

HDMECs and RT4 cell line81 Detection of ORM inFigure 39:

for ORM in HDMECs.82 and gene silencing via siRNAOverexpression Figure 40:

Endothelial tFigure 41: ube formation assay...83

on85 antibody on endothelial tube formatiEffect of ORM and anti-PAI-1 Figure 42:

Figure 43: Effect ORM and anti-PAI-1 at VEGF-induced endothelial tubes..86

iPAI-1 combination on endothelial Effect of VEGF, ORM and antFigure 44:

..................87 ...formation tube

type endothelial cells after n of ORM on wildLocalizatioFigure 45: tube

ssay.88 a formation

on VEGF-induced endothelial tubes89 ining for ORM ImmunostaFigure 46:

on endothelial tubes treated with Localization of ORM Figure 47:

antibody...90 anti-PAI-1

helial tubes treated with ORM in Localization of ORM on endotFigure 48:

91 low concentration and anti-PAI-1antibody

Figure Index

Figure 49:

Figure 50:

helial tubes treated with ORM in Localization of ORM on endot


.92 h anti-PAI-1 antibodyhigh concentration wit

hin the uPAR-uPA-PAI-1 systems and Important interactions wit

the role of ORM..102

Table Index

Table Index

Table 1:

Table 2:

Table 3:

Table 4:

Table 5:

Table 6:

Table 7:

Table 8:

Table 9:

Table 10:

Table 11:

Table 12:

Table 13:

Table 14:

Table 15:

Table 16:

Table 17:

Table 18:

Table 19:

Table 20:


Sources of urinary proteins.13

Staging of bladder cancer17

AJCC stage groupings.18

Primary antibodies32

Secondary HRP antibodies.32

Cell lines.32

edium for cell lines.32Cultivation m

Bacterial strains.33

leotides used for in PCR..33Oligonuc

leotides used Oligonuc .33for in siRNA construction

Urine samples34

es.36 IPG strips used for 2-DE analys

Applied voltage steps for IEF..37

riction digest reaction.45 Composition of rest

of PCR reaction.46 Composition

M.47 PCR conditions for OR

.52 The stimulating factors used for tube assay

Proteins identified by MS analyses....67

of protein levels of identified Semiquantitative determination

proteins in urine samples by 2-DE.....68

Determination of tube lengths.93


ySummar is currently the method of choice for 2-dimensional gel electrophoresis (2-DE)


separation of complex protein as in cell and tissue extracts or body hmixtures suc

s a central role in clinical urine playuding urine or serum. Human lfluids inc

anc of diseases such as cdiagnosticser and inflammation. Researchers and

of a map of the human proteome but in scientists are working on the development

urinary proteomics and theiraboutare only a few investigations the literature there

first aim of this study was to view the tumor. The behaviour in the case of bladder

t their functions. For this aim, fourtyfive whole proteins present in urine and to predic

cancer of different stages, patients on les from patients with bladderpurine sam

ed by 2-DE. At the beginning of this volunteers were analysfollow-up and healthy

study the 2-DE protein patte unknown because of lackrn of human urine was almost

s prior to the 2-DE. Thus, the studyof appropriate preparation of urine sample

samples providing the best preservationfocused on methods for preparation of urine

optimization of 2-DEof urinary proteins. After experimental , usable 2-D protein

oteins related to bladder cancer using patterns of urine were analysed to identify pr

and/or conventional immunoblotting and subsequent mass spectrometric analyses

to other cancer types, the bladder ochemical methods. In comparison immunohist

and ninth in womausing death in man carcinoma is the seventh camong malignant en

n- as the transformation of superficial notumors. The growth and metastasis as well

invasive tumors to an invasive tumor phenotype are closely associated with

tion of tumor tissue. Thisaovascularisactivation of angiogenesis resulting in ne

process is regulated by a net balance between angiogenic activators and inhibitors.

RM) and human zinc-alpha-2-glycoprotein In the present study, orosomucoid (O

2-DE of urine sed in(ZAG) have been identified to be increasmples of patients with a

e urine samples of healthy volunteers.bladder cancer in comparison to th

a part of oin addition to cancer cells alsImmunohistochemical results let assume that

ndothelial cthe tissue resident inflammatory cells and e lls of tumor associated bloode

vessels may serve as source for this increase of ORM in urine samples of patient

ein and increased in acute infection, with bladder cancer. ORM is an acute phase prot

inflammation, and cancer. Recent studies show that ORM forms a complex with the

active form of plasminogen activator inhibitor-1 (PAI-1) in thymosin β4 (Tβ4)-

it was aimed to determine a lls. Therefore e endothelial cactivated but not in quiescent

is. The findings presented in this giogenespotential role of ORM up-regulation in an



ition to the cancer cells,rensson showed, that, in addstudy and results reported by So

ORM endogenously. For cells (HDMECs) produce human vascular endothelial

al cells, ORM-gene in vascular endothelifunctional characterization of ORM

ng were perfromed. Employing the sion and ORM-gene silencioverexpres

supernatants of HDMECs-ORM and HDMECs-ORM-siRNA in in-vitro angiogenesis

ed endothelial tube upports the VEGF-inducassays, it was found that ORM s

ORM was potentiated by co-treatment of formation. This supportive effect of

and anti-PAI-1 antibody. HDMECs with VEGF, ORM

hat ORM is increased demonstrates for the first time tThis study in urine of patients

c supporting the tube forming effects of with bladder cancer and acts pro-angiogeni

icantly increased by additive blockage of VEGF. This supportative effect was signif


seems mM and PAI-1 and anti-PAI-1 systeIn summary, the interaction between OR

in the -mediated angiogenesis and probablyto be essentially involved in the VEGF

cancer. vascularisation of urinary bladder

sung smmenfaZusa


Zusammenfassung Bei der 2D-Elektrophorese (2-DE) handelt es sich zurzeit um die Methode der Wahl
usammensetzungen von Zell- und für die Trennung komplexer Proteinzn wie Urin oder Serum. Die Untersuchung rakten sowie KörperflüssigkeiteGewebeextankheiten wie Krebs gnostik von Krentrale Rolle in der Dia eine zvon Urinproben stellt ung eines humanenten an der Entwicklund Entzündungen dar. Wissenschaftler arbei h nur wenige Untersuchungentur sind jedocGesamtproteomenmusters, in der Litera beschrieben. Die Harnblasenkarzinomzu Urinproteomics und ihrem Muster beim von nuchung des Proteinmusters im UriZielsetzung dieser Arbeit war die Unterszu normalen Patienten. Dazu wurden 45 enkarzinompatienten im Vergleich HarnblasPatienten und gesunden en, Follow-up-arzinompatientUrinproben von Harnblasenkmente i Beginn dieser Experysiert. ZuProbanden mittels der 2D-Elektrophorese analder angemessener und fehlennproben aufgreinmuster in Uriwar das 2-DE ProtVorbehandlung der Urinproben für die 2D-Elektrophorese kaum bekannt. Daher
bereitung der Urinproben, chst auf Methoden zur Aufwaren die Untersuchungen zunä fokussiert. Nach gewährleisten,ung oteinerhaltdie die bestmögliche Pr rophorese wurden 2D-Proteinmuster dererung der 2D-Elektiexperimenteller Optimektrometrischer Analysen, Western Blot-Urinproben analysiert, um mittels massenspr die mit ne zu identifizieren,Analysen und Immunhistochemie Protei es sich Beim Harnblasenkarzinom handeltenkarzinomen assoziiert sind. Harnblasum die siebthäufigste Krebstodesursache beim Mann und die neunthäufigste
asentumorwachstum- und metastasierung ursache bei der Frau. HarnblKrebstodesinvasiven in einen invasiven - nichtsowie der Übergang von einem oberflächlichen,Aktivierung der Angiogenese und somit ark verknüpft mit der tTumorphänotyp sind sss wird geregelt durch ein Gleichgewicht erung. Dieser Prozeider Tumorvaskularis toren und Inhibitoren.achen Aktivzwischen angiogenetislpha-2-aomucoid (ORM) und humanes Zink-In der vorliegenden Arbeit wurden Orosktrophorese als im Urin von Glykoprotein (ZAG) anhand der 2D-Eleleich zu normalen Urinproben erhöhte enkarzinompatienten im VergHarnblasche Untersuchungen lassen stochemisMarkerproteine identifiziert. Immunhivermuten, dass zusätzlich zu Tumorzellen auch ein Teil der gewebaansässigen
er Blutgefäße die QuelleEndothelzellen tumorassoziiertinflammatorischen Zellen und enten darstellenenkarzinompatinblasfür den ORM-Anstieg in Urinproben von Har ute-Phase-Protein, welches bei akuten könnten. Bei ORM handelt es sich um ein Ak



rankungen erhöht ist. In neuerenInfektionen, Entzündungen und Tumorerk

aktivens ORM einen Komplex mit derdasPublikationen konnte gezeigt werden,

Form von Plasminogen-Aktivator-Inhibitor-1 (PAI-1) in Thymosin- β4 (T β4)-

aktivierten, aber nicht in ruhenden Endothelzellen eingeht. Unsere weitergehenden

ner möglichen Rolle von ORM in der Untersuchungen zielten auf die Ermittlung ei

ierten Ergebnisse als auch Daten derAngiogenese. Sowohl die hier präsent

neben Tumorzellen auch humane vaskuläre son zeigen, dass Arbeitsgruppe Sorens

ORM endogen exprimierEndothelzellen (HDMECs) en. Zur funktionellen

Charakterisierung von ORM in Endothelzellen wurde ORM überexprimiert und

in-vitro-gesilenct. Anhand von ys mit Überständen ORM-aAngiogeneseass

e gezeigt werden, dass ORM encter HDMECs konntüberexprimierender und gesil

eigert. Diese unterstützendermierung stierte endotheliale Tubefodie VEGF-induz

r eden durch simultane Behandlung dWirkung von ORM konnte potentziert wer

und PAI-1-Antikörper. HDMECS mit VEGF, ORM

ist in Urinproben vonmalig, dass ORM erhöht Die Daten zeigen erst

Harnblasenkarzinompatienten und proangiogenetisch wirkt durch die Verstärkung

terstützende Effekt konntenDieser udes tubeformierenden Effektes von VEGF.

signifikch zusätzliche Blockade von PAI-1. ant gesteigert werden dur

zwischen ORM und PAI-1 und dem Anti-Zusammenfassend scheint die Interaktion

PAI-1-System entscheidend involviert zu sein in die VEGF-vermittelte Angiogenese

sierung des Harnblasenkarzinoms. herweise in die Vaskulariund möglic

s nAbbreviatio

Table of Abbreviations 2 dimensional gel elec 2-DE complex Avidin-biotin ABC American Joint Committee on Cancer AJCC Ammonium per sulphate APS lignment search tool l aBasic loca BLASTmembrane Basement BM Coomassie blue brillant CBB ntary Compleme cDNA3-[(3-Cholamidopropyl)diCHAPS 3,3-Diaminobenzid DAB modified eagle medium Dulbeccos DMEM lfoxide uDimethylesDMSO ucleic DNA Deoxyribon
uclease DNase Deoxyribon
leosid DeoxynucdNTP ol Dithiothreit DTTcells Endothelial EC EscherichE.coli coli a iEthylendiamine EDTA MoleculaEuropean EMBL Fetal calf serum FCS Fibroblast growth factor FGF minute,second Hour, h,min,sec Human dermal microvascular endothelial cells HDMECsHuman umbilical cord vein endot HUVEC focusing Isoelectric IEF pH gradient dImmobilize IPG Kilobasepairs kb Kilodalton kDa Liter l broth Luria LB Liter Mol/ M Miliamper mA

trophoresis 2 dimensional gel eleccomplex Avidin-biotin American Joint Committee on Cancer Ammonium per sulphate lignment search tool l aBasic locamembrane Basement Coomassie blue brillant DNA ntary Complememethylammonio]-1-propanesulfonate 3-[(3-Cholamidopropyl)diin-tetrahydrochloride 3,3-Diaminobenzid modified eagle medium Dulbeccoslfoxide uDimethylesDeoxyribonucleic acid
uclease nyriboxDeotriphosphate leosid Deoxynucol Dithiothreitcells Endothelial Escherichcoli a itetraacetate Ethylendiamine r Biology Laboratory MoleculaEuropean Fetal calf serum Fibroblast growth factor minute,second Hour, Human dermal microvascular endothelial cells helial cellsHuman umbilical cord vein endotfocusing Isoelectric pH gradient dImmobilizeKilobasepairs Kilodalton Liter broth Luria Liter Mol/ Miliamper


s nAbbreviatio


mM mmol mRNA


Mu m/z


ng OD



rpm RT


r desorpsion/ ionization Matrix assisted lasetime of flight ion/ ionization-Matrix-assisted laser desorps Minimum essential medium -3Milliliter Milligram (=10(=10-3l) g)
-6Microliter Microgram (=10(=10-6l) g)

Liter / lMillimoMillimol (=10-3mol)
RNA Messenger

spectrometry Mass

unit Mass Mass to charge ratio

body Multivesicular National Center for Biotechnology Information

Nanogramdensity Optical

Orosomucoid Plasminogen activator inhibitor e-antiperoxidase Peroxidasbuffer Phosphate

Phosphate buffered saline chain reaction ePolymeraspoint Isoelectric ing complex ed silencRNA-inducRiboacid ic nucle

Revolutions per minute temperature Room

Receptor tyrosine kinase

Sodium dodecyl sulphate SDS gelelectrophoresis SDS-PAGE SDS-polyacrylamide siRNA Small interfering RNA

Tris-acetate-EDTA TAE

phosphin Tributyle TBP


s nAbbreviatio
















Tris buffered saline with tween



etic acid buffer Trifluoroac

Buffer tion Transforma



e violatUltra


Vascular endothelial growth factor

growth factor receptor endothelial Vascular


World Health Organization


zinc-alpha-2-gylcoprotein Human


ction Introdu


on i1 Introduct ingly important part of life sciences, The field of proteomics has become an increas

especially after completion of the human genome sequence. Proteome analysis
includes separation, identification, and quantitation of proteins from biological

from prognosis of many types of cancer samples with the purpose of revealing the function of living cells. over drug development to monitoring ofApplications range

environmental pollution. The study of genes cannot provide much information on the properties of proteins,
e because the molecules responsible for cellular functions (e.g. signal transduction) ar

post-translational 200 different types of proteins. Proteins may impose more than phorylation, glycosylation, modification, including phosacetylation, deamination,

farnesylation, myristolation, palmitoylation, and proteolysis (Krishna and Wold, 1993).
quences. e be predicted purely from DNA sSuch a wide range of modifications cannot

eins themselves their characteristics and functions can Only through the study of protbe expounded. From the data obtained from studies of the genome project, an

wever, it isthe genome are known. Ho estimated number of proteins encoded bydifficult to predict the actual numbers of a number of reason (Eisenberg et al., 2000)proteins enc. Firstly, the exonoded based on-intron cannot be genomic data, for
(Dunham et al., 1999) i.e. genomic accurately predicted from genomic DNA

ained from protein studies to confirm information needs to be integrated with data obtlicing of a transcript can , alternative spgene. Secondlythe presence of a specific yield more than one protein product (Newman, 1998). Therefore, the direct analysis

Thirdly, as a result of coof mRNA or genome does not rempartmentalization and translocaflect the exact number of prtion, the same protein can otein products in a cell.

(Fig. 1) ons in different locationsunctibe found with different properties and f999). (Colledge and Scott, 1

These problems can only be solved by proteomics, which can directly identify the
ation of he appropriate integron by tproteins and provide the genomic informati

technology to solve problems which cannotgenomic and proteomic data (Fig. 2). Scientists worldwide are be resolved by traditional methoapplyids. ng proteomic

ction Introdu


leadinFigure 1g: Th to physically ande flow of ge/or functionomic infornalmly variable ation to prformotein prs of protoducts. ein isofoDifferermnst epigen from a sinetic procgle genessees
sequence. The concept of one-gene to one-protein is over-simplified since an RNA can be
than 20differentially spli0 different typeced and can s ofpr post-tranoduce slatvariouional modis proteifin producations. cts.TComphe prartmentaliotein zcaatin beon an expod transesld to moocatioren
(Lau et al., 20result in the same p03). rotein can be found with different properties and functions in different locations
1.1 Proteomics a genome, and thus ed by eins expressThe term proteome refers to all the protproteomics involves the identification of proteins in the body and the determination of
ocesses. their role in physiological and pathological pr

appliFigure 2: cations of proteoProteomicsmics, and th and genoe bmics integrenefits of integratination. g Reprotpreseneomic and gtation ofenomi categoc riesdata (Lau et , potential., al

ction Introdu


the characterization of all or selected The studies of proteomics give a chance for can outline the flow of information withinproteins expressed within a given cell and It is estimated that more than 500,000 .etricoin et al., 2004)that protein network (Pproteins comprise the human proteome, derived from ~35,000 genes in the human
Proteomics offer more complexity but.genome (Banks et al., 2000; Stein, 2004) of proteomics canThe studythan examining genes alone. potentially more specificity generally be divided into two categories: (i) the characterization of protein expression
) the characterization of protein function. iiand (Expression proteomics evaluate cellular protein production encoded by those genes
gan. It exposes the differential expression esent in the target oractive in a cell and prstates. The technologies applied to of proteins between healthy and diseased expression proteomics allow investigation of protein expression in multiple sources
including tissue as well as serum and urine. and/or interactions between he activation state oft Functional proteomics evaluatensive network of signaling pathways in a proteins and can be used to map the extecell. Mapping protein-protein interactions can be useful due to discovery of new
binding interactions which may give insight into new proteins that are involved in
provide evidence of common downstream cancer as well as novel oncogenes or information of how varioussignaling networks. Further events shared by two distinct ns directed againsttherapeutic interventiopathways intersect can help in developing these pathway targets (Cai et al., 2004). in proteomics. The first technology isThere are several major technologies used l l polyacrylamide geher. Two-dimensionaotnecessary to separate proteins from each thod of choice for separation of complex esis (2-DE) is currently the meelectrophornd tissue extracts or body fluids. protein mixtures such as in cell a 1.2 Technology of proteomics: 2-Dimensional Gelelectrophoresis (2-DE)
itative analyses of r qualitative and quantotein databases fo2-DE can provide prochemical, biological and biomedical protein expression in a wide range of bins in two steps, chnique separates proteiinvestigations (Celis et al., 1989). This terst-dimension is isoelectric focusing operties: the fi two independent proaccording tto their isoelectric points (pI); the second-(IEF), which separates proteins according parates eectrophoresis (SDS-PAGE), which sacrylamide gel eldimension is SDS-polyproteins according to their molecular weights (MW). In this way, complex mixtures

ction Introdu


and the relative amount rent proteins can be resolvedconsisted of thousands of diffeof each protein can be determined.

preparation 1.2.1 Sample

Appropriateprocedure must be determined empirically sample preparation is ess foential for good 2DE results. The optimalr each sample type. An ideal process
should result in complete solubilisation, disaggregation, denaturation, and reduction
the final 2-DE resultof the proteins in the sample. It i when developing s important to have a cleara sample preparation strategy. Different idea of what is desired in
samples; some proteins are naturally ftreatments and conditions are required to ound in complexessolubilise different types of protein with membranes, nucleic
acids, or other proteins; some proteisome proteins precipitate when remons form various non-specifved from their natural environment. The ic aggregates; and
the choice of cell disruption method, effectiveness of solubilisation depends on protein concentration and discomposition of the sample solution. The solution methods, choice ofcomposition of the sample s detergents, and olution is
bilisation treatments for the first-dimension critical for 2-DE, because soluparticularlyconductive solution. In general, cseparation must not effect isoelectric pointoncentrated (pI), nor leave the surea as well as one aor more detergents mple in a highly
is used.

extraction 1.2.2 Protein

equires the disruption of the cell membraneThe extraction of proteins from a cell rained in the cell and the cell membrane. An and the solubilization of the proteins contids ure them, and exclude nucleic acact all proteins, denatextraction buffer should extrd widely used solubilising buffers contain and cell debris. The most successful anchaotropic agents that will disrupt the hydrogen bonding, a combination of several
de chainsiions between hydrophobic sdetergents that prevents hydrophobic interactand helps solubilize the proteins, and a reducing agent that breaks the disulfide
residues (Chevallet et al., 1998; Tastetbridges initially formed between two cysteine uption, removal of interfering ll disre ceet al., 2003). Such detergents will achievagents, and solubilization and denaturing of proteins (Gorg et al., 2004; Herbert,

ction Introdu


agent Chaotropic disrupting the hydrogen bonding (RabilloThe use of urea and thiourea (Fig. 3A,B) increase the solubilityud et al., 1997). Urea solubilises and of proteins by
denaturates proteins, unfolding them to expose internal ionisable amino acids.
ration can be increased to 9 or 9.8 M. 8 M urea is used, but the concentCommonlyUsing thiourea in addition to urea improves solubilisation, particularly of hydrophobic
Rabilloud, 1998). membrane proteins (Molloy, 2000;

for the proteiFigure 3: Structurn extractioe of Urn. The presenea and Thiourea.ce of the Two sche aotropitwo rec reageagents, urents in the lysis buffer alloa (A) and thiourea (B)ws for th usede
solubilizatiooxygen of the carbn of non-soluonyl groble proteiup of an amins by no aciddisrupting the with the hydro hydrogegen n boof an amino nding that argroue formed bp of anotetweeher amin the no
acid. Detergents hains from side c between hydrophobic interactionsDetergents prevent hydrophobicproteins. SDS is considered the most fility ooccurring and allow for better solub ie et al., 1995; Harder etbilisation of proteins (Boucherefficient detergent for the soluc nature, it may interfere with the first dimension of al., 1999), but because of its ionireaks on the gel (Gorg et al., zontal st2-DE and it has been reported to cause hori ylglucopyranoside) and zwitterionic2004). Non-ionic detergents like OG (Octfonate) are lholamidopropyl)dimethylamino]-1-propanesureagents like CHAPS (3-[(3-cbeing favoured over SDS use (Kersten et al., 2002; Rabilloud et al., 1997; Rabilloud
et al., 1999) (Fig. 4A-C). A combination of several non-ionic and zwitterionic
bilization ct for the solue no one detergent is perfedetergents should be used becausoteins (Schuck et al., 2003). of most of the hydrophobic pr

ction Introdu


(SDS), an aFigure 4: Strnion ucturdee of detergent; terge(B) Onts uctyl seglucopyranod in solubilisation oside (OfG) a prnon-ioteins. oni(Ac d) etergeSodium Dodent; and (C) cyl Sulfate 3-[(3-
cholamidopropyl)dimethylamino]1 propanesulfonate (CHAPS) a zwitterionic detergent.
agents Reducing

To obtain well resolved spots, disulfide bridges should be cleaved to achieve total

protein. This can be done using a reducing agdenaturation of the

l (DTT) (Fig. 5). oDithiothreit

ent like

Figure 5: Structure of dithiothreitol (DTT). A reducing agent used to cleave the disulfide bridges
that can form between two cysteine residues to achieve complete denaturation of proteins and
r solubility. increase thei

ate to the basic region of the IPG strip r, DTT is a weak acid which will migreHowev

when the sample is loaded for isoelectric focusing (IEF) and will only reduce proteins

ction Introdu


al., 2002). Tributyl phosphine in that region, necessitating the introduction(TBP) (Fig. 6) was reported of excess DTT at the cathode (Hoving et as an alternative reducing
agent (Herbert et al., 1998).

Figure 6: Structure of tributylphosphine (TBP). A reducing agent used in 2-DE process.

oelectric focusing (IEF) First dimension: Is1.2.3

proteins according to their isoelectric IEF is an electrophoretic method that separateseither positive, negative, or eric molecules; they carry points (pI). Proteins are amphotthe pH of their surroundings (Fig. 7). The net charge zero net charge, depending on ges of its amino acid side harall the negative and positive cof a protein is the sum of chains and aminoand carboxyl-termini. The isoelectric point (pI) is the specific pH at
e positively charged at pH otein is zero. Proteins arwhich the net charge of the pre their pI. If the net at pH values abovively charged values below their pI and negatpH of its environment, the resulting curve charge of a protein is plotted versus the of a pH gradient is (Fig. 7). The presence intersects the x-axis at the isoelectric pointcritical to the IEF technique. In a pH gradient, under the influence of an electric field,
ent where its net charge is zero. A protein a protein moves to the position in the gradi ly lessethe cathode, becoming progressivwith a positive net charge migrates toward reaches its pI. A gradient until itpositively charged as it moves through the pH the anode, becoming lesscharge migrates toward h a negative net protein wit If a protein should diffuse reaches zero net charge. negatively charged until it alsoy gains charge and migrates back. s pI, it immediateltaway from iThis is the focusing effect of IEF, which concentrates proteins at their isoelectric
ed on the basis of very small charge oteins to be separatpoints and allows prdifferences.

ction Introdu

Figure 7:

Str es of pructurhoteric moleotein as ampctric point ocules, isoele proteins.f


the electric field the pH gradient and The resolution is determined by the slope of

ically in excformed at high voltages (typstrength. IEF is therefore per ess of 1000 V).

When the proteins have reached their final positions in the pH gradient, there is very

little ionic movement in the system, resulting in a very low final current (typically

a given electrophoresis system is generallybelow 1 mA). IEF of a given sample in

of Volt-hours (Volt-hour (Vperformed for a constant number h) being the integral of

the volts applied over the time).

ghest resolution and the conditions gives the hi denaturing IEF performed under

achiesolubilization isuration and cleanest results. Complete denatved with a mixture

of urea and detergent, ensuring that each protein is present in only one configuration

and aggregation and intermolecular interaction is minimized.

covalenAn immobilized pH gradient (IPG) is created byating a gradient of ly incorport

ylamide gel at the time it is cast. The cra into a polyacidic and basic buffering groups

Immobiline reagents is: general structure of

ction Introdu


R = weakly acidic or basic buffering group.


Figure 8:matrix with attac Structurhee of pd bufferiolyng groacrylupsamid. e gel matrix. Graphical representation of the polyacrylamide

Immobilized pH gradients are formed using two solutions, one containing a relatively

acidic mixture of acrylamido buffers and the other containing a relatively basic

mixture. The concentrations of the various buffers in the two solutions define the

range and shape of the pH gradient produced. Both solutions contain acrylamide

on, the acrylamide portion of the buffers is. During polymerizattmonomers and catalys

bisacrylamide monomers to form a copolymerizes with the acrylamide and

polyacrylamide gel. The graphical representation of polyacrylamide matrix with

attached buffering groups is shown in figure 8.

he 2-DE process. The proteins should be The IEF is the most critical step of t

solubilised without charged detergents, usually in high concentrated urea solution,

reducing agents and chaotrophs. To obtain high quality data it is essential to achieve

it self. Since different types of samples rength conditions before the IEF low ionic st

to adjust the IEF buffer and the electrical ntent, it is necessaryodiffer in their ion c

profile to each type of sample.

ction Introdu

SDS-PAGE mension: di1.2.4 Second


ed vertically according to protein molecules are separatIn the second dimension, the proteins (that is, ate treatment denatures their molecular weight. Sodium dodecyl sulfit unfolds them into long, straight molecules) and coats all proteins essentially in
proportion to their mass. ft proteins according to size because of the second dimension gel siThe poresoportion to their mass. The ially in prlphate coats all proteins essentudodecyl sellipsoids with a uncoating of the protein in negativiform negative charge-ely-charged SDS to-mass ratio, with mobilityallows the proteins to migrate as related
logarithmically to mass. Proteins are therefore separated horizontally based on their
isoelectric point and vertically based on their molecular mass. r, forms globular micelleshat, when in solution in wateergent, tSDS is an anionic detcomposed of 7080 molecules with the dodecyl hydrocarbon moiety in the core and
proteins form complexes the hydrophilic shell. SDS andthe sulfate head groups in with a necklace-likshort flexible polypepte structuride segments (Ibel et al., 1990)e composed of protein-decor. The result of the necklace ated micelles connected by
structure is that large amin a ratio of approximately ounts of SDS are inco1.4 g SDS/g protein. SDS rporated in the SDmasks the chargS-protein complex e of the
c complexes have a roughly constant net proteins themselves and the formed anioninegative charge per unit mass. Besides SDS a reducing agent such as dithiothreitol
inkages present in the proteins. When (DTT) is also added to break any -S-S-lelectrophorproteins are treated witetic separation with bothin a polyacrylamide gel h SDS and a reducing agent, the degree of depends largely on the
y linear relationship there is an approximatel In fact, molecular weight of the protein.lative distance of migration the re the molecular weight andbetween the logarithm of of the SDS-polypeptide complex.second-dimension SDS-PAGE is the tris-r The most commonly used buffer system foli, 1970). This buffer system separates tem described by Laemmli (Laemmglycine sysion and advantage of minimal protein aggregats the proteins at high pH, which confer. The Laemmli buffer system vy protein loadsclean separation even at relatively healimited gel shelflife. has the disadvantage of a

ction Introdu

1.2.5 Equilibration


quired for p with the SDS buffer system reriThe equilibration step saturates the IPG st contains buffer, urea, n. The equilibration solutionthe second-dimension separatio tional equilibration step replaces theeductant, SDS, and dye. An addiglycerol, ron introduces reagents essential for the reductant with iodoacetamide. Equilibratisecond-dimension separation. Urea (6 M) together with glycerol reduces the effects of electroendosmosis by
increasing the viscosity of the buffer (Gorg et al., 1985). Electroendosmosis is due to
ectric field and can interfere l on the IPG strip in the ethe presence of fixed chargessecond-dimension gel. the IPG strip to the with protein transfer fromGlycerol (30%) together with urea reduces electroendosmosis and improves transfer
second-dimension (Gorg et al., 1985). of protein from the first to the oteins. of denatured, unalkylated prpreserves the fully reduced state DTT Sodium dodecyl sulfate (SDS) denatures proteins and forms negatively charged
protein-SDS complexes. The amount of SDS bound to a protein, and therefore the
n. Thus, mass of the proteidirectly proportional to the harge, is additional negative celectrophorproteins on the basis of molecular mass. esis of proteins through a sifting gel in the presence of SDS separates
preventing their reoxidation during alkylates thiol groups on proteins, Iodoacetamide an result in streaking and dation during electrophoresis cesis. Protein reoxielectrophorto prevent point streaking also alkylates residual DTT other artifacts. Iodoacetamide and otherequilibration step. The second equilibration silver-staining artifacts. Iodoacetamide is intr with iodoacetamide is also used tooduced in a second
dues (i.e. when mass spectrometry is tominimize unwanted reactions of cysteine resibe performed on the separated proteins). esis. allows monitoring of electrophor(bromophenol blue)Tracking dye

Identification of proteins 1.2.6

,ns are detected by color, immunoanalysisAfter first and second dimension the protei staining intensity.e quantified by their or prebound fluorescent dyes, unique spots arto protease digestion, and sed from gel and subjected Spots of interest are exciMass Spectrometry. Mass spectrometry ing ined uspeptide fragments can be exameins as they migrate through ionized protmeasures the mass-to-charge ratio (m/z) of are analyzed and identified based on unique an electric or a magnetic field. Proteins

ction Introdu


structural features on offeatures allow identificatispectrometric signatures. These such as phosphorylation or methylation. Trypsin digestion of proteins yields peptides
another method byng MS with MALDI isthat are amenable to MS sequencing. Coupliwhich proteins can be identified. 1.3 Urinary proteomics

1.3.1 Formation of urine

plasma to eliminate waste by ultrafiltration from the Urine is formed in the kidney . Although the kidney accounts for onlyproducts, for instance urea and metabolites of plasma (350-400 ml/100 g tissue/min) 0.5% of total body mass, a large volume of ultrafiltrate (150-180 l/day) under flows into the kidney, generating a large amountultrafiltrate such as water, glucose, in the normal physiologic conditions. Componentsamino acids, and inorganic salts are selectively reabsorbed, and less than 1% of
ultrafiltrate is excreted as charges at the glomeruli (Haraldsurine. Serum proteins are filtson and Sorensson, 2004). ered based on their sizes and
After passing through glomerimmunoglobulin light chain, transferrin, vitamin D binding proteiuli, abundant serum proteins such as albumin,n, myoglobin, and
receptor-associated protein are reabsorbed, mainly by endocytic receptors, megalin,
d Gburek, 2004; Maunsbach,enal tubules (Christensen anand cubilin in proximal rurine is1997). Thus, protein concentration in normal (less than 100 mg/l very low s than 150 mg/day. etion is lesrprotein excwhen urine output is 1.5 l/day), and normal with other body fluids such as plasma. s compared This is about a factor 1000 lesday protein is defined as proteinuria and is indicative Excretion of more than 150 mg/eabsorption dysfunction. of glomerular or r

1.3.2 Sources of urinary proteins

components of solid phase luble proteins and protein Urinary proteins include soents consist of sediments that can be elements of urine (Tab. 1). Solid phase elemprecipitated at low centrifugation speeds and exosomes that are of very low density
and sediment only with ultracentrifugation. be useful as a means of enriching for markers of partiPrefractionation of thescular types of disease. A study e components can
that, of the total urinary l human adult subjects indicated of urine collected from normaremaining 3% was in exosprotein excreted, 48% was omes (Zhou et al., 2006).contained in sediment s, 49% was soluble, and the

ction Introdu



Normally present (<150 mg/day)
Defects in glomerular filter increase high
molecular weight protein (e.g. albumin)
n excretio Defects in proximal tubule reabsorption or
abnoplasma prmal proteirons iductioncrease ln of low moleow moleculacular r weiweigght ht
light chainprotein (e.g. sβ2, retinol-microgl-binobding ulin, immunoprotein,globulin and
n ) excretioscidaminoa

glycosVia exocytoylphosissphatidylino (e.g. episitol-andermal gcrhoowthred fa proteinctor) or
detachment (e.g. Tamm-Horsfall protein)

Increased cell number compatable with several
diserenalases in tubulcle celluding ac shediute tubulang) andr ne glcrosis omerular (e.g.
diseases (e.g. podocyte shedding).
Coulapoptoticd be du procese to nonses. specific, nephrotoxic, or

Sources of urinary proteins Comments
ns iSoluble Prote • Glomerular filtration of plasma proteins Normally present (<150 mg/day)
Defemoleculacts in r weigglomerularht protein (e.g. filter increasealbumin high)
n excretio abnoDefects inrmal p roductioproximal tubn of low moleule reabsocularpr weigtion or ht
plasma proteins increase low molecular weight
light chainprotein (e.g. sβ2, retinol-microgl-binobding ulin, immunoprotein, and globulin
aminoacids) excretion
• Epithelial cell secretion of soluble proteins. glycosVia exocytoylphosissphatidylino (e.g. episitol-andermal gcrhoowthred fa proteinctor) or
detachment (e.g. Tamm-Horsfall protein)
Solid phase components
• Epithelial cells Increased cell number compatable with several
a. Whole cell sheding diserenalases in tubulcle celluding ac shediute tubulang) andr ne glcrosis omerular (e.g.
diseases (e.g. podocyte shedding).
b. compoPlasma menent sheddimbraneng and intracellular Could be due to nonspecific, nephrotoxic, or
apoptotic processes.
c. Exosome secretion Normal process
• Other cells cellIn certain dis, or tumor cellseases, red bls (e.g.ood bladdecells, whir cantcer ae bloodnd
lymhome) can be present in urine.
Table 1: Sources of urinary proteins. (Pisitkun et al., 2004).
glomerular filtration. Thee are derived largely from in urinThe soluble proteins proteins. ssage of high molecular weight filter effectively retards paglomerularHowever, even with very low sieving coefficients, proteins that are abundant in the
erular filter in he glomlins can pass tma such as albumin and various globublood plas the nephron. Beyond this, peptides andsubstantial amounts to enter the lumen ofsmall proteins (10 kDa) are freely filtered by the glomerulus. Most of the proteins and
ed in the are scavenged and proteolyzular filterhat pass the glomerpeptides tproximal tubule by highly specialized apical uptake processes that involves receptor-

Normal process

cellIn certain dis, or tumor cellseases, red bls (e.g.ood bladdecells, whir cantcer ae bloodnd
lymhome) can be present in urine.

ction Introdu


, 2001; ntensen and Birypeptide molecules (Chrislike recognition of the polotein that ount of a given soluble prChristensen, 2002). Thus, a change in the am a change in its concentration in the bloodreaches the final urine can result from , or an alteration in the ilteron of the glomerular fplasma, a change in the functiproximal tubule scavenging system (Fig. 9).

Figure 9: Mechanism of urinary exosome formation and excretion. Apical membrane proteins
undergo endocytosis followed by targeting to the.multivesicular body (MVB). The membrane proteins
are segreinvagination, encapgated initially isulatingn cytosoli the MVB c proteins in the outer membprorane acess. After nd thenaccum are internulatioalizen of numerod by muse internmbrane al
vesicles, the outer membrane of the MVB fuses with the apical plasma membrane releasing its
internal vesicles, called exosomes, into the urinary space. Exosomes contain both membrane and
cytosolic proteins (Hoorn et al., 2005).

Based on t ion rate of specific urinary proteinshese mechanisms, changes in excretproximal tubule, rescan be indicative of systemic disease, glomerpectively. Some of the soluble proteins in urine originate asular disease, or diseases affecting the
ically cleaved from their membrane membrane-bound proteins that are proteolytattachments.

ction Introdu


cancer Bladder 1.4 ccuring worldwide. The rate he most common cancer type oBladder cancer is one of ttries, where they rank as the sixth most loped counefor these tumors is highest in dev rrently ranks as the fourth most common cause ofufrequent neoplasm. The disease cdeaths from bladdercancer death in men and the eighth in cancer are 13,180 in women. Estimated new casthe United States ein 2005 (American s are 63,210 and
bladder cancer increases with age 2005). The incidence ofCancer Society, Atlanta, before age fourty. The xty and is uncommon high after age of si particularlyand isurinary bladder has a flat, smooth, shiny, watertight lining consisting of layers of cells
ng of the bladder can be imagined to be h each other. The linitightly connected witsimilar to the lining in the oral cavity (mouth). Underneath this lining is the muscle
the time out the urine ate is responsible for pushing tissue of the bladder. The muscl in The bladder liesound 350-400 cc in adults.of voiding. The capacity of bladder is arthe pelvis anterior and inferior to the peritoneal cavity, and posterior to the pubic
bones. In the female, it rests directly on the muscular pelvic floor, while in the male
der and separates it the base of the blad attached directly tothe prostate gland isfrom its muscular support. The cells grow abnorBladder cancer is an abnormal growth or tumomally fast causing a tumor r arising from the linto sprout up from the flat lining into a ing of the bladder.
he bladder cavity. Bladder cancer occurs within growth projecting into the interior of tent types of etiological factors such the domain of human neoplasms by many differ -naphtylamine, benzydine, 4-nitro diphenyl),βas some aromatics (i.e. xylamine, coffee, artificial sweetWynder and Stellman, 1977). Most eners and smoking of bladder cancers present(Cohen et al., 1979; Morrison et al., 1982; as superficial disease
The rate of recurrence on by endoscopic resection.iand are amenable to local excisogression in stage is from 15% to 25%. ranges from 50% to 75% and the rate of prwith biopsyCystoscopy is the gold standard for routine of suspicious lesions and histsurveillancopathologic evaluation e for bladder tumor recurrence, required to confirm
the diagnosis (Tsihlias and Grossman, 2000).

Staging of bladder cancer 1.4.1

the bladder is determined by the depth of The clinical staging of carcinoma of aluation (Fig. 10). Based on morphologic ev the bladder wall by the tumor invasion ofing assified into two groups havl been cand natural history, urothelial neoplasms have

ction Introdu


) low-grade tumors (always papillary and usually idistinct behavior and prognosis; (superficial) and (ii) high-grade tumors (either papillary or non-papillary and often
mors (stages Ta, Tis, and T1) account for). Clinically, superficial bladder tueinvasiv75% to 85% of neoplasms, whereas the remain (T2, T3, eing 15% to 25% are invasivT4) or metastatic (N+, M+) lesions at the time of initial presentation (Rabbani and
CC) haserican Joint Committee on Cancer (AJAmCordon-Cardo, 2000). The er (American Joint define bladder cancodesignated staging by TNM classification tAJCC stage grouping is shown in table 3. (Tab. 2). Committee on Cancer, 2002)

Figure 10based-on the: Repr depth of tuesentamor invation of bladdsioner canc into the bler satages. dder wall (FScherom Hame for blautmanddn er tuand Hulamor clnad, ssifUrolicatiogy, on
r). isheer-PublSpring

classification 1.4.2 Cellular

transitional cell carcinomas derived from More than 90% of bladder carcinomas are the uroepithelium. About 6% to 8% are squamous cell carcinomas and 2% are
arcinomas may be either of urachal adenocarcinomas (Mostofi et al., 1988). Adenoctype is generally thought to arise from origin or of nonurachal origin; the latter metaplasia of chronically iPathologic grade, which is based on cellurritated transitional epitheliumlar atypia, nuclear abnormalities, (Wilson et al., 1991). and the
ic importance. Resection of the tumor in number of mitotic figures, is of great prognosthis entirety, with inclusion of the lamina propria and muscularis propria, and biopsy of

17 ction Introdued erytematous areas) are us areas (i.e., nontraumatizopically suspicioall endosc ensure proper staging.generally performed to ons TNM DefinitiTX Primary tumor cannot be assessed
mor ce of primary tunNo evideT0 noma illary carcipapnvasive Ta NoniTis Carcinoma in situ (i.e., flat tumor)
T1 Tumor invades subepithelial connective tissue
T2 Tumor invades muscle
pT2a Tumor invades superficial muscle (inner half)
pT2b Tumor invades deep muscle (outer half)
T3 Tumor invades perivesical tissue
PT3a Microscopically
PT3b MacrTumor invaosdecopically (extras any of the followinvesical masgs) ; prostate, uterus, vagina, pelvic wall, or
T4 nal wall abdomi PT4a Tumor invades the prostate, uterus, vagina
PT4b Tumor invades the pelvic wall, abdominal wall
mph nodes (N) al lyRegionNX Regional lymph nodes cannot be assessed
NO No regional lymph node metastasis
N1 Metastasis in a single lymph node, =2 cm in greatest dimension
N2 Metastamultiple lympsis in h nodea singls, =5 cm in greatee lymph node, >2 cm but =5 cm ist dimension n greatest dimension; or
N3 Metastasis in a lymph node, >5 cm in greatest dimension
Distant Metastasis (M)
MX Distant metastasis cannot be assessed
is sstant metastaNo diMO metastasis M1 Distant Table 2: StaJoint Committee on Canging of bladcder caer (AJCC) ncer.using TNM cl Definition of bladdassification. (Ler cancamer whim, 1994).ch wa s designed by American
e adjacent or distant mucosa if urineDirected biopsies may be performed to evaluatt in the emenurethra to evaluate its involv positive. Biopsy of the prostatic cytology isoverall tumor diathesis (especially in the setting of CIS) is also important (Lee and
Droller, 2000).

ction Introdu

e groupings agtCC sAJ


Ta, N0, M0 Stage 0a N0, M0 ,TisStage 0is T1, N0, M0 Stage I T2a, N0, M0; T2b,N0, M0 Stage II T3a, N0, M0; T3b, N0, M0; T4a, N0, M0 Stage III Stage IV T4b, N0, M0; Any T, N1,M0; Any T, N2,M0; Any T, N3,M0; Any T, any N,M1

CancTable 3: AJerCC s (AJCC) (Lamm, 19tage groupings. 94). Classification of bladder cancer by American Joint Committee on
Clinical proteomics and biomarkers 1.5 iology, sough severe changes in human phyMany diseases manifest themselves thrwhich forms the basis for clinical chemistry and confers its value in diagnoses and
subsequent therapeutic interventions (Bischoff and Luider, 2004). Clinical proteomics
eins expressed by the genominclude the analysis of prot e of an organism, with thetypical aim being the evaluation of quantitative changes that occur as a function of
e . Proteomics strategies aronment (Somiari et al., 2003)disease, treatment, or envirbeing used to identify disease-specific protein markers called biomarkers that could
new diagnosis methodologies, treatments, provide the basis for the development of 000; Somiari et al., of cancer (Hanash, 2and early detection of diseases particularly identification using followed by protein 2003). Already for a number of years, 2-DEtechnique for biomarker discovery in mass spectrometry has been the primary org et al., 2000; Hanash, 2000).conventional proteomic analyses (G 1.6 Urinary biomarkers
Because urine can be collected noninvasively in large amounts, it provides an
(Thongboonkerd et al.attractive alternative to blood plasma as , 2004). The use of urinary biomarkers a potential source of disease bto diagnose disease is a iomarkers
omarkers of disease in urine have beenlong-standing practice. Studies to identify bith century and igative medicine throughout the 20ing component of investan underlyearly 21st century. These studies have been based on knowledge of the
al ould be tested in clinicidentify biomarkers that cpathophysiology of disease to mples has revealednormal human urine sag of trials. Large scale proteomics profilinducts and many more peptide otein gene prothe presence of at least 1000 different pr(Castagna et al., 2005; Jurgens et al., 2005; Oh et al., fragments of larger proteins 2004; Pisitkun et al., 2004; Smith et al., 2007; Sun et al., 2005). There is hope,

ction Introdu


therefore, for discovery of urinary protein excretion profiles that can be used clinically
tion of diseases particularly of cancer, caifor tasks such as early detection and classif and monitoring of a particularsessment of prognosis, agents, aschoice of therapeuticbeen identtherapeutic regimen. Several ified, including nuclear matrix potential diagnostic markers for blprotein 22, bladder tuadder cancmor antigen, and er have
telomerase. angiogenesis 1.7 Tumor ancer growth is the development of a newOne of the critical events required for cvasculogenesis (Folknetwork of blood vesmsels. This is provian and D'Amore, 1996)ded by angiogenesis as . Angiogenesis is defined as the well as postnatal
sprouting of new vessels from pre-exisCarmeliet, 2004), while postnatal vasculogting blood vessenesis desels (Folkman, 2003; Luttun and cribes the formation of new
d or derived from ooating in peripheral blvessels by endothelial precursor cells circul Gehling et al., 2000; Rafii et al., the bone marrow (Asahara and Kawamoto, 2004;ly demonstrated (Zengin et al., 2006). 2002) from the vessels wall as recent ated by angiogenicis are regulsculogenesAngiogenesis as well as postnatal vaman, 1996). The structural formation and activators and inhibitors (Hanahan and Folk a veryis and angiogenesis issculogenesmaturation of blood vessels during va iferation and tube steps including prolcomplex process that runs in successive the basement membrane, integration of formation of endothelial cells, construction of, and embedding of blood vessels into the peri-endothelial cells into the vascular wall1996). Numerous angiogenic fperi-vascular tissue (Ergun et al., 2006; Foactors including vasclkman et al., 1989; Foular lkendothelial growth factor man and D'Amore,
(VEGF), fibroblast growth factor-2 (FGF-2), angiopoietins (Ang1, Ang2), and their
receptors, which belong to the receptor tyrosine kinase family, are involved in several
aisonpierre et al., 1997). steps of this process (M and acts asnesisand pathological angiogeVEGF is the key regulator of physiological , both in vitro and in vivo (Ferrara, 2005). a survival factor for endothelial cells (EC)The biological effects of VEGF(RTKs), vascular endothelial growth fact are mediated by two receptor receptor-1 (VEGFR-1) and VEGFR-2 or tyrosine kinases
F with its receptor VEGFR-2 , the interaction of VEGularly(Ferrara et al., 2003). Partic

ction Introdu






with a deFigure 11: Vascular nse BM (gray) enclodestabilization sing both endothand initiationelial cell of angios (gregenesis.en) and peri A normal stabilicyte (red) (Azed capillary ) will be
destaendothebilized lial by action of fenestrationpro (fn), o-angiogepennic factoing of inters as r-eexemplandothrelial contactsily shown by VEGF and An (iec), develg2opment leading toof
endothtransendelial lothelayer (Bial gaps (g). Thaese mp), degorpradhogenetiation of BM ac events are nd finally daccoempanietachmd byent of an abn pericyteormsal vascula from ther
and ileakinnestabss. Thle bloode furthe vesselr durats (C), aion of pro- process dangiogeefinednic a acsti angioon wogeneuld finasis (Adolly lead to sppted from Ergürouting of nen et al., Canw nascentcer
Encyclopedia, 2007, in press).
(known also as KDR in human or Flk-1 in mouse) intiates the angiogenic activity by
causing the structural destabilization of the vascular wall with subsequently increased
vascular leakeness (Ergun et al., 2006; Thurston et al., 2000). Also angiopoietin-2
and both Ang1 and Ang2 act in-1 (Ang1)of the angiopoiet(Ang2), a partial antagonist e in the-2, is essentially involvedvia the same tyrosin kinase receptor Tidestabiliazion of the vascular wall as shown in figure 11. enzymes degrading he extravasation of mal vascular leakeness results in tThe abnore enzymes is the uPA, the urokinase the vascular basement membrane. Among thes volved in the remodelling extracellularnogen activator, a key enzyme initype plasmthe angiogenic activity of on of endothelial cells and thus matrix supporting the migratihe action of this enzyme is ., 2003). T (Agirbasli, 2005; Rakic et althese cellsr-1) which has been shown to otor inhibitamodulated by the PAI-1 (plasminogen activinder et Bor the receptor of uPA, uPAR (have a dual role in the interaction with uPA al., 2007). The concerted action of the mentioned factors above howeever results in
the complete disintegration of endothelial cells of pre-existing blood vessels with
and finally in the of endothelial cells,oliferation subsequent migration and prels. outgrowth of new vessels from pre-existing blood vess proteins: Orosomucoid (ORM) yOne of urinar1.8 ha1-acid glycoprotein (AGP), was firstly alpOrosomucoid (ORM), also known as Schmid, 1953). It belongs to a group of described in 1950 by Schmid (Schmid, 1950;

ction Introdu


acute phase proteins (APPs) and plays a role in the modulation of the immune
functions (Schmid K, 1975; Bennet M, response to stress, along with many other ORM in the serum of healthy humans is 1980). The relatively high concentration of conditions such as acutee to differentfold in responsknown to rise two- to fiveer (Schmid, 1975). s and cancive disorderinfection, inflammatory and lymphoproliferata polypeptide chain ed and is composed of The structure of ORM is well characteriz of fucosylic and sialice including a large amount containing about 45% carbohydratacid. Thus, its proposed immunomodulatory activities have been attributed to its
fucosylated and sialylated ORM glycoforms glycosylation pattern, since the strongly inhibit complement activation (De Graaf ethave the ability to bind E-selectin and to al., 1993). ent component 3, serum amyloid A, C-Type 1 APPs, including ORM, complemegulated by IL-1, IL-6 and in and hemopexin, are rreactive protein, haptoglobglucocorticoids and type 2 APP, including the three chains of fibrinogen and several
proteases inhibitors, are regulated by IL-6- type cytokines and glucocorticoids
types e.g. granulocyt(Baumann et al., 1989).es (Fournier T, 2000) ORM is synthesiz. ed in livIL-1, IL-6 and glucer and various extrahepatic cell-ocorticoids are the
et al., 1989; Baumann and Gaulmajor modulators of ORM gene expression in liver cdie, 1990). In most instells ances, a strong synergistic(Alam et al., 1993; Baumann
action is achieved by the combination ofBaumann et al., 1987). An induced expression of sialyl the three factors (Alam et al., 1993; Lewis X (sLeX) on ORM
influencduring acute the E- or P-selectin-e inflammation has been reported, mediated inflleading tux of sLeX-expressing leukoco the speculation thatytes into it might
inflamed areas. It has been suggested that ORM could have a feedback inhibitory effect an increaon the extravasation of leukocsed level of sLeX-expressing ytes, by
competition for the E-selectmodify the permeability of the vascular endin adhesion molecules (Lasky,othelium, possibly by interacting with the 1992). ORM is thought to
endothelial glycocalyx (Curry and Michel, 1980). Thus far it has been shown that
ORM binds to the vascular endothelial cell surface (Fig. 12) and then causes
intercellular junction (Predescu et al., e cell without passing the transcytosis across th1998).



Figure 1transmigratio2: Hyn of leukopothesis focr thytes to a site oe inhibitif inflaon of leukmmation are ocyte extravsummariseasationd. ORM e byx ORM.pressin Events lg sialeadinyl Lewisxg to
interacts with E-selectin expressed at the surface of endothelial cells and compete with leukocytes
expreleukocytessisng ES may then bL-1, the ligeand of E-sel inhibited. (From E.C. ectin. ConseHavenquently, rolliaar, Thesisng, ad, Vrije hesion and extravaUniversiteit, Amsterdam,sation of

stem The plasminogen activation sy1.9 e the formation of the serine proteasThe plasminogen activation system regulates

arts with s of fibrinolysis sthe procesplasmin and subsequent fibrinolysis. T

plasminogen, a 92-kDa single-chain oenzyme, conversion of an inactive pr

to the active enzyme, plasmin (Forsgren glycoprotein consisting of 791 amino acids in

net al., 1987). Plasmin is, in turdegrade fibronectin, laminin, vitronectin, , able to

proteoglycans, as well as fibrin and activate latent collagenases. Angiogenic growth

factors induce the expression of tissue-type plasminogen activator (tPA) and

PA) on the surface of endothelial cellsvator (uiurokinase-type plasminogen act

e serine-Pepper et al., 1991). Both tPA and uPA ar(Flaumenhaft et al., 1992;

ymogen plasmin by proteolytic cleavage of its zproteases that can generate

plasminogen. Plasminogen, like fibrinogen and other plasma components of the

ction Introdu


provisional matrix, is synthesized in th response to e liver and deposited in is essential for invasion and migration ofhyperpermeability. The formation of plasmin minogen activation scularized. The plasendothelial cells into the tissue to be vasystem is not limited to endothelial cells. While tPA is almost exclusively expressed
by endothelial cells (Mandriota and Pepper, 1997), uPA also facilitates migration of
ts and tumor cells (Del et al., 1990; fibroblas like epithelial cells,other cellsMacDonald et al., 1998). A variety of cell types can bind components of the
fibrinolytic system, including plasminogen (Miles and Plow, 1985), plasmin (Correc et
PA (Hajjar et al., 1987). There are two lli et al., 1985) and taal., 1990), uPA (VassPAI-1 and PAI-2. Plasminogen activator minogen activator inhibitors, main plastor of plasminogen activators (Carmeliet et inhibitor type 1 (PAI-1) is the main inhibiminogen-ents 60% of plasThe PAI in blood repres al., 1993; Fay et al., 1992).(Fig. 13). PAI-1 inhibits both tPA and uPA, activation inhibitor activity in plasma only uPA.swhereas PAI-2 inhibit



t-PA /u-PA

Fibrin degradation


Cellular ECM migration remodelling Angiogenesis

moleculeFigure 13: s is the maiPlasminogen plasn activminogen-actiation syvator inhistem. bitor in The pla blood asmind nogenit is impo-artantctivator inhibito for fibrin der 1gradatio (PAI-1) n,
remodelling of ECM and angiogenesis (Agirbasli, 2005).
which only a signaling peptide and a short Unlike the simple digestive proteases, inytic proteases bear c region, the fibrinolactivation domain are attached to the catalytiatalytic regions (Patthy, 1985). large nonc ch as "kringle", "growthonally autonomous modules, suThese regions contain functi outside the serine ns, which also occure" and "finger" domaifactorlike" or "EGFlikprotease family. A graphic representation of the domain arrangement in plasminogen,
uPA and tPA is shown in Fig. 14.



Figure 14: Graphical representation of the protein domains of plasminogen, tissuetype
EGFlike plasminoge(EGF) and n activator (tPA) ancatalytic (CD) domaid urokinase ns are marke(ud. PA) proThe activatenzyimes.on site The krins are igle (Kndicated by a), Fingen arrow. r (F),
lfide bond-linked polypeptide uof two dise plasmin consists The serine proteinas

The C-five so called kringle domains. chains. The N-terminal A chain contains

terminal B chain contains a typical serine proteinase domain, which is responsible for

olysis of peptide bonds on the s the hydrelyzcatalytic activity (Fig. 15). Plasmin cata

and ArC-terminal side of Lys asminogen to plasmin cang residues. Conversion of pl

her uPA or tPA. be catalyzed by eit

an exposed center loop that is availableThe active form of PAI-1 is thought to have

renc (Lawfor interaction with proteinasesrence, 1997). The e et al., 1995; Law

ating this surface-res incorpo form involvconversion from active PAI-1 to its latent

-sheet of the protein βve center into the central exposed loop containing the reacti

1992). ORM, one of the major acute phase (Lawrence et al., 1994; Mottonen et al.,

ct with PAI-1 and stabilizeproteins, can intera

tory activity towardi its inhib



plasminogen activators (Boncela et al., 2001). The ORM-PAI-1 co-localization was

found in thymosin β4 (Tβ4)-activated but not in quiescent HUVECs.

Figure 15: Schematic presentation of uPA and its interacting partners. uPA consists of a serine
proteinase domain (SPD), a linker, a kringle (K), and a uPAR-binding epidermal growth factor-like
and 3 domain m(E). uake contacPAR has thret with uPA. e domainPlasmins an had is ans a sechorinered proteinase do to the membrane by a GPmain (SPD) andI ancho five krir. Dongles (Kmains 1)
and binsite of uPA. Vitroneds to pericellular prctin, in the suboteins stratwith C-terminal lysium, has anne N-termins. The al PAI-1- aRCL of PAI-1 is abnd uPAR-binding sole to bind to the active matomedin
B domain (SomB) next to an integrin-binding RGD sequence. α and β integrin subunits have
transmembrane helices (TM) and are anchored to the cytoskeleton. Integrins can interact with uPAR
(Andreasen et al., 2000).

udy The aim of st

The aim of study2


urine proteins present in) to analyse the whole pattern ofiThe aim of this study was: (

urine samples of healthy persons in comparison to that of patients with bladder

r etion between urine proteomics and bladdcancer, and to find out the potential rela

ORM, as one of urinary proteins ) to explore the potential effect ofiicancer stages. (

found as an increase amount in cancer, in vascular and urine of patients with bladder

the interaction between ORM ) to study potential effect ofiiicapillary morphogenesis. (

. and capillary morphogenesisand PAI-1 in angiogenesis

s Methodnd Material a

Methods and 3 Material 3.1 Materials

Chemical and Consumables 3.1.1



Sigma Acetonitril Serva Acrylamide PeqLab Agarose Sigma Ampicillin Merck APS SiTrypsin Bovine LadderBench Mark Prestained Protein CHAPS PureCol Collagen, Coomassie Brillant Blue G-250 Gibco DMEM Merck DMSO BioRad DTT Ethidiumbromide
Gibco FCS ThermoShandon, Histomount Iodacetamide Sigma AgarLB Gibco L-Glutamin (NP-40) Nonidet Gibco MEM Normal Swine Serum (NSS) Normal Rabbit Serum (NRS) Nitrocellulose-Membrane otides (Primer,siRNA) leOligonuc Seromed cco lbeDuPBS Penicillin/Streptomycin
ktail Protease Inhibitor Coc KodakX-Röntgenfilms Sigma Solution Silvernitrate

(Taufkirchen) Sigma (Heilderberg) (Erlangen) (Taufkirchen) Sigma (Darmstadt)gma-Aldrich (Steinheim) Holland) Invitrogen (Groningen, LadderSigma (Taufkirchen) Inamed (Nutacon,Finnland) BioRad (München) (Karlsruhe) BRL (Darmstadt) (München) Serva (Heidelberg) (Karlsruhe) BRL USA) (Pittsburg, Sigma (Taufkirchen) (Taufkirchen) (Karlsruhe) BRL tadt) Calbiochem (Darms (Karlsruhe) BRL Dako, (Hamburg) Dako, (Hamburg) Schleicher & Schuell BioScience (Dassel)
MWG Biotech (Ebersberg) (Berlin) Gibco BRL (Karlsruhe) Sigma (Taufkirchen) AR5 Kodak (Stuttgart) (Taufkirchen)

28s Methodnd Material aSDS-PAGE marker wide range Sigma (Taufkirchen)
T4 DNA Ligase New England Biolabs (Schwalbach/Taunus)
(München) BioRad TBP (Taufkirchen) Sigma TEMED (Taufkirchen) Sigma Thiourea Sigma (Taufkirchen) X-100 Triton Trypsin/EDTA (1x) Gibco BRL (Karlsruhe)
(Taufkirchen) Sigma Urea Mowiol Calbiochem (Darmstadt)
Falcon (Heidelberg) Cell culture flasks, plates, and tubes (Neustadt) edium) Cryo-Save1 (conservation m were purchased from following umablesals and consUnless otherwise stated chemicreiburg), BioRad (München), Biozym ences (Fi Amersham Biosccompanies:nnheim), Merck (Darmstadt), Roche (Ma Holland), (Hameln), Invitrogen (Groningen, unus), Promega (Heidelberg), RocheNew England Biolab (Schwalbach/Taberg) and Sigma-Aldrich (Taufkirchen). rva (Heidele(Mannheim), Roth (Karlsruhe), S3.1.2 Kits Vector (USA) x (ABC) kit Avidin-Biotin Comple Pierce (USA) Protein Assay Kit BCAQiagen (Hilden) RNeasy Mini Kit(Freiburg) Biosciences t Amersham ECL Plus Western Blotting Detection KiAmbion (Darmstadt) on Kit nstructincer siRNA CoSileSA) Amaxa-Biosystem (UHMVEL-L Nucleofector Kit Holland) trogen (Groningen,Invi TOPO TA Cloning kit®Macherey-Nagel (Düren) Plasmid Kit NucleospinQiagen (Hilden) ation Kit EndoFree Plasmid Maxi PreparQiagen (Hilden) Gel Extraction Kit(Freiburg) Bioscience t Amersham el Band Purification KiGFX PCR DNA and GMillipore/Amicon (Bedford, USA) it Microcon YM-10 Centrifugal Filter UnMillipore/Amicon (Bedford, USA) Unit Centricon YM-10 Centrifugal Filter BioRad (München) ReadyPrep Sequential Extraction Kit BioRad (München) 3-10 IPG Strips (11,17 cm) pHBioRad (München) Criterion Precast Gel

s Methodnd Material a

Stock Solutions and Buffers 3.1.3


epared according to standard proceduresStandard media and stock solutions were prusing deionised water. Solutions were sterilised by autoclaving (25 min/ 121ºC/ 2
ock solutions, filter-sterilised tpared as ss were prebars). Heat sensitive componentm) and added to the medium/buffer after cooling to 50ºC. μ(0.2 Tris-HCl 5x SDS-PAGE Running 125 mM Buffer (TGS Buffer)
SDS Glycin 0.5% 960 mM (w/v)
2D-PAGE Rehydration Buffers: equential Extraction Kit)Reagent 3 (ReadyPrep SUrea Thiourea 5 2 M M
CHAPS SB 3-10 2% 2% (w/v) (w/v)
Tris Biolyte 3-10 40 2% (v/v) mM
TBP(fresh) DTT(fresh) 2 0.5% mM (w/v)
Bromphenolblue trace Urea 8 Reagent 2 (ReadyPrep SM equential Extraction Kit)
SB 3-10 CHAPS 4% 2% (w/v) (w/v)
Tris Biolyte3-10 40 0.2% mM (v/v)
DTT (fresh) TBP (fresh) 2 mM 0.5% (w/v)
Bromphenolblue trace Urea 5 2D-PAGE EquilibrationM Buffer I Urea 5 2D-PAGE EquilibrationM Buffer II
SDS Tris-HCl (pH 8.8) 20% 1.5 M (w/v) SDS Tris-HCl (pH 8.8) 20% 1.5 M (w/v)
Glycerol DTT(fresh) 0.5% 50% (w/v)(w/v) I Glycerol odacetamid(fresh) 2.5% 50% (w/v) (w/v)
Fixer Solution I for gel staining Fixer solution II for gel staining
Methanol Acetic acid 50% 10% (v/v) (v/v) Methanol Acetic acid 5% 7.5% (v/v) (v/v)

s Methodnd Material a



Sodiumcarbonate Developer Solution 6% (v/v) Coomassie BB G-250 Coomassie Staining Solution 0.1% (w/v)
HFormaldehyde 6 mM (NH3PO4)42 SO4 2% 8% (v/v(w/v) )
5x Laemlie Protein Loading Buffer RIPA Lysis Buffer
Glycerol Tris-HCl (pH 6.8) 30% 50 mM (v/v) NaCl 150 Tris-HCl (pH 7.4) 50 mM mM
SDS 4% (w/v) NP-40 1% (v/v)
mM 1 (w/v) EDTA 0.5% Bromphenolblue DTT (fresh) 0.1 M SDS (fresh) 0.1% (w/v)
nhibitor cocktail (fresh) I Protease Triton X-100- SDS Lysis Buffer 10x Blotting Buffer
SDS Triton X-100 1% 1% (v/v) (w/v) Glycin Tris-HCl 1 1.93 M M
10x TTris-HCl 200 BS Buffer, pH 7.6 mM TBS-T Buffer1x TBS (pH 7.6)
NaCl 1.37 M Tween-20 0.1% (v/v)
(w/v) 0.5% SDS rylamide GelsclyaSDS Po Tris; pH 6.80 5 M Running Gel (12%):1.5 M Tris; pH 8.8 Stacking Gel (4%):SDS 4% SDS 4%
Stripping Buffer RNA/DNA-Loading Buffer
βTris-HCl (pH 6.7) -Mercapthoethanol 100 62.5 mM mM XyBromphenlencyanol olblue 0.4% 0.4% (w/v) (w/v)
SDS 2% (w/v) EDTA 1 mM
Glycerin 50% (v/v)
RbCl TFB1 (pH 5.8) 100 mM MOPS TFB2 (pH 6.8 by KOH) 10 mM
MnCl2 Potassium acetate 50 30 mM mM RbCl CaCl2 10 15 mM mM
CaCl2 10 mM Glycerol 15% (v/v)
(v/v) 15% Glycerol 0.1 M PBS (pH 7.4) 0.1 M PB (pH 7.4)
NaNaCl 2HPO 0.2 4 0.2 M M Na KH22POHPO4 4.2H2 0.018 O 0.082 MM
Antibody dilution buffer (PBS/ BSA/ NaN3) 50x TAE Buffer (pH 7.5)
BSA 0.1 M PBS (pH 7.4) 0.2% (w/v) Tris-BaseGlacial acetic acid 2 M 5.7% (v/v)
NaN3 0.1% (w/v) EDTA 50 mM


s Methodnd Material a

Glucoseoxidase solution (for 50 ml)DAB 0.063 PB 0.1 M (pH 7.4) M 1 45 ml ml
NHNiSO44 Cl 3.35 M 0.05 M 900 100 µl µl
Glucose (10%) Glucose-oxidase 179 U/ml 900 150 µl µl
l Mixture (for 10 ml)eCollagen GSterile wat10x MEM Medium er 3.95 1 ml ml
L-Glutamin 200 mM Sodiumpyruvate 100 mM 100 µl 100 µl
Collagen 4 Sodium bicarbonate (7.5%) 500 µl ml
40 µl Sodium hydroxyle 0.1 M

Equipment and Applications 3.1.4

: Amaxa., BioSystems NucleofectorCameras: ProgResTMC10plus, Jenoptik

: Phase contrast microscope (Zeiss, Jena); Axiovert 25 (Zeiss, Jena); Microscopes

SM-Lux (Leitz)

: 5415D Centrifuge (Eppendorf) Centrifuges

: Hera Cell 240 (Thermo) Incubator

mocyclerPCR-Ther iometra): T3000 Thermocyler (B

artSpec 3000 (BioRad) m: UV-Spectrometer, SPhotometer

Axima CFR (Shimadzu Biotech) :MALDI-TOF spectrometer

Criterion Dodeca Cell, Protean :electroblotting equipmentGel electrophoresis and

Equilibration Tray (BioRad), Mini protean and ion/IEF Cell, Disposable Rehydrat

Trans-Blot-System (BioRad).


s Methodnd Material a

3.1.5 Antibodies


Antibody Source Working dilution Supplier
Anti-Alpha1 acid glycoprotein Rabbit 1:200 Dako, Hamburg
GP) (AAnti-Zinc Alpha Glycoprotein(ZAG) Goat 1:200 Santa Cruz, USA
Vimentin Mouse 1:1000 Dako, Hamburg
Table 4: Primary antibodies used for western blot and Immunohistochemistry

Antibody Working dilution
Anti-mouse IgG 1:8000 Sigma,
1:30,000 Sigma, t IgG -rabbiAnti1:30,000 Sigma, t IgG -goaAntiTable 5: Secondary HRP antibodies used for western blot

3.1.6 Cell lines and medium for cultivation of cell lines

Supplier en rchTaufki en rchTaufkien rchTaufki

ons and supplements used in cell culturechased sterile, all media, solutiUnless purwork were sterilised with a 0.2 µm filter, strored at 4°C and prewarmed to 37°C prior
to use. Source cs acteristiCharCell Lines HDMECs CellHumas (HDMECn Dermal Microvas) desrived from dermicular Endots helial PromoCell, Heildelberg
Obtained from Department of RT4 Human Bladder Cancer Cells UrolHamburg-Eppendogy, University Hospitalorf
d cell lines UseTable 6:

Medium Cell LinesSupplemEndothelial entCell GMix, the conrowthcentration Medium s ofMV grow (Promth faoctCeors are; ll): After ad0.4% ECGS/H; ding the
HDMECs Hydrocortison; 1ng/mL ba5% Fetal Calf Serum; 0.1 ng/mL Epidermsic Fibroblast Factor al Growth Factor; 1µg/mL
RT4 peniMcCoys Mecillin/ streptomycin dium (Gibco,USA): 10% Fetal Calf Serum; 1% Glutamine; 1%
ation medium for cell lines CultivTable 7:

strains 3.1.7 Bacterial

and XL1-Blue were used. Ligation α DH5ations, the E. coli strainsFor cloning applicstrain; plasmids requiring digestion with reactions were transformed into XL1-Blue strain. PCR αstriction enzymes were transformed into DH5emethylation sensitive rproducts were cloned using the TOPO TA cloning kit (Invitrogen) with the supplied
Top10F' bacteria.

s Methodnd Material a


Strains Genotype Source
DH5α F' endφA1 80hslacdZ∆R17(rM15 k-∆(,mklac+) ZYphA-oA argsupF)U16E44 9ג - deothi-R 1 regyrcAA1 96 Invitrogen (Groningen,
Holland)A1 rel XL1-Blue recA1, endA1, gyrqA96, thi-1, hsdR17, RsupE44, relA1, Stratagene(Heildelberg)
lac {F', proAB, lacIZΔM15, Tn10(Tet)}
F- {lacIq Tn10 (TetR)} mcrA ∆(mrr-hsdRMS-mcrBC) Invitrogen (Groningen,
Top10F' φgal80U lacgaZl∆K rpM15 s∆L (StrlacR) X74 endrecA1A1 nuparaG D139 ∆(ara, leu)7697 Holland)
Table 8: Bacterial Strains. Names, genotypes and suppliers of the bacterial strains used.

3.1.8 Primers

leotides used for PCR and contructOligonucion of siRNA were purchased from MWG Biotech.

NamPrimer e Line Sequence ( 5→3 ) NumGenbaber nk Tm (°C) PCR cnumbers ycle
Table 9: Oligonucleotides used for in PCR

Primer Name Line Sequence ( 5→3 )
Table 10: Oligonucleotides used for in siRNA construction

) (°CTm65.3 65.3 63.9

s Methodnd Material a


samples 3.1.9 Urine h bladder cancer of different stages witSpontaneous urine samples of 45 patientss were lthy volunteer and heaGIII), patients on follow-up(pTa, pT1, pT2-3, GI, GII, collected and after centrifugation at 5000 rpm for 5 min stored at 20°C (for a longer 80°C) until their use. time than 2 months at

(n=45) ts cer Patienr CanBladdePT1(n=8) PT2-3(n=8)
Age, 60-80 Age, 60-80
Female, n=2 Female, n=1
Male, n=7 Male, n=6 GI, n=GII, n=1 3 GIII, n=8
4GIII, n=

(n=45) ts cer Patienr CanBladdepTa (n=12) PT1(n=8) PT2-3(n=8)
Age, 60-80 Age, 60-80 Age, 60-80
Female, n=7 Female, n=2 Female, n=1
Male, n=7 Male, n=6 Male, n=5 GI, n=4 GI, n=1 GIII, n=8
GII, n=8 GII, n=GIII, n=4 3
Patients on follow-up (n=10) Healthy Volunteers (n=7)
0 30-50 Age 60-8n=3 Female n=4 n=7 Male n=6 Table 11: Urine samples used in 2-DE analyses and western blotting
Tissue samples 3.1.10 Tissue samples were provided from the Department of Pathology of the University
esamples from human bladder werHospital Hamburg-Eppendorf. Normal tissue d tumor cancer, anobtained by biopsy or by cystoprostatectomy because of prostate undergone surgical om patients who hadtissues of bladder cancer were obtained frment ofage was made by the Departtherapy. The determination of tumor st classification. Theealth Organization) Pathology according to the WHO (World Htissue blocks were fixed in formaldehyde and embedded in paraffin and subsequently
sectioned for use in immunohistochemistry. 3.2 Methods es syanal3.2.1 Protein Determination of total protein assay kit which ismined by BCA protein The concentration of proteins was deteration of lorimetric detection and quantithoninic acid (BCA) for the cobased on bicinc

s Methodnd Material a


total protein. The complex giving colour exhibits a strong absorbance at 562 nm. BSA
andard. 2 ml of the diluted and used as st serially o(Bovine Serum Albumin) was als standard tubes. After aadded to each sample and to mix was BCA working reagent 37°C for 30 min. For each dilution, short vortex the tubes were incubated aterage was taken for the ate and the avd in duplicmeasurements were performeotein concentration. calculation of the pr Two dimensional polyacrylamide gel electrophoresis (2D-PAGE)
protein mixtures due to differences in Two dimensional gelelectrophoresis separates cular edimension and subsequently by their molrst their isoelectric point (pI), in the fiesented in figure 16. ension reprweight (MW) in the second dim

two dimenFigure 16sions ho: Schematicrizontallayl repres by iso-eleentatioctric poin of twnto (pI) a dimend vnsional sepertically byaration. moleculaProtr weins aeight (Mre sepaW). rated in
urine samples for 2-DE of Preparation l protein was applied containing 200 µg totaAfter protein determination, urine volume to remove molecules smaller than 10 kDaonto a Centrifugal Filter Column (Amicon) such as urea, electrolytes, salts and to increase the protein concentration of the urine
was centrifugated at speed mn containing urine volumehe centrifugal colusamples. T at 4°C until complete urine volumeseased from 3000 rpm to 6000 rpm rlinearly incwere filtered through twith 50 µl distilled water he filter membrane. Remained protand centrifugated at 6000 rpm foein molecules were washed r 30 min. Filtered urine
samples were diluted in rehyration buffer 2 (3.1.3). After fresh adding of TBP, DTTaotein lysand trace of bromophenolblue, pr tes were centrifugated at 15,000 rpm for10 min at 4°C to remove insoluble particles. Supernatant containing urinary proteins
was used for sample loading step of 2-DE procedure.

Methodnd Material as

nge IPG Strip Length / pH Ra11 cm, pH 3-10, nonlinear (NL)
) r (L 3-10, LineaH17 cm, p

otein amount Loaded pr µl volume) 00200 µg (in 2 µl volume) 00300 µg (in 3


Table 12: IPG strips used for 2-DE analyses
Additionallysamples to precipitate all proteins present , TCA (tricholoroaceticacid) precipitation was pein urine. Shortly, an equal volumerformed on the urine of 20%
TCA were added to the urine samples centrifugation at 13,000 rpm for 10 min at 4°C, the sand incubated on icupernatant was remoe for 30 min. After ved and
effective for 2-DE analysis; it led to vertprotein pellets were diluted in rehydration buffer2-3 (3.1.3). But,ical streaks due to remained T thiCs method was notA particles
protein present in urine, To precipitate a specific ).(data was not shown r, the 2-DE results ofed on urine samples. Howeveimmunoprecipitation was perform informative due to remained IgGimmunoprecipitated samples were notwas not shown). Therefore ata antibody (dom secondary (Immunglobulin G) coming frheir use in 2-DE process. r all urine samples prior tultracentrifugation was used fo dration y3.2.1.4 Reh The PROTEAN IEF cell with integrated power supply was used for rehydration and
IEF protocols. Diluted protein samples were loaded onto IPG strips in IEF focusing
strip gels were placed side t dimensional separation. IPG tray for rehydration and firsrehydration solution. Fdown in the channel of a focusior urine samples mostng or rehydration tray thatly 11 cm nonlinear IPG strips with 3-10 contains the sample in
pH range were used and for a large scale used (Tab. 12). Strips were covered with 2 mlof gel the strips wit of mineral oil to prh 17 cm length wereevent evaporation
which causwith sample in a focusing tray were activees the urea to precipitate as it becomes more rehydrated by running concentrated. IPG strips of IEF cell under
low voltage as 50 V overnight. ion: Isoelectric focusing (IEF) First dimensional separat3.2.1.5 After the strips have rehydrated the endswicks were inserted between the strip and the of each strip was lifted and wet electrode electrodes. Strips were covered with
mineral oil before starting the focusing run to prevent evaporation and carbon dioxide

s Methodnd Material a


increased fabsorpsion during focrusing. Isoelectric foom 250 to 5000 V during the first 5 h, followed by 8000 V for a total of cusing was performed at voltage linearly
70000 Vh/h (Tab. 13). C ûTemperature 20Current max. 0.05 mA per strip strip, 300 µl for 17 cm strip Sample volume 200 µl for 11 cm

Time / Volt-hour d30 min. rapi w60 min. slo w60 min. slo w60 min. slo w60 min. slo w60 min. slo w60 min. slo w4 h sloear 70,000 Vh lin3 h linear

Voltage Step Time / Volt-hour d30 min. rapi150 V 1 3 2 1000 V 300 V 60 min. slo60 min. sloww
w60 min. slo1500 V 4 w60 min. slo2000 V 5 w60 min. slo3000 V 6 8 7 8000 V 5000 V 4 h slo60 min. slow w
ear 70,000 Vh lin8000 V 9 3 h linear 500 V 10 Table 13: Applied voltage steps for IEF Equilibration lfhydryl groups of ates the resultant suThis process reduces disulfide bonds and alkylthe cysteine residues. Rehydration/equilibration trays sized for each size strip were
used for equilibration. Each strip was moved to equilibration tray and the channels
re first incubated in DTT e. Strips w with the equilibration bufferswere filledoups for 15 min at RT, than the hat reduces sulphydryl grequilibration buffer-I (3.1.3) tchannels were refilled with iodacetamide equilibration buffer-II (3.1.3) which alkylates
sulfhdryl groups and incubated for 15 min at RT. For the strips with 11 cm length, 3
rips with 17 cm length 5 ml of each ml of each equilibration buffer and for the stbration, IPG strips were removed and equilibration buffer were used. After equilidimension gel. embed into the prepared second- mensional separation Second di3.2.1.7 rried outaoelectric focused proteins was cThe second dimension electrophoresis of isfrom BioRad (Criterion Precast Gel crylamide gels obtained aon pre-prepared poly were placed on top of the The equilibrated IPG gel strips 10% Tris-HCl, 1.0 mm).e prepared in SDS-ed agaroslaid with 2 ml 0.5% meltpolyacrylamide gel and over

s Methodnd Material a


s added to the agarosel amount of Bromophenolblue waPAGE running buffer. A smaloverlay to track the ion front during the run. The SDS gel running was performed with
BioRad Dodeca Cell 150 V for 20 min. a voltage setting of 120 V for 45 min and r proteomics analyses. To r run was mostly used fowhich accommodate 12 gels peestimate the molecular mass of each spot, marker proteins (SigmaMarkerTM, wide
molecular weight) were placed on a filter strip left to the strip on the gel.
ant Blue (BioRad) and/or Coomassie BrillerPolyacrylamide gels were stained with silvaccording to the procedures described previously (Merril et al., 1981). detection Protein To visualize proteins after separation in 2-DE gels it is necessary to use an adequate
e of the in characteristics and abundanc variation staining method. The enormousstaining technique. The most important dual proteins put high demands on the iindivgh reproducibility and dynamic range, hinearrequirements are high sensitivity, high li s of protein stainingtrometry. Major developmentcompatibility with mass specs. Since silver staining is the most he last few yearmethods have occurred during tly e and the costs for reagents are relativeve detection techniquisensitive non-radioactlow when compared to fluorescence techniques, it is still widely used. There are
staining protocol of the original silverpresently more than 100 different modifications In this study silver staining and .that was introduced by (Merril et al., 1981)used to visualize the protein spots. coomassie blue staining methods were staining Silver s were taken out and incubated first with ion the gelAfter second dimensional separat 10% acetic acid for 30 min methanol andfixer solution-I (3.1.3) containing 50% following second incubation with fixer solution-II (3.1.3) containing 5% methanol and
fixation, the gels were washed twice with 7.5% acetic acid for 10 min at RT. After distilled water for 5 min at RT on a shaker. Gels were then incubated with 0.002%
led water for 1 min. Forn and washed twice with distilsodiumthiosulphate for 1 mi eed. The gels werate solution was usstaining, fresh prepared 0.2% silver nitrat RT. After a washing step as for 30 min trate solution in darkincubated with silver nideveloper solution (3.1.3) containing 6% incubated with fresh e2x1 min, the gels werr 5-20 min until the protein spots de fosodium carbonate and 6 mM formaldehy

s Methodnd Material a


acid particles were added to stop the tric ied. A few solid cbecame visualizage analysis. m gels were scanned for the idevelopment of the gels. After washing, staining e Coomassi For coomassie staining, the gels were incubated with fixer solution-I (3.1.3) for 30
with distilled water for 5 min, gels weree min at RT on a shaker. After washing twicstained by incubation using coomassie solution (3.1.3) for overnight. Stained gels
ed water to remove remained coomassie were washed several times with distillcrystals and excess dye from gel. Image and data analysis d using PDQuest software programmeThe resulting 2-DE images were analyse(BioRad). Gel analysis includes spot detection, spot quantitation, gel comparison,
d the protein spotsee automatically detectand statistical analysis. Before the softwar corrected and background was subtracted.of a 2-DE gel, the raw image data were and bioinformatics ryspectromet3.2.1.12 Mass Some of the visible protein spots on the stained gels have been identified by mass
titute of Hamburg University. Coomassie -Pette-Insh in Heinricspectrometric analysisBrilliant Blue-stained protein spots (Coomassie G250) were excised, cut in 1 mm3
r and destained with 50% v/v acetonitrile pieces, washed twice with distilled wate15 min, shrunk by dehydration in e for 2x(MeCN) in 25 mM ammonium bicarbonat uum centrifuge for 10 min. The gel piecesacetonitrile for 15 min, and dried in a vacwere reswollen in 10-20 µl of 25 mM ammonium bicarbonate, containing 200-400 ng
of bovine trypsin (proteomics sequencing grade; Sigma-Aldrich) at 4°C. After 60 min,
sary to keep the gel es added if nece were10-20 µl of 25 mM ammonium bicarbonatovernight). The peptides were extracted during tryptic cleavage (37ºC, pieces wetTFA, 20% MeCN/ 0.1% TFA 1% TFA, 10% MeCN/0.1% r, 0.esequentially with watThe separated liquids were combined h. and 40% MeCN/0, 1% TFA for 30 min eacredissolved in 4 µl matrix solution uum. The peptides were and dried under vaccontaining 10 mg dihydroxybenzoic acid, 5 mg diammonium hydrogen citrate, 0,1%
TFA, and 0,05% phosphoric acid in 50% MeCN. For MS-analysis 1 µl were spotted
MS measurements were performed on an onto the target plate and air-dried. MALDI-

s Methodnd Material a


in positive mode with ometer (Shimadzu Biotech)R MALDI-TOF spectrAxima CFized for 2500 mu. Dependent on spectra reflectron, delayed extraction was optimquality 200-400 lasspectra processing was done with the Axer shots were accumulatima CFR soed. Control of the ftware V2.2.1. Mass spectraspectrometer and
Angiotensine III andagments of trypsin, were calibrated using known autolytic fracetylated Insulin B chain as internal standards. The peptide masses were measured
as monoisotopic masses.

NaNattiivvee U Urrineine

CeCeFFiltiltntrifugationntrifugationrarattiion on aandnd

SeSepapararationtion of u of urrinainaryrypprrootteeinsins

DeDetteeccttioionnoof prf proteinoteinssppootsts
((CCoooomassmassieie/ Sil/ Silvveerrststaiainniinngg))

IdentificaIdentificationtionof pof prroteinsoteins
urineFigure 17: E samplexs. Native uperimental florinwe containi diagrang m. a certain amThis diagram shoount total wprots the experimeein was centntal steprifugated ans of analysed filtrated. s of
silver After firstand/o anr d secoomcondassie dime blue nsional stainingse prparation uotocols to visuing 2-alizeDE techni protein quespot, the 2s. The-DE sp gels ots of iwere nterestainst weed ure sincutg
out from gel and identified by mass spectrometric analyses.
using the programng the NCBI database Proteins were identified by searchiMASCOT (Matrix Science;
ons acetamide on ifollows: the modificateters for the search were as The param

Material as Methodnd


re considered as a partial modification cysteine residues and methionine oxidation we .and three was used as the maximum missed tryptic cleavage sites SDS-polyacrylamide gelelectrophoresis
li, 1970) under denaturing conditions (in One dimensional gel electrophoresis (Laemme of their ns on the basr separation of the proteiopresence of SDS) was used fs prepared as a separating gel (sometimes molecular size. The polyacrylamide gel wa gel) topped by a stacking gel and secured in ancalled resolving or running r sample proteins are solubilised by atus from BioRad. Afteesis apparelectrophorlution is applied to a gel an aliquot of the protein soe of SDS,boiling in the presenc y of theed electrophoretically. The mobilitl proteins are separatline, and the individua heir molecular mass. SDS isonal to the logarithm of t inversely proportiproteins is deins, to dissociate protein complexes anemployed to effect denaturation of the proties proportional to negative charge densitto impart upon the polypeptide chains net existing as DTT was used to reduce anythe length of the molecule. A reducing agent oteins was detected usingmensional separation the prdisulphide bond. After one di immunoblotting for and byable to detect all proteinsstaining methods which is ific protein. detection of a spec blotting Western ylamide gels onto retentive membranes wasElectroblotting of proteins from polyacron of accumulated proteins using a ectiperformed for immunoblotting following det electrophoreticallyed by SDS-PAGE and specific antibody. Proteins were separattransferred from the polyacrylamide gel to a nitrocellulose membrane at 350 mA
constant current for 1 hour. lamide gel which was prepared according SDS-AcryProteins were separated on 12% om BioRad. For preparation of gels 30%ruction frtto the Mini Protean Manual Insused. 25 µl of different protein samples acrylamide solution obtained from Serva was of sample loading buffern were denaturated by adding containing 50 µg total protei95ºC for 5 min. Protein samples were (3.1.3) including DTT and following heating at lt for 90 min. Prestainedrunning was performed at 120 voo the gel and loaded ont size of separated ogen was used to estimate molecularprotein marker from invitr om SDS gel to nitrocellulose membrane,proteins. For transferring of proteins frrding to oained from BioRad was used accequipment as Mini Trans-Blot-System obt

s Methodnd Material a


blotting nitrocellulose membranesthe manufacturers recommendations. For (Schleicher & Schuell, BioScience) were cut in size as 7x9 cm and wetted by transfer
pre-wetted with 1x and filter papers werebuffer shortly before placing. The fiber padsTransfer buffer. Gel containing proteins and nitrocellulose membranes were allowed
ber pad, a filter paper, gel, ing in order as a fil placto contact by a sandwich mode rom cathode plate to anodea filter paper, a fiber pad (fnitrocellulose membrane, beside pack was puthe chamber and a colded in tplate). The cassettes were placophoretically transfer was er buffer (3.1.3). Electrfilled with pre-chilled 1xTransf andellulose, transfer onto nitroc on a stir plate. After the performed 1 h at 350 mA currentthe blots were taken out and washed with distilled water. To prevent unspecific
bindings, blots were incubated with blocking buffer containing 5% non-fat milk
day the blots were incubated r (3.1.3) overnight at 4ºC. NextBS-T buffesolution in 1xT min washing RT. After 2x10BS-T solution for 1 h at in 1xTwith the primary antibodyubated for 1 h at RT buffer the membranes were incBS-Tin 1xTwith the horseradish blocking buffer. Finally, the membranes e-coupled secondary antibody in peroxidis BS-T buffer. The antigen-antibody1xTwere washed 2x10 min and 2x20 min with complex was detected by enhanced chemiluminescence using ECL reagents
(Amersham-Pharmacia) and visualized by auroradiography. The X-ray films were
the bands using thec quantification of digitalized for subsequent densitometriTMSA). (Seattle, Umorphometric program Optimas

methods 3.2.2 Molecularbiological Cultivation and storage of E. coli
)-broth or on LB-agar plates at 37°C. ForBacteria were cultivated in Luria-Bertani (LBlied with 50 mg/l re suppteria, broth and plates weselection of transformed bacampicillin. hen stored at 4°C. ne month woximately oFreshly plated bacteria were viable for apprFor long-term storage, glycerol stocks were prepared by mixing 500 µl of an
l. The stocks were stored at e with 500 µl of 15% sterile glyceroovernight liquid cultur80°C.

s Methodnd Material a


Preparation of competent cells DH5-α bacterial were obtained from Invitrogen and competent E. coli cell stocks were
cellsαiously (Sambrook, 1989) 200 µl of DH5-prepared using protocol described prevedium without antibiotic. After m LB-e and were mixed with 10 mlwere thawed on icat 225-250 rpm, 200 µl of mixture were overnight incubation at 37°C with shaking plated on an agar plate without antibiotic and incubated overnight at 37°C. A single
After overnight .hcolony was picked and 10 ml LB medium was inoculated wited to 100 ml pre-warmed LB overnight culture was addincubation at 37°C, 1 ml ofed at 37°C until an and shak incubated The culture wasmedium without antibiotic.OD600 OF 0.5 was reached (approximately 90-120 min). The culture was cooled on
ound-bottom centrifuge tube. The cells were ice for 5 min and transferred to a sterile rm, 4°C). After supernatant min, 1500 rp5 low speed (collected by centrifugation atwas removed, the cells were resuspended in cold TFB1 buffer (3.1.3) (30 ml for a 100
ml culture) and suspension was incubated on ice for 90 min., followed centrifugation
were resuspended in 4 ml ice-cold TFB2 for 5 min at 1500 rpm at 4°C. The cells ed andile microcentrifuge tubes were preparbuffer (3.1.3). Aliquots of 100 µl in ster were stored at -80°C. ent cellsfrozen in liquid nitrogen. The compet transformation Bacterial plication was performed using needed for cloning apBacterial transformation which is competent cells were thawed on and XL1-Blue (Tab. 8). 50 µl ofα strains DH5E. coli of cellseaction was added to one aliquotion rice. 1-5 ng purified DNA or 1-5 µl of ligat e incubated on iceng cells and DNA werand mixed by pipetting. The tubes containi bath for 45 seconds, followed 42°C waterfor 30 min, and then were heat-pulsed in a h to eac 500 µl pre-warmed LB mediumincubation on ice for 2 min. After addingg at 225-250 rpm. for 1 hour with shakinsample, the tubes were incubated at 37°C ead on LB agar plateson mixture were sprAfter incubation 50-500 µl of transformatire incubated overnight at 37°C. Single containing 50 mg/l ampicillin. The plates we medium containing ampicillin at 37°C colonies were picked and incubated with LB using either Plasmid Maxi Kitisolatedmid DNA was overnight with shaking. The plasion, EndoFree Plasmid Maxi Kit (Qiagen). at(Qiagen) or for further transfection applicl stock at 80°C. oure with sterile glycerxtTransformed bacteria were stored as 1:1 mi

s Methodnd Material a


Purification of DNA from solution and gel bands ct and/or agarose gel bands, ion of DNA from PCR produicatFor isolation and purif ciences) was used. Kit (Amersham BiosGFX PCR DNA and Gel Band Purification ates proteins, dissolves agarose, and that denaturThis kit uses a chaotropic agentilobase-pairs) pairs to 48 kof double-stranded DNA (100 base-promotes the binding ovided DNA was performed using the protocol pr fiber matrix. Purification of to a glassband purification, denatured and for gel by GFX purification kit. First DNA was re passed through the GFX column ed. DNA samples weagarose was dissolvprovided with kit to capture the DNA onto the glass fiber matrix. Then Matrix-bound
remove salts and other contaminants. h an ethanolic buffer to DNA was washed witth buffer (TE pH 8.0, a low ionic streng in nPurified DNA was eluted from GFX colum10 mM Tris-HCl pH 8.0, or water). ion of plasmid-DNA Minipreparat For small-scale preparation of plasmid DNA, Nucleospin® Plasmid Kit (Macherey-
protocol fromwas performed according to used. Isolation of plasmid DNA Nagel) wasNucleospin® Plasmid kit. The pelleted bacteria were resuspended and plasmid DNA
was liberated from the E.coli host cells by SDS/alkaline lysis. SDS precipitate and
onto a upernatant was loadeda centrifugation step, the sed by cell debris were pelletNucleospin® Plasmid column. Pure plasmid DNA was finally eluted under low ionic
-Cl, pH 8.5) which provided slightly alkaline buffer (5 mM Trish strength conditions witmeasured by UV-spectrophotometer. with kit. Concentration of DNA was ion of plasmid-DNA Maxipreparat mid Maxi Kit (Qiagen) was in high culture volume, PlasFor isolation of plasmid DNA used. The purification protocol of kit is based on a modified alkaline lysis procedure,
n under ito Qiagen Anion-Exchange Resfollowed by binding of plasmid DNA ning Ampicillin LB-Medium containd pH conditions. 100 ml of appropriate low-salt agar plate and then from a selective angle colony iwas inoculated by a picked sC on a rotation shaker. Plasmid DNA was over night incubation at 37°cultured byted in 200 µl TE buffer or in ol from Plasmid Maxi Kit and diluisolated using the protoc determined by UV entration wasDNA concbuffer containing 10 mM Tris-Cl, pH 8.5. on an agarose gel. Plasmid DNA which isspectrophotometry and quantitative analys

s Methodnd Material a


was further transfected into the cells was isolated using EndoFree Plasmid Maxi Kit
(Qiagen) due to endotoxin effects efficiency of gene transfer. Restriction digest of DNA were obtained from NEB. Restriction Restriction enzymes and appropriate buffersDNA were performed according to the striction digest of edigest and double r ction digestion of DNA isgeneral protocol for restrimanufacturers' protocol. The shown in table 14. Components Concentration / Volume
Restriction DNA Enzym e 3 1-3 U/ µgµ g DNA
10x Distilled Buffer wat er 2 ad µl 20 µl
Table 14: Composition of restriction digests reaction
After incubation of the reaction mixture at 37°C for 1 h per 5U of restriction enzyme,
incubation at 75°C for 20 min. the reaction was inactivated by l electrophoresis eAgarose g3.2.2.8 separated on 1-2% allyDNA were electrophoreticPCR fragments and/or digested AE buffer containing 0.1 g/ml ethidiumepared in T(w/v) analytical agarose gels prbromide. 1x TAE buffer (3.1.3) was used as running buffer. DNA samples were mixed with 6x
gel. 500 ng of DNA molecular markerloading buffer (3.1.3) and loaded onto the egrity. The DNA was and intsample size(Invitrogen) were used for evaluation of at 302 nm and documented by a gelvisualised on an UV transilluminatorre cut out from agarose as). Interested DNA bands wedocumentation instrument (Int ed from cut gel using GFX PCR DNA kit oring a sterile scalpel. DNA was isolatgel usion and purification, DNA on Kit (Qiagen). After isolatin some cases Gel Extractisamples were electrophoretically separated on agarose gel for controlling. After that,
DNA concentration was measured by UV-spectrophotometry. ing of DNA cSequen3.2.2.9 he sequence data d by MWG Biotech. T performeAll sequencing of plasmids wasl (Basic Locapared using sequence comparing programme, BLASTwere com

s Methodnd Material a


Laboratory)-Databank n Molecular Biology Alignment Search Tool) in EMBL (Europea). http://www.ncbi.nlm.nih/gov/Blast/( ation rDetermination of DNA concent3.2.2.10 SmartSpec3000 UV-Spectrometer (BioRad)entration For determination of DNA conc o1:100 with distilled water, which used alsles were diluted as was used. DNA samps were placed into micro-quartz cuvetes. as blank and 100 µl of diluted DNA sample and DNA using UV-light by 260 nmre was measured for double stAbsorbancwavelength. DNA3.1(-)/ORM expression vector Construction of pc3.2.2.11 s via nucleofection technology, the ECFor overexpresssion of ORM in HDMtructed. For this, the full-length cDNA DNA3.1(-)/ORM was conson vector pciexpressencoding ORM was cloned into the XhoI and HindIII restriction sites of the
Hygro(-) (Clontech). Firstly the cDNA encoding for ORM pression vector pcDNA3.1/ex using gene specific human granulocytes gene was amplified by PCR from cDNA of® vector. he PCR product was ligated into the 2.1-TOPOprimers. Subsequently t merase chain reaction (PCR) yPol3.2.2.12 Polymerase chain reaction (PCR), a procedure for rapid in vitro enzymatic
, was used for the amplification of cDNA amplification of a specific segment of DNApartment eom the Dobtained as a gift frencoding ORM. cDNA of granulocytes which of Clinical Chemistry of University Hospital Hamburg-Eppendorf, was used directly as
ytes. e ORM genes present in granuloca template for PCR to amplificat Components Concentration / Volume
dNTP DNA 1 10 µl mM
M Primer gCl2 20 50 mM pmol each
Taq DNA polymerase 1 U
Distilled 10xPCR watBuffer er 1x ad 25 µl
Table 15: Composition of PCR reaction

s Methodnd Material a

sec 45 sec 45 min 1 10 min


Step Temperature [°C] Time
Initial denaturation 95 10 min
35 Cycles:
Denaturation 95 45 sec
Primer annealing 60 45 sec
Elongation 72 1 min
Final elongation 72 10 min
for ORMns: PCR conditioTable 16 ubes (Biozym), using a PCR ml PCR tThe reactions were performed in 0.2 imers annealing to the vector backbone thermocycler (MWG Biotech). Specific prwere used for PCR reaction (Tab. 15). GAPDH was used as control reaction.
The PCR products scribed in table 16. Thermocycler conditions were used as deied using GFX PCR DNA and agarose gel ( and purif sed on a 1%were analyham Biosciences) ( Gel Band Purification Kit (Amers Ligation of PCR product into pCR® 2.1-TOPO® Vector
purified using GFX PCR DNA and Gel The PCR product coding for ORM gene was Taqences). For direct insertion of Band Purification Kit (Amersham Biosci plasmid vector, TOPO TA cloning Kite-amplified PCR product into a polymeras(Invitrogen) was used. The pCR® 2.1-TOPO® vector provided with kit was used as
) residueshanging 3´-deoxythymidine (Tlinearized with single 3´-thymidine (T) overto ligate with the vector. rts ws PCR insellowhich a o vector was performed according tInsertion of PCR product into the TOPO PCR product and TOPO vector were t.manufacturers protocol provided with kimixed and incubated at RT for 5 min. TOPO cloning reaction was transformed into
and single colonies were selected to isolate DNA. chemically competent cellsThe selected single colonies were cultured in LB-medium overnight and DNA was
® DNA concentration Plasmid kit. Afterisolated using protocol from Nucleospin restriction site and XhoI and HindIIImeasurement, DNA samples were digested at elected ones was s One of positive cletically separated on agarose gel.electrophorinto the expression vector as next step. O/ORMTOPand used for subcloning of

s Methodnd Material a

Subcloning into pcDNA3.1(-)


ansient rned for high-level stable and tThe pcDNA3.1(-) is 5.4 kb vector desig

expression in mammalian cells. The pcDNA3.1(-) expression vector and TOPO/ORM

plasmid vector were digested at HindIII and XhoI restriction site and after extraction

om agaroof digested pcDNA3.1(-) and ORM insert frse gel ligation reaction were

ernight at 16 °C tion was incubated oveac Ligation rperformed using the T4-Ligase.

and directly transformed into E. coli strains DH5α or XL1. Single colonies on LB-agar

plates were selected and analysed for the presence of insert by HindIII and XhoI

ect restriction pattern wasrh has correstriction digestion. A transformant whic

M gene was cloned in confirm that ORselected and sequenced by MWG Biotech to

the proper orientation.

truction scon3.2.2.15 siRNA

mcing via sFor ORM gene silenall interfering RNA (siRNA) th

Kit (Ambion) was used. Construction

Figure 18: Mechanism of gene silencing via siRNA

e Silencer

iRNA s™

s Methodnd Material a


post-ele-stranded RNAs, which inducThe siRNAs are 21-23 nucleotides doubng the siRNAs into the cells, they were lencing. After introduciitranscriptional gene sg complex (RISC). ed silencina RNA-inducassembled with protein components into vates the RISC, which in turn binds to An ATP-generated unwinding of the siRNA acties the mRNA. This interaction and cleavthe homologous transcripts by base pairing sults in gene silencing (Fig. 18).adation of mRNA repecific degrsequence scted from the cDNA of the siRNA sequences are seleFollowing target regions for hir et al. (Elbashir et al., 2001). Elbasaccording to the guidelines described by ORMferase gene was chosen as a control for Target sequence (cDNA) from the firefly luciORM silencing studies.

methods 3.2.3 Cellbiological

General cell culture work was performed under sterile conditions under a laminar flow
hood using disposable plastic ware. cells of Culturing Cell lines (Tab. 6) were placed in polystyrene culture flasks 25 cm2 (T25) and 75 cm2
ophobic vent caps and maintained in a(T75) (Falcon) provided with 0.2 µm hydrCell 240) at 37°C. All culture atmosphere in an incubator (Hera d 5% COhumidifie2 ed by heating at Glass wares were steriliz (Tab. 7) were sterilized bylaved at 120°C and 1.2 bar for 20 min. 180°C for 8 h, and plastic wares were autocluence of 70-90%. Medium was removed lls were cultured until a confeAdherent ccubated with trypsin for one lls were ining, the ceand after two times PBS washtion of double used trypsin addihe reaction was stopped byminute at 37°C and tn was harvested by centrifugation (1200 volume of culture medium. Cell suspensio were washed bywas removed and cellsrpm /5 min/ RT). The supernatant equent centrifugation. For subculturing cells wereubsresuspension in PBS with sfold with fresh medium. diluted approximately 1:5 ing of cells Freezing and thaw3.2.3.2 500,000 viable human dermal microvascular endothelial cells (HDMECs) (Tab. 6),
freezing medium, after eserved in 1ml serum-free derived from dermis and cryoprcultured and passaged until re PromoCell. HDMECs wethawing were obtained from

Material as Methodnd


HDMECs were not used after passage passage four, and then used for experiments.ents thawed and viable ozen and stored. For all experim not froeight and were alsfrom PromoCell. ed HDMECs were shipp6 cells/ml, washed with ed at a concentration of 0,5x10RT4 cells (Tab. 6) were harvestPBS and resuspended in 1 ml ice cold cryo-conservation medium. The cryovials
placed in a -80°C freezer and transferred (Nunc) with 1 ml cell suspension each were to liquid nitrogen for long-time storage. bath, transferred to 20 ml r eed rapidly in a 37°C watCryopreserved cells were thawSupernatant was discarded rifuged (1200 rpm/ 5 min). prewarmed medium and centand cells were seeded at high density in culture flasks (25 cm2 and 75 cm2,
respectively). Determination of cell number For determination of cell number 10 µl of the cell suspension was mixed with 90 µl
trypan blue solutions. After 3 minutes of staining, only dead cells turn blue, while
were counted in a Neubauer chamber. The tained. The latter living cells remain unsnumbers of cells were calculated according to the equation below. Number of cells/ml = number of cells over a large square x dilution factor x 104
extraction Cell Cell lysates of HDMECs and RT4 cells were prepared by chemical lysis. The cells in
culture were washed two times with cold-PBS and after third adding of cold-PBS the
cells were scraped using a scraper on ice. Cell suspensions were collected into a
upernatant was removed andr 6 min. Sfalcon tube and centrifuged at 1200 rpm focell pellets were washed three times more with ice-cold PBS. RT4 cell pellets were
s pellets were lysed in Triton X-100-SDS PA-Lysis buffer (3.1.3). HDMEClysed in RIspensions uadding lysis buffer onto the cell pellets, cell sLysis buffer (3.1.3). After ing. Cell lysates were incubated on ice were mixed first by pipetting followed vortexmin. After that, the cell ed at RT for 10 rtex, incubatfor 30 min and after a short vo ant were collectedmin at 4ºC. Supernatlysates were centrifuged at 15,000 rpm for 15 and stored at -80ºC until further analyses. Protein concentration was determined
using BCA protein assay kit (Pierce).

s Methodnd Material a


HDMECs via Nucleofector of Transfection the Nucleofector technology (Amaxa ansfection was performed using rHDMECs t a non-viral alternative to primary cell Biosystems). Nucleofector technology offersal parameters and cell-ination of electrictransfection, which bases on a unique combtype specific solutions. HDMECs were transfected using HMVEC-L Amaxa
Nucleofection Kit along with program S-05 on the Nucleofector device according to
cy, the neaching 70% conflueanufacturer (Amaxa). After rthe optimized protocol of mtion. The cells after passage ten were cells were passaged 3-4 days before nucleofecfer efficiencies and gene transad to lowertion because it may lenot used for nucleofecy damage the cells. For transfection, amtrypsin treatment is more difficult and nization and count to determine cell density.cultivated cells were harvested by trypsi5x105-1x106 cells per nucleofection were centrifuged at 1200 rpm for 10 min and
resuspended in 100 µl Nucleofector solution and mixed with 2 µg DNA or siRNA. Cell
suspension was transferred into an Amaxa certified cuvette and inserted into the in the Nucleofector, 500 µl of full cuvette holder. After running the adequate program could be transferred into a preparedmedium was added to the cells and the sampleculture flask. in HDMECs g of ORMsilencingene Overexpression/ To overexpress ORM in HDMECs the cells were transfected with the expression
bp full-length cDNA of ORM was cloned vector pcDNA3.1(-) ORM, in which the 770into the Xhol and HindIII restriction sites of the expression vector pcDNA3.1/Hygro(-)
(-) was used as a negative control. (Clontech). Empty vector pcDNA3.1.2.15). The siRNA-ed siRNA-ORM (3.2ing construct silenced usORM gene was e 2 µg of plasmid DNA or siRNA were control.luciferase was used as negativ.3.5). Transfected ector technology (3.2transfected into the HDMECs using nucleofHDMECs were cultured for 1-2 days and used for further studies. After 2 days llected and stored at -20°C and for later were cosupernatant of transfected cellswere later used for tube formation assay Supernatants which .western blot analyses6 min to remove dead cells. Supernatant (3.2.4) were centrifuged at 1200 rpm for was taken and sterilised using 0.2 μm filter. To see if overexpression and gene
ormed for were perfn level WB analysesing are effective at the proteisilenctransfection, cells were after Two daysfected cells.supernatant and extract of trans

s Methodnd Material a


ses ( After protein analylysed ( and used for western blottotal protein were loaded onto SDS-concentration measurement, same amount of PAGE to compare ORM level for each sample. The same blot was then striped by ibody as control for same bated with anti vimentin antfer (3.1.3) and incustriping bufoaded protein. amount of l

3.2.4 Endothelial ube formation assayt

was carried out using three-diThis assay mensional type I collagen gel (PureCol,r tissue culture dishes as described ustelInamed) which was prepared in 48-well cpreviously (Ergun et al., 2000; Oliveira-Ferrer et al., 2004; Pepper et al., 1990).
ice-cold collagen gel mixture (3.1.3) and 48-well plates were coated with 190 µl of ymerization of the gel, wild type ORM- at 37°C. After polincubated over nightoverexpressolidified gels at a csing and ORM-silenced endothelial oncentration of 4x104cells cells /well in 300 µl of growth medium (HDMECs) were seeded onto
luence, the medium was replaced by basal medium containing 5% FCS. At confcontaining 2-4% FCS without further supplements. After 24 hours VEGF-A was
3 days after taking photographs with ery added to the wells and was renewed evphase conttubes of interest were fixed rast microscopy (Zeiss, Jena). overnight with Bouin´s fixativeThe collagen gels containing endothelia solution following overnightl
incubation with 70%left out from 48-well using a scalpel ethanol and two times ethanol incubation and embedded in paraffin to use for for 1 h. The gels were
ochemical studies. immunohist

Stimulating Factors Description Working concentration Supplier
ORM Orosomucoid , human serum 2. 300 ng/ml 1. 100 ng/ml Calbiochem
Oxford AntiPAI-1 Anti-humaantibody (fron PAI-1 monom mouse) clonal 15 µg/ml Biomedical
Re hrcseaVEGF-A Recombinant Human VEGF-A 50 ng/ml Immunotools

VEGF-A Recombinant Human VEGF-A 50 ng/ml

Table 17: The stimulating factors used for tube assays
unction modulating and the ORM-PAI-1 complex has any fTo find out if PAI-1 or/ was seeded intobes, endothelial cellseffects on VEGF-A induced endothelial tucollagen gel and incubated until confluent. Afterwards cells were left untreated or
stimulated with; (i) VEGF-A (positive control), (ii) ORM, (iii) VEGF-A together with
ORM, (iv) VEGF-A and anti-PAI-1 antibody, (v) VEGF-A, ORM and anti-PAI-1

s Methodnd Material a


antibody, (vi) anti-PAI-1 antibody alone (Tab. 17). Endothelial cells and VEGF-A
by day aphed days examined and photogrube formation wainduced endothelial tJena) equipped with a digital camera using phase contrast microscope (Zeiss,(ProgResTMC10plus, Jenoptik).

methods 3.2.5 Histological

ples and cells ng for tissue samFixation and HE staini3.2.5.1 Paraffin-embedded bladder tissue sections obtained from patients who have clinically
cancer as well as normal bladder tissue, obtained been diagnosed to have bladder The bladder tissue unohistochemical biopsy, sections were used for immsections used in this study were obtained from department of Pathology of University
ctionseunohistochemical staning the sHospital Hamburg-Eppendorf. Before imm on tissue sections. Eosin to confirm presence of cellsmatoxylin-ed with Hewere stainSlides containing paraffin sections were deparaffinised by incubation in order as
% ethanol, and lately deionized water for lene, isopropanol, 96% and 70three times xyxylin after a short washing ons were stained with Hemato5 min. After that, the sectiwith water, destained with acid ethanol and washed again with water. For
treated with Eosin and dehydrated by 96%cytoplasmatic staining the sections were isopropanol incubation. ethanol and subsequently y3.2.5.2 Immunohistochemistr Each section was deparaffinised (rehydrated) in descending alcohol row and
ed to immunohistochemistry. he sections were subjectincubated in 1xPBS. Then tmal swine serum to block the unspecific After treatment with normal rabbit or norubated with the corresponding were incbackground for 30 min at RT, the sectionsprimary antibodies against ORM and/or ZAG diluted in PBS-BSA-NaN3 buffer (3.1.3)
10 min and exposed to shed with 1xPBS for 3x waefor 24 h at 4°C. The sections werthe secondary antibodies for 1 h at RT. Then washed again and treated with the
eat RT. Subsequently, the sections werperoxidase-anti-peroxidase (PAP) for 30 min treated with ABC-complex (Vector) for 30 min at RT, washed again for 2x10 min with
1xPBS and transferred to the solution for the immunostaining development. For the
tibody. nubated only with secondary a were inccontrol reaction, same samples

s Methodnd Material a


the modified nickel-oped by means of ed and devele activity was enhancPeroxidasglucose oxidase technique (Ergun et al., 1997). Glucoseoxidase solution was
prepared freshly in dark according to the receipt described in section 3.1.3. After
washing with 1xPB buffer (3.1.3) for 10 min, the sections were incubated with
at RT. Developing was followed by glucoseoxidase solution for 10-30 min ear, the reaction was stopped by removingmicroscope. After the staining became clthe developing solution followed washing 3x5 min with PBS.
hed 2x5 sec at RT and was0m Red for 2Sections were counterstained with Calciumin with distilled water. The sections were dehydrated by incubation in order as
three times xylene for 5 min. After deionized water, 70%, 96%, 100% ethanol and don). In some cases Mowiol hermoshanomount (Tthat, were embedded using histm red staining to embed the sections. after Calciu(Calbiochem) was used directlyUsing the microscope system equipped with a digital camera the immunostaining
were studied and photographed. ochemistryt3.2.5.3 Immunocy Wild type cells were seeded anparaformaldehyde for 15 min and washing 3 d cultured in chamber slidestimes with 1xPBS, the cells were . After fixation with 4%
eunspecific background. The cells werincubated in normal serum to block the incubated with biotinincubated with primary ylated secantibodyondary ant for 24 h. After washiibody. Afterwng in 1xPBS the cells were ards, they were washed in
PBS and incubated with peroxidase (PAP). After washing the slides were incubated
or). The immunostaining were developed (ABC Kit, Vectin avidin-biotin complexusing Nickin immunohistochemistry section. If cel enhanced-glucoseoxidase technique (Davounterstaining is needed the sections were idoff et al., 1990) as described
stained in Calcium Red for 1-2 min. Using a microscope system equipped with a
method we have studied the digital camera the immunostaining werelocalization of ORM and know studied and photographed.n angiogenic fac Using tthe sameors and
o after ORM overexpressiontheir receptors on endothelial tubes induced in vitrusing Bouin´s fixative overversus ORM silencing in HDMECs. Endot nighthelial tubes on colla and embedded in paraffin. On 5-7 µm thick tissue gen gel were fixed
e. performed as mentioned abovsections immunhistochemistry were

s sultRe

4 Results 2D gel electrophoresis proteins byProfiling of urinary4.1

4.1.1 Optimization of the sample electrophoresis on human urine preparation method for 2D gel


cancer, 2-DE analysIn order to determine the ces were performed on urhanges in urinary protein patteine samples of fourtyfive patients with rns by presence of bladder
(n=8), GI, GII, GIII), 12), pT1 (n=8), pT2-3 bladder cancer of different stages (pTa (n=lunteers (n=7). The details of 2-DE patients on follow-up (n=10) and healthy voanalyses were described in the section of material and methods. Protein spots were
silver staining. Obtained 2-DE images visualized using Coomassie Brillant Blue or (BioRad). Several protein spots in sed by PDQuest software programmewere analyadder cancer were identified using a patient with blDE gel ofcoomassie stained 2-It is well kmass spectrometric analyses, which were nown that urine contains trace amounts of protein orignot present in normal urine samplesinating from blood.
and amounts of body filtrates such asplasma, the kidneys and the urogenital tract, waste products (Anderson and Anderson,water, salts, electrolytes and nitrogenous mple preparation of urine samples is2002; Beetham and Cattell, 1993). Thus, the sapreliminary screening of biomarkers. very important for protein analysis and les several techniques including protein In order to optimize 2-DE for urine sampimmunoprecipitation, ultracentrifugation and precipitation with TCA or in some cases ecipitation led to the vertical TCA prfiltration were used for sample preparation.streaks on 2-DE gel probably due to residual TCA in protein sample and difficulties in
resolubilization of proteins (data was not shown). Immunoprecipitation was used for
ose proteins whicermine changes in thdetection of a specific protein and to deth suppose to be related to bladder cancer. However, immunoprecipitated proteins
caused horpresence of remained immunoglobulin G leadiizontal streaks and dark background on 2-ng to ovDE geer loading of protein amount. ls probably due to the
Ultrafiltration was performed on native urine samples using Centrifugal Filter Column
rfering molecules containing salts, inte(Amicon) to remove high abundance ofwaste products, which disturb the electrolytes and/or some nitrogenous t dimension of 2-DE. After that, different esis process especially in the firselectrophorrehydration buffers were used to solubilise filtrated and concentrated urine proteins.
methods and different rehydration buffersThe efficiency of each buffer was compared to were applie each other. Ultrafiltrad on urine samples and tion, precipitation

s sultRe


urine hod for sample preparation of to find the best metcompared each otheree revealed preparation methods (ssamples. The use of optimized sample and reduced background and streaks.clear detectable protein spots ficial bladder cancerof a patient with superThe 2-DE protein pattern of urine sample length and 3-10 pH range wasp with 17 cm IPG striis exemplary shown in figure 19. staining ing silverpots were visualized usused for this urine sample and the protein s in thens and focused particularlying different proteiprotocol. Many spots representstained 2-DE gel. seen in the silver pH-spectrum ranging from pH 5 to 8 were pHpHpHMWMWMW333444555676767888999101010

66 k66 kDDaa

55 k55 kDDaa

45 k45 kDDaa
36 k36 kDDaa

29 k29 kDDaa

24 k24 kDDaa

UrinFigure 19e sampl: e contaiProtein paning 2tter00 µg n in urine sample of a total proteins were apatiennalyset d wby ith 2-DE ubladder casncer of ing 17 cm strip with pH ranthe stage
3-10. Silver staining revealed numerous spots, particularly focused in the pH range 5-8.
4.1.2 Determination of urinary protein pattern in relation to bladder cancer by
2-DE Urine samples were subdivided into three groups: urine samples of (i) healthy
persons, (ii) patients with bladder cancer of different stages, and (iii) follow-up

s sultRe


hanges in whole protein pattern of urine cFirstly, it was aimed to find out possible

. To this aim the urinary ncerbe related to bladder casamples, which thought to

hat of patients with bladdterns of healthy volunteers to tprotein patr cancer of e

pT3, as well as of patients on follow-up different stages such as pTa, pT1, pT2 and

which were present in urine samples ofwere compared to eachother. Several spots,

urine up, were not present in follow-patients with bladder cancer and in those of

ted with a were indichese protein groupssamples of healthy persons (Fig. 20-25). T

circles and arrows.

th pTa iversus urine of patients wProtein pattern of normal urine 4.1.3

persons (n=7) and patients with bladder om healthyNative urine samples obtained fr

ed by 2-DE technique. Urine samplesre analysecancer of stage pTa (n=12) w

on IPG strips with 11 cm length and 3-10 containing 200 µg total protein were loaded

ed protocol. Protein spots were visualiz(NL) pH range for the first dimension of 2-DE

using silver staining (

healthy volunteers (Fig. 20A) and patients Comparison of urinary protein pattern of

n20B) revealed big differences in patterwith bladder cancer of stage pTa, GI (Fig.

and intensity of protein spots. 2-DE images of urinary protein of different healthy

shown in figure 20A, 21A,he same protein pattern as volunteers revealed almost t

sizes of 55 and 66 kDa. Using the same 22A. The strongest spots were seen at the

sulted ea patients rDE analyses on a urine sample of pTprotocols and conditions, 2-

protein spots (Fig. 20B). Compared to in protein patterns showing many additional

healthy urine, the number and the density of detectable protein spots were higher in

urine samples of pTa patients. The proteins which were seen as faint spots in normal

patients but in significurine were also present in urine sample ofantly higher amount.

s sultRe


6666 k kDDaa
5555 k kDDaa

6666 k kDDaa
5555 k kDDaa
4545 k kDDaa
3636 k kDDaa
2929 k kDDaa22












BB Figure 20: Comparison of protein pattern in normal urine (A) and in urine of patients with
bladder cancer of the stage pTa,GI (B). Urine samples containing 200 µg protein were loaded on 11
cm IPG strips with pH range 3-10 non linear (NL). Protein spots were visualized by silver staining. 2-
DE image of urine sample of a healthy volunteers (A) and urine of a patient with bladder cancer of the
stage pTa, GI (B) are shown. The protein spots at 55 and 66 kDa indicated by circles are similar in
both urine samples, but significantly stronger in urine samples of bladder cancer (B). In urine samples
of bladder cancer patients there are numerous additional spots as numbered (1-6).
th pT1 iversus urine of patients wProtein pattern of normal urine 4.1.4 urine, containing eange, nativNL pH rUsing IPG strips with 11 cm length and 3-10 adder cancer of pT1 stages (n=8) were of patients with blg total protein,μ200 ng protocol. Protein ned using silver stainianalysed by 2-DE. The 2-DE gels were stai a healthy person revealed two different mple obtained fromapattern of the urine s

s sultRe


ilar to healthy urine which was shown in ig. 21A) simFprotein spots at 55 and 66 kDa (pT1,GIII refigure 20A. The urinary protein pattern ofvealed several additional protei a patient with bladder cancer of stage n spots, compared to normal urine (Fig.


6666 k kDDaa

5555 k kDDaa

pHpH3434MWMW6666 k kDDaa
5555 k kDDaa
4545 k kDDaa
3636 k kDDaa
2929 k kDDaa22












55BB Figure 21: Comparison of protein pattern in normal urine (A) and in urine of patients with
bladder cancer of the stage pT1, GIII (B). Urine samples containing 200 µg total proteins were
loaded on 11 cm IPG strips with pH range 3-10 NL. Protein spots were visualized by silver staining.
The protein spots indicated by cycles in healthy urine (A) and in urine of patient with bladder cancer of
the stage pT1, GIII (B) are similar. There are additional spots which were numbered and indicated by
doted sequares (1-7) in urine samples of patients. Among marked protein spots, especially the protein
groups at 55 kDa are stronger in urine samples of patients (B).

s sultRe


in normal urine, were seen in ent kDa, which were also presThe spots at 55 and 66 urine of pT1 patients too. But the protein group at 55 kDa was present in urine of pT1
patients in a significant higher amount than that in normal urine. Spot 1 and spot 2,
detected in urine of pTa patients, were not present in urine of pT1 patients and also
6 were detected in urine samples of both were not present in normal urine. Spot 3- much stronger present in bladder cancer but they werepatients, either pTa and pT1 y pattern of pT1 patients, inarin figure 20B. In ururine of pTa patients shown pot 7 representing severaled as 7 were detected. Sadditional protein spots numbera patients. esent in the urine of samples pTdifferent proteins, at 35-45 kDa, was not pr

th pT2 iversus urine of patients wProtein pattern of normal urine 4.1.5

from patients with ein were obtained Urine samples containing 200 µg total protbladder cancer of invasive stages as pT2-3 (n=8) and were separated using 2-DE and 3-10 NL pH range ps with 11 cm length technique. For the first dimension IPG strisualized using silver staining i were vwere used. The protein spots on 2-DE gels thy urine (Fig. 22A) revealed the similarprotocol ( Protein pattern of healurine samples of healthy volunteers in protein spots at 55 and 66 kDa as shown for of urine sample of a image obtained from analyses figure 20A and 21A. The 2-DEprotein spots at 55 and 66 age pT2, GIII revealed tncer with spatient of bladder caa well as in urine of pT present in normal urine, askDa (Fig. 22B) which were alsoand pT1 patients, but in comparison to the stages pTa and PT1, these protein spots
were weaker in the urine samples of pT2 patients. The protein spot at 55 kDa was
rmal urine. Spot 1, which patients with pT2 than the nostronger in urine samples ofs, was present in the urine pT1 patientwas not present in the urine samples of tients. Spot 2 was not present in urinary samples of pT2 patients as well as in pTa pa ents. Spot 3 and 5, with that of pT1 patiprotein pattern of pT2 patients, in similarity detected were also,ine samples of pTa and pT1 patientswhich were present in the urble in urine of pT2 4 and 6 were not detectain the urine samples of pT2 patients. Spot detection of these spots in urine samples of pTa and pT1patients in contrast to the patients. Spot 7, which was seen only in the urine samples of pT1 patients (Fig. 21B),
8,spots numbered as2 patient. Additional was not present in the urine samples of pT 9 and 10 were present only in the urine samples of pT2 patients.

s sultRe


6666 k kDDaa
5555 k kDDaa

pHpH3434MWMW6666 k kDDaa
5555 k kDDaa
4545 k kDDaa
3636 k kDDaa
2929 k kDDaa














3636 k kDDaa99
22882929 k kDDaa33
4545BB Figure 22: Comparison of protein pattern in normal urine (A) and in urine of patients with
bladder cancer of the stage pT2, GIII (B). 200 µg urinary proteins were loaded on 11 cm IPG strips
with pH range 3-10 NL. Protein spots were visualized by silver staining. The protein spots at 55 and 66
kDa indicated by circles are similar in both urine samples, healthy urine (A) and urine of a patients with
bladder cancer of the stage pT2,GIII (B). In urine samples of patients with bladder cancer of the stage
were detected. 1, 3, 5, 8, 9,10 s and numbered asspotpT2,GIII additional protein es of follow-up versus urine samples of Protein pattern of urine sampl4.1.6 bladder cancer pateints patients on follow-up (n=10) rn of the urine samples of In the next step, protein patte 3) were compared., pT1, pT2-der cancer of different stage (pTaand patients with blads on follow-up, IPG strips samples obtained from patientFor 2-DE analyses of urine

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length and 3-10 NL pH range were used. Native urine containing 200 µg with 11 cmsualiztotal proteins were loaded and the gels were vied using silver staining protocol.

pHpHpH3434MWMWMW66 kDa66 kDa

55 kDa55 kDa

29 kDa29 kDa


66 kDa66 kDa

55 kDa55 kDa45 kDa45 kDa36 kDa36 kDa29 kDa29 kDa













BB bladder cFigure 23ancer of: Comparison o the stfage pTa, GI (B). protein pattern in urine 200 µg of paproteins, involvtients oed in un follorinw-e saup (A) anmples, wed patiere loadents wd onith
spot11 cm IPG s, at 55 strips and 6with p6 kDa,H whirange ch a3-1re marked 0 NL. Protein with spciotrcles were vis, are seen in sualized by silvurine er stsamples aining. Tof patients ohe proteinn
follow-up (A) and patients with bladder cancer of the stage pTa, GI (B). Additionally, the protein spot
numbere21B, 22B, 23B) is also pred as 3, which wass deteent in uricted inne sampl urine samplee of patients on folloof patients with bladdw-up. er cancer (shown in figure
ed with doted circles were detected in 2-DE spots groups at 55 and 66 kDa markurine of follow-up patients with similarity to all urine samples used including urine
h bladder cancer. However, the density samples of healthy persons and patients wit

Res sult


of these proteins wasdifferent stages and patients on follow-up t stronger in urine sahan those in urine of healthy persons. mples of patients with bladder cancer of


kDa66 kDa66

kDa55 kDa55

kDa29 kDa29


kDa kDa6666

kDa55 kDa55 kDa45 kDa45 kDa36 kDa3629 kDa29 kDa














BB bladder Figure 24cancer of the : Comparison ofstage pT1, GIII (B). protein pattern in urine Urine sampleof pas contatients oining n follo2w00 -uµgp (A) an proteins wed patiere nts usewd ithfor
2-DE staininanalyg wases uses. 11 d. The spcm IPG stripots grosup with spH ra, at 55 and 66 kDnge 3-10 NL were ua, indicatesed. For d by circldetees care sition of proteimilar in urinans, silveryr
protein pattern of patients on follow-up (A) and patients with bladder cancer of the stage pT1,GIII (B).
22B), is alThe protein spots numso present in uberine samred as 4, plewhis ocfh was see patients on follow-up. n in urine sample of pTa and pT1 patients (Fig. 21B,
ients on follow-up (Fig. 23A, 24A, 25A) and In comparison of urinary pattern of pat(Fig. 23B), pT1,GIII stage such as pTa,GI patients with bladder cancer of different different spots in urinary revealed similar but also ig. 25B) F(Fig. 24B), pT2,GIII (

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mples of patients on follow-up, there are few protein patterns. In 2-DE gels of urine saein spots in addition to protein spot groups marked detectable and faint stained protby doted circles.

4.1.7 patients of tumor staProtein pattern of urine samples ofge pTa patients on follow-up versus that of

As mentioned above the protein spots at 55 and 66 kDa were present in urine
doted circles (Fig. 23A). The densities of sample of follow-up patients as marked by these protein groups seen in urine samples of patients on follow-up were as strong
onal protein spot numbered as ing bladder cancer. The additias in that of patients havs on follow-up, which was not present in 3 was detected in urine samples of patientients with bladder cancer ent in urine samples of patesnormal urine but prominently pr pots representing different proteins, asof stage pTa (Fig. 23B). Many additional sshown and numbered for the same image in figure 20B, were detected in urine
to urine samples of patients on follow- patients with pTa, in comparisonsamples ofup.

4.1.8 patients of tumor staProtein pattern of urine samples ofge pT1 patients on follow-up versus that of

urine samples of another kDa were present also in The protein spots at 55 and 66 patient on follow-up as shown in figure 24A. These spots were marked as doted rked as number 4 was detected in urine pot at about 30 kDa macircles. The protein spatients with bladder cancer of pT1 (Fig. sample of pateints on follow-up and also 24A,B). The same protein spot was also present in urine sample of pTa patients and
higher ed with the same number in figure 20B. The density of spot 4 waswas indicatage pT1 (Fig. 24B) then the ients with bladder cancer stin urine sample of the patein spots, which were numbered for the follow-up patients. But the additional protine samples of patients with y in the ursame image in figure 21B, were seen onl bladder cancer.

4.1.9 Protein pattern of urine samples ofpatients of bladder cancer stage pT2 patients on follow-up versus that of

The protein spot at 66 kDa as marked by doted circle was not present in urine
in figure 25A. But the spot at 55 kDa wassample of this follow-up patient as shown present in urine sample of a patient on follow-up (Fig. 25A) in a higher intensity as in

65s sultRethe urine sample of a pT2 patient (Fig. 25B). The protein spots numbered as 3, 4, s on follow-up and those with th patient present in urine samples of boeand 5 werbladder cancer stage pT2/GIII.


kDa66 kDa66 kDa55 kDa55


kDa66 kDa66 kDa55 kDa55 kDa45 kDa45 kDa36 kDa36 kDa29 kDa29














BB Figure 25: Comparison of protein pattern in urine of patients on follow-up (A) and patients with
11 cm stbladder caripnscer of with pHthe range 3stage pT2, GIII-10 NL. (B). Silver staini Urinne sampleg was contas used for ining 20detec0 µg tion protein of proteiwen re loadespots. Thd one
protein spots seen at 55 kDa which marked by circles are similar in both sample, urine of follow-up
patients (A) and and patients with bladder cancer of the stage pT2, GIII (B). The protein spot at 66
kDa, which was seen in other 2-DE images is not visible in urine sample of this follow-up patient (A).
The pAnother protein rotein spspot marked byot in figure B marked by fat s a fat square in figure A isquare i not pres not psernt in urine esent in urinsamplee sampls of pT2 es ofpatie follow-unts (B). p
patients. The protein spots numbered as 3, 4, and 5 are present in both urine samples.



up (Fig. urine sample of a patient on follow-The spot marked as fat square in 2-DE of er stage pT2 (Fig. patient with bladder cancmple of a25A) was not present in urine s t square, present in urineprotein spot ,marked by fa25B). On the other hand, another ig. 25B) was not seen in urine sample of sample of a patient with tumor stage pT2 (F at 55 erent groups of spotsfmmary, two difa patient on follow-up (Fig. 25A). In suand 66 kDa were present in all urine samples (Fig. 20-25), but they were particularly
tients on follow-up. h bladder cancer and paprominent in urine samples of patients witNumerous additional proteins were detected in urine samples of patients with bladder
persons urine samples, including healthycancer in comparison to other groups ofre analysed in this study. and follow-up patients, which we

th bladder iurine samples of patients wIdentification of protein spots in 4.1.10 cancer GI were stage PTa/s with bladder cancer patientProteins from urine samples ofh both silver and CBB (Fig. 26). separated on a 2-DE gel and stained witpHpHpHMWMWMW333444565656797979888111000

6666 k kDDaa
5555 k kDDaa

3636 k kDDaa
2929 k kDDaa
2424 k kDDaa










the staFigure 26ge : PpTa/GI. rotein ideThe ntiurinarfication y patternfrom of a p 2-DE imaatientge with of blurine addof a per caancer tient of with bladdthe stage pTer canca/GI ewar of s
chosen for protein identification. Nine protein spots indicated by quadrates in Coomassie stained 2-
DE gel were cut out and subsequently underwent mass spectrometric analyses.

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Several protein spots observed in urine samples of patients with bladder cancer of
and underwent mass ained gel oomassie stthe stage PTa/GI were cut out from the cspectrometric analys , several proteins called as uromodulin,es. After these studiesalbumin (HSA), orosomucoid1 (ORM), DNA-binding glycoprotein, human serum been en (HC) havx-forming glycoproteihuman zinc-alpha-2-glycoprotein and compleidentified (Tab. 18).

NumSpot ber Match to Score NCreferBI datencabae se CovSequencerage (e %)
1 Urourommoducoid (Homo saulin; Tamm-Hopienrsfas) ll glcoprotein; 38 gi | 4507833 11
2 Nuclear DNA-binding protein (Homo 36 gi | 37550855 -
s) ensapi3 Ribosomal Protein S19 (Homo sapiens) 39 gi | 3164200 -

4 Human Serum Albumin (Homo sapiens) 164 gi | 4389275


5 Human Serum Albumin (Homo sapiens) 229 gi | 4389275 74
6,7 Orosomucoid 1 precursor (Homo 152 gi | 20070760 38
s) ensapi8 Human Zinc-Alpha-2-Glycoprotein 212 gi | 4699583 72
ns) o sapiem(Ho9 Complex-forming glycoprotein HC 94 gi | 223373 41
ns) o sapiem(Ho Table 18: Proteins idendified by MS analyses. For spot 2 and 3 no sequence coverage was
determined. These protein spots were predicted to be Nuclear DNA-binding protein (spot2) and
Ribosomal protein S19 (spot3).

uromodulin, is one ofOne of these proteins abundant , tamm-horsfall glycoproteiprotein in normal urine. n (Fig. 26, spot 1), also referred to asIn 2-D images the spot
kDa in figure 20-25) wasmentioned as the spot at 66 (belonging to uromodulin cancer but almost patients with bladderprominently present in the urine sample ofvolunteers and patients on follow-up. DNA-equally present in the urine of healthy as shown in figure spot group at 66 kDa binding protein (Fig. 26, spot 2) included inalmost equally20-25 was particularly pres prominentent in urine sample in urine samples of patis of patients on follow-up and healthyents with bladder cancer but
volunteers. The spots 4 and 5 (Fig. 26) were identified as human serum albumin,
which were present in all urine ssimilarity with spots marked by doted circleamples ans for the protein at alysed in this study. These spot55 kDa as shs were in own in
ticularlyman serum albumin were parpots representing hufigures 20-25. The sThe proteins indicated as prominent in urine samples of patients witspot 6 and 7 in figure 26 wereh bladder cancer and pa identified as orosomtients on follow-up. ucoid 1

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s with bladder cancer (showns of patientand they were present only in urine samplein figure 20B as spot 2), but were absent in urine samples of healthy persons and h the number 8 in figure 26 was identified patients on follow- up. The spot marked witominently present in the urine samples as human zinc-alpha-glycoprotein and was prof patients with bladder cancer (Fig. 20-25, included in spot 3) and in those of
patients on 9 in figure 26 was identified as But it was absent complex-forming glyin urine samples of healthy persons. The coprotein (alpha-1-
with bladder cancer, particularly smicroglobulin, also called HC). This protein trong in the was found in urine sstage of pTa, and visiable as a faint spot mples of patients
23-25). llow-up (Fig.mples of patients on fo(Fig. 20-25, included in spot 4) in urine saIn contrast, it was not detectable in urine samples of healthy persons. The spot 3 in
figure 26, which was identifieurine, but no considerable changes were sd as ribosomal protein S19,een for this protein in urine samples of was not present in normal
patients witmarked by doted circle at 55 kDa (Fig. 20-h bladder cancer and patients on follow-up. It is 25). The changes in densincluded in the spot group ity of identified
proteins in urine samples were summarized in table 19.


pT2-3 -/+

number Spot Idendified Proteins Healthy Follow-up pTa pT1 pT2-3
1 Uromodulin -/+ -/+
2 DNA-binding protein -/+ -/+
3 Ribosomal Protein S19 -/+
4,5 Human Serum Albumin -/+
6,7 Orosomucoid 1 --- ---
8 HumaGlycopn Zinroteinc -Alpha-2---- ---
9 gylcopComplex-frorotein ming --- -/+ -/+
Table 1samples b9: Sy e2-DE. miquantitative determination of protein levels of identified proteins in urine

4.1.11 Human serum albumin in normapatients with bladder cancer and follow-up l urine versus in urine samples of

The 2-DE results showed that most abundant proteins in urine samples, which were
ese protein spots, a. One of thD and 66 kanalysed in this study, were detected at 55albumin using mass spectrometry (Tab. at 55 kDa, was identified as human serum the amount of human serum albumin in normal urine 18). To view changes in

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samples and urine samples of patients with bladder cancer the urinary protein
mples were compared to each other. patterns of all groups of urine sa


kDa55 kDa55

kDa55 kDa55

kDa55 kDa55







HuHumaman Sn Seerruum m AAllbumbuminin

HuHumaman Sn Seerruum m AAllbumbuminin

HuHuman Seruman Serumm AAllbubummiinn

CC Figure 27: Comparison of protein spots indicating human serum albumin (HSA) in all urine
as Humsample grouan Seps.rum The prot Albumin. The ein spotproteis at 55 kDa, whichn pattern of heal are prthy uriesne en(A), urint in all urine sae of patients mples, were ion follow-up dentified (B),
shoand pwna as tients exemplawith blardder y) (C) were comcancer with dpared ito eafferent stach otges (uriher for thnary e sppatots tern of showina patig HSA. Theent with propTa,tein spot GI was s
compof HSA are arisonstronger to healthy person in urine s. samples of patients with bladder cancer and patients on follow-up in

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Human serum albumin was present in all urinary protein patterns (Fig. 27 A-C), but
urine of patients witthe strongest protein spots which belong toh bladder cancer (Fig. 27C) and human serum albumin were detpatients on follow-up (Feig. 27B)cted in
when compared to those in healthy urine (Fig. 27A).

ptides for identified proteins Mass spectrums and matched pe4.1.12

he NCBI database as shown for all identified Proteins were identified by searching t patients with bladder cancer proteins in urine samples of(Fig. 26). Spectrum of mass given for spot 1 which wase werspectrometric analyses and matched sequencesilar to and 3 which were identified as sim; spot 2 identified as uromodulin (Fig. 28)ein (Fig. 29A-B); spot 4 and 5 which ein and ribosomal protDNA-binding glycoprot(Fig. 30A,B); spot 6 and 7 which were were identified as human serum albumin 8 which was identified as zinc-alpha-2- 31); spotidentified as orosomucoid1 (Fig.mplex-forming glycoprotein (Fig. 32A,B). glycoprotein, and spot 9 identified as Co


Figure 28: MS identification of Uromodulin (spot 1). Matched peptides are in red.


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Figure 29: MS identification of DNA binding glycoprotein and ribosomal protein. Spot 2 was
idendified as DNA binding glycoprotein (A) and spot 3 was identified as Ribosomal protein (B).
Matched peptides were not given for this two proteins.


Res sult





Figure 30: MS identification of Human Serum Albumin (HSA). Spot 4 (A) and spot 5 (B) were
identified as human serum albumin. Matched peptides are in red.

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2201 01 SS
Figure 31: MS identification of orosomucoid 1. Spot 6 (A) and spot 7 (B) were identified as
orosomucoid. Matched peptides are in red.

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Figure 32: MS identification of Zinc-alpha-2-glycoprotein (ZAG) and Complex forming
glycoprotein. (spot 8, 9). Spot 8 (A) was identified as Human zinc-alpha-2-glycoprotein and spot 8
(B), as Complex forming glycoprotein (HC). Matched peptides are in red.

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4.2 Idendification of two urinary proteins using Western blot analyses
4.2.1 Detection of orosomucoid (ORM) patients with bladder cancer and patients on followin urine samples of healthy-up persons,
r stages classified as pTa, pT1, pT2, pT3 In urine samples (n=45) of all bladder tumoORM was detected at the size of 41 kDa by Western blot analyses using the
M was also found in the urine samples ig. 33A). ORpolyclonal anti-ORM antibody (F, but prominently in the urine samples y volunteershof patients on follow-up and healtof patients with bladder cancer, particularly in the invasive tumor stages as pT2-3.
y three times in 85 % of urineeased b volunteers ORM was incrCompared to healthysamples of bladder cancer patients on follow-up, by seven times in 83 % of patients
pT1, and twenty times in 71 % of patients with pTa, 16 times in 85 % of patients with with pT2-3.

4141 k kDDaa

4545 k kDDaa











Figure 33: Detection of ORM and ZAG in urine samples. Western blot analyses using polyclonal
anti-Ofollow-up RM antand in higheibody revealst ameount id ORM (An urin) ate sampl the referrees of d patients withsize of 41 invasive kDa in uribladdene sampler cancer stas of pages pTtients of 2-
ZAG at the expected 3. In similar pattern of ORsize of 45 kDM, westerna in urine blot analsamyseples usis (B) of patieng a polyclonal antints on follo-ZAw-up and pG antibody revealeatients withd
er canbladder. c

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4.2.2 healthyDetection of zinc-alpha-2-gly persons, patients with blcoprotein (ZAG) in urine samples of adder cancer and patients on follow-up

he size of 45 kDa in urine samples-glycoprotein (ZAG) was detected at tZinc-alpha2(n=45) of all bladder tumor stages classified as pTa, pT1, pT2, pT3 by western blot
anti-ZAG antibody (Fig. 33B). ZAG was also found in analyses using the monoclonal but prominently in eerson follow-up and healthy volunturine samples of patients er, particularly in invasive tumor stages h bladder cancurine sample of patients wit mples of healthy volunteers, ZAG wasasuch as pT2-3. Compared to urine ssample of bladder cancer patients on follow-increased by four times in 85 % of urine five times in 85 % of those s with pTa, by those patientup, by sixteen times in 75 % ofof patients with pT1, and twenty times in ients with pT2-3. 71 % of those of pat

4.3 bladder Expressiotisnsue pattern of ORM and ZAG in bladder cancer versus normal
an urinary bladder tissue comparing tumor e ORM and ZAG proteins in humTo localizes were performed on paraffin analysand normal tissue areas immunohistochemicalsections of bladder tissue. onsiderable staining in normal areas ofImmunohistochemistry for ORM revealed no c ge and small blood vessels larhereas endothelial cells ofbladder tissue (Fig. 34A) wy of tumor tissue (Fig. 35A) e proximitwithin tumor tissue (Fig. 34B) or in closexhibited ORM. In addition to the blood vessels, also some single cells, probably
inflammatory cells (Fig. 35B), within or around the tumor tissue were strongly positive
cularly those of invasive r cells exhibited ORM, partifor ORM. Also a part of tumo ZAG immunostaining was found at the tumor stages as pT2 and pT3 (Fig. 35A). 36A) which disappeared in superficial .luminal surface of normal urothelium (Figurrently, one or two cell rows at the basal bladder tumors as pTa (Fig. 36A). Concterestingly, the ZAG immunostaining was d ZAG (Fig. 36A). Inea expressside of pTmost prominent in tumor cells located at the invasive front (Fig. 36B) where tumor
ll marking the transition of tumor from de the lamina propria of bladder waacells invpTa to pT1 stage.

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there is nFigure 34: Lo consiocalizaderable tstaining for ORion of ORM in normal and bladder tumoM, neither in transitional r tissue. epithelium (TIn normE) nor in bloal bladdod vesseler tissues
(A). In contrast, blood vessels (arrows) of tumor tissue are strongly positive for ORM (B).

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Figure 35: Localization of ORM in bladder tumor cells. Vascular endothelial cells (arrows) and
exhibit ORMpositive for OR immunoM in closstaie ning (Aproximity of bladde). Inflammatory r tumor cellstissu (arrows) e, but also some tumwithin the tuor cellmor tissue s (arroalwso sho heads)w
). staining (BMRpositive O

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Figure 36: Localization of ZAG in bladder tumor tissue. ZAG immunostaining is localized at the
luminal suepithelium rfa(acrroe w headof normal transitios) when supenal eprficial blithelium (aadder trrowumor s) whisucch ah switchs pTes a is to thseee ban (A). Thsal side of e strongebladder st
staining of ZAG is found in tumor cells located at the invasive front (B).

4.4 immunocytochemistry Localization of ORM on endothelial cells (HDMECs) b


To determine whether the ORM staining in vascular endothelial cells due to

helial binding of ORM produced byendogenous production or only due to the endot

ltivated human primary microvascular immunostaining for ORM on cuother cells

These studies confirmed the resultsendothelial cells (HDMECs) were performed.

er tissue and revealed a strong stainingfrom immunohistochemistry on bladder canc

lls were mostly negative for ZAG (Fig. for ORM in HDMECs (Fig. 37A) while the ce

37B). ORM immunostaining was also present in cultivated bladder cancer cell line

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t, ZAG was not detectable in ECs (Fig. 38A). In contrasRT4 but weaker than in HDM

RT4 cells (ig. 38B). The presence of ORF

western blot analyses (Fig. 39A) whereas

extract of RT4 nor in that of HDMECs.



med by was confirM in endothelial cells

ZAG was neither detectable in protein

Figure 37Cultivated hu: Immunocyman dermal microvastochemical staining for cular endothlelial cellocalizats (HDMEion Cof Os) are RM anstrod ZAG in ngly positiveHDME for ORMCs.
(arrows, A), but are mostly negative for ZAG (B).





line. Figure 38ORM staining is al: Immnucytso preochemical staisent in bladder ning for locacancerliz ceation oll line RT4, f ORM and but its expreZAGssion i in bladder cs weakaerncer c than inell
HDMECs (arrows, A). ZAG is not detectable in RT4 cells (B).


4141 kD kDaa


kDa55 kDa55



BABA Figure antibody 39: coDetecnfirm thet immuion of ORMnohi in HDMstochemical ECs and RTdata and4 shcell ow Oline. RM Western at the blot aexpenacted lyses usize ofsing 41anti O kDaR iMn
perfoHDMECrmed bys (A). immunobl ORM was notting usiot seen in blng adder caanti-vimentin antibody (B). ncer cell line RT4. The control of protein loading was

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versus ORM gene silencing in HDMECs nORM overexpressio4.5 us ORM esis, ORM overexpression verss the role of ORM in angiogenTo addres The effeciency was confirmed by gene silencing were performed in HDMECs. and protein extracts of were performed on supernatantwestern blot analyses which at 41 kDa in lysate of . ORM protein was detectable (Fig. 40)transfected HDMECs . 40A) and their supernatant (Fig. 40C)ng endothelial cells (FigiORM overexpresscted endothelial cells (Fig. ibody. Empty vector transfeonal anti-ORM antlusing polycin the literature hed data publiseviously40A, Lane1) produce ORM as described in pr the gher amount of ORM was detected inicantly hi(Sorensson et al., 1999). A signifprotein extract of ORM-overexpressing HDMECs in comparison to empty vector
transfected HDMECs (Fig. 40A, Lane2). But ORM expression was almost completely
or plus texpressing vec simultaneously with ORM reduced in HDMECs transfected gmed usinM siRNA was confirORM-siRNA (Fig. 40A, Lane3). The specificity of ORsiRNA for luciferase, which did not cause any significant reduction of ORM in
On the other hand, as shown in figure contrast to ORM siRNA (Fig. 40A, Lane4). HDMECs was detected inafter its overexpression in40C, the majority of ORM (Fig. 40C, Lane2). ORM was detectable ng HDMECsisupernatant of ORM expressctor transfected cells (Fig. 40C, Lane1). as a faint band in supernatant of empty ve


4141 k kDDaa


a55 kDa55 kD



a41 kDa41 kD



B B Figure 40cells (HDME: OvCs). Therexpression and gee ORM amount in necell extra silencing vcit of ORM-a siRNA overexfor pressiORM in humng HDMECs (A, Laan dermal ene2) indothse onlylial
slightly higher as in the control. The use of ORM-siRNA reduces the ORM amount significantly below
the endogenous level (A, Lane1). No change is seen by application of luciferase siRNA used as
negative control (A, Lane4). Control of protein loading for cell lysates was performed by
immunoblotting using anti-vimentin antibody (B). Immunoblotting using anti-ORM antibody reveals
high amount of ORM at the size of 41 kDa in the supernatnats (C) of ORM-overexpressing HDMECs
(C, Lane2), which is sucsessfully reduced by additional transfection with ORM-siRNA (C, Lane3) while
luciferase siRNA used as control did not change the ORM amount (C, Lane4).

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ed ORM protein amount (Fig. siRNA reducThe transfection of HDMECs with ORM 40C, Lane3) while luciferase siRNA which used as a negative control did not change
the ORM amount (Fig. 40C, Lane 4). in-vitro angiogenesis assays via studies 4.6 Mechanistic

4.6.1 ORMsilenced In-vitroHDMECs endothelial tube formation using ORM-overexpressing versus

M in angiogenesis and capillary morhogenesis,To find out the potential effects of ORin-vitro endothelial tube formation assay was performed.





enhaFigure 4nced by 1: combiEndothelial ned aptubeplicatio formatin of VEGF-A anon assay. d VEGF-inthe supernataducedn endt of ORothelial M-overexpressitubes (arrows; A) areng HDMECs
(arrows; B). The treatment with VEGF-A and supernatant of HDMECs transfected with ORM
expressing vector plus ORM-siRNA reduced the tube formation (C) in comparison to VEGF-A alone
(A) and VEGF-A plus supernatant of ORM overexpressing HDMECs (B). Untreated endothelial cells
a negative control (D). swere used a nd ORM-silenced HDMECs were used in The supernatant of ORM-overexpressing are transfected with empty vector, ORM endothelial tube formation assay. HDMECs weexpressing vector, and ORM siRNA using nucleofector technology as described in

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itive control which was used as a posmaterial and methods ( VEGF-A as expected. The number and the ube formation (Fig. 41A)induced endothelial ted endothelial tubes were increased by combined network of VEGF-A inducapplication of VEGF-A and the supernatant of ORM-overexpressing HDMECs (Fig.
ECs transfected with supernatant of HDM41B). Combined application of VEGF-A,ORM expressing vector and ORM siRNA simultaneously did not affect VEGF-
ed as a treated endothelial cells were usinduced endothelial tubes (Fig. 41C). Un negative control and did almost not induce tubes (Fig. 41D).

nd PAI-1 in endothelial tube formation The interaction between ORM a4.6.2

l -1 and ORM on the endotheliaed of PAI a complex composSince it was reported that endothelial cells, it was aimed to study es the behaviour ofcells surface influency between ORM and PAI-1 in capillarpotential effect of the interaction re cultured and at weendothelial cellsmorphogenesis. To this aim, untreated ed until confluence. llagen gel and culturpassage 4 the cells were seeded onto coAfterwards, endothelial cells were stimulated with: (i) ORM, (ii) VEGF-A, (iii) anti-PAI-
ion of these factors. Two different alone and with different combinat1 antibodyre used for stimulation of HDMECs. concentration of ORM as 100 and 300ng/ml wedium were used as negative control and Endothelial cells exposed only to basal meused as a positive control and induced did not form tubes (Fig. 42A).VEGF-A was endothelial tube formation as also mentioned in tube assay studies above (Fig. 42B).
most not induce tube formation as also ORM, at 100 ng/ml concentration, alone did al h anti-PAI-1 antibody. 42C). Treatment witobserved in cases of untreated cells (Figk PAI-1 protein, induced the formation of a few ed to blocalone, which was usig. 42D). To test the effects of the endothelial tubes in contrast to ORM alone (F stimulated with these nd anti-PAI-1 the cells werecombined application of ORM afactors alone or in combination with VEGF-A. The application of ORM at 100 (Fig.
eased the VEGF-A A simultaneously incr43A) and 300 ng/ml (Fig. 43B) plus VEGF-depending on the applied concentration of ORM. induced endothelial tubes PAI-1 increased the network and the Combined application of VEGF-A and anti-number of VEGF-induced tubes too (Fig. 43C). Treatment of endothelial cells with
few tubes (Fig. 43D). The combined ORM at 300 ng/ml and anti-PAI-1 induced a lyM, anti-PAI-1, and VEGF simultaneousapplication of all three factors as ORtube formation. The application of anti-exhibited the strongest effect on endothelial

s sultRe


in combination with VEGF-A increased PAI-1 and ORM at 300 ng/ml (Fig. 44B,C)

n of VEGF-A and 100 ng/ml of ORM with than the combinatiomore endothelial tubes

anti-PAI-1 (Fig. 44A).





to untreFigure 42ated : EffeHDMEct oCf Os, as RMnegative and ancontti-PAIrol (A-1 an), andtibody VEGF-on endoA treatetheliad celll tube s, as poformsitive coation. ntrol In com(B), OparisRonM
(C) did not effect endothelial tubes, while treatment of antibody against PAI-1 formed several
endothelial tubes (D), but it is not a considerable effect. Wild type HDMECs were stimulated with 100
ng/ml ORM protein and 15 ng/ml of anti-PAI-1 antibody.

Res sult






appliFigure 4cation of3: Effect o VEGF-A andf ORM a ORM proteind an,nti-PAI-1 at two diffeat VEGF-indurent concceentrad endotions asthelial tubes. 100 (A)and 30 By c0 ngomb/ml (B), ined
the numbers of VEGF-induced endothelial tubes are significantly increased in correlation with applied
ORM VEGF-indconcuecentration. Thd endotheliae treatment with l tubes in comparisVEGF-A pluon to OsRM appli anti-PAI-1 acation (arrontibody induws, C). Combinces a higheed ar increpplicationase of
of 300 ng/ml of ORM and anti-PAI-1 antibody does not effect endothelial tube formation (D).

s sultRe





Figure 44VEGF-indu: ceEffecd endotht ofelial VEGF, ORM tubes are eaxnd antiteremly incr-PAI-1 combinaeased by cotmbined ion on endappliocthelial tubation of Oe RformatioM, as 10n. 0
ng/ml (aVEGF-indrroucews, A) and 3d endothelial 0tubes 0 ng/ml cocorrelncentration ates with OR(aM rrowconcens, B,C) witration. th anti-PAI-1 antibody. Increase of
dothelial tubes -vitro induced enImmunolocalization of ORM in in4.7 Endothelial tubes on collagen gel which were formed by wild type HDMECs after
re fixed eing factors such as ORM, VEGF-A and anti-PAI-1, wtreatment with stimulatOn paraffin sections of 5-night and embedded in paraffin. using Bouin´s fixative over 7 μm thickness obtained from paraffin embedded tissue block of in-vitro induced
performed using polyclonal anti-ORM endothelial tubes immunhistochemistry was a weakcells and tubes on collagen gel revealedantibody. Untreated endothelial staining for ORM (Fig. 45A) in comparison to endothelial tubes induced by VEGF-A
(Fig. 46A). There is no immunostaining in control sections exposed only to secondary
1ubes induced by treatment with anti-PAI-antibody (Fig. 45B; Fig. 46B). Endothelial tORM (Fig. 47A) in comparison to tubes alone exhibited stronger immunostaining for VEGF and anti-PAI-1 (Fig. 47B). induced by combined application of

s sultRe




UnFigure 45stimulated: Localiza wild type tionHDMECs of ORM o(arron wwis) in ld tcollypeag endothen gel erevealed a lial cells afteweak str tube formatiaining for ORM (Aon assay). In.
control section, no staining is seen (B).
induced by treatment ion of endothelial tubes ed in control sectNo staining was detectlication of ORM at by app Endothelial tubes induced with anti-PAI-1 alone (Fig. 47C).taining (Fig. 48A) in comparison to mmunos100 ng/ml alone exhibited stronger ORM ium (Fig. 45A). But the treatment of ediendothelial cells treated only with basal m and anti-PAI-1 revealed strongest ORM endothelial cells with ORM at 100 ng/mlimmunostaining on endothelial cells (Fig. 48B,C). Interestingly, in the case of
entrationh the same combination but a higher concwittreatment of endothelial cells on endothelial tubes was almost weakerof ORM as 300 ng/ml ORM immunostaining ,B). The control section of endothelial than the use of ORM at 100 ng/ml (Fig. 49Aed only to the PAI-1 which was expostubes treated with ORM at 300 ng/ml and anti-unostaining (Fig. 49C). Immunostaining secondary antibody did not show ORM imm hl tubes treated witobtained from endotheliafor ORM was most prominent in sections PAI-1 antiobody and VEGF-A simultaneously (Fig. 49D). ORM at 300 ng/ml, anti-network of endothelial tubes which were To define exact changing in number and g on -1 plus VEGF-A, during their growin treatment with ORM plus anti-PAIinduced byeasured. After the cells have endothelial tubes were mcollagen gel, the length of been stimulated with the factors ment of wellsioned above the photographse contrast taken every day using phascontaining endothelial collagen gel were microscope

s sultRe



immunoFigure 46staini: Ing for mmunostainORM on ening for OdothelRial cellM on VEGFs form-induceing VEGF-id ennduced tubdothelial tues,bes. whi
positive control (arrows, A). Control section is negative for ORM (B).


cTheh rwee ires po used asitives

th of each (Zeiss, Jena). The endothelial tubes were marked with arrows and the leng

ed by a factor or a of all tubes inducendothelial tube was measured. Total lengths

combination of factors were determined

summarized in table 20.

for each s

mple and the values werea

s sultRe





Figure 47: Localization of ORM on endothelial tubes treated with anti-PAI-1 antibody. ORM is
strongly positive in endothelial cells forming tubes by blocking of PAI-1 (arrows, A) in comparison to
combined application of VEGF-A and anti-PAI-1 antibody (arrows, B). The control section shows no
ng (C). stainiific cspe

The quantification of mentioned above revealed that the endothelial tubes as

ount as ml tubes in considerable aapplication of VEGF alone induces endothelia

al medium as al cells treated with basexpected (Tab. 20, column2) while endothelia

lial tubes (Tab. 20, column1).negative control did not form endothe

s sultRe





anti-PFigure 4AI-18: L anocalizatibodyt. ion ofEndothelial ORM oncell ens formingdothelial tubetubs byes tr ORMea ted stwimuilth ORation (100M in lo ngw c/ml) aonrcene sigtratnion anificantlyd
againspositive for Ot PAI-1 shoRM (aws stronrrows, A), whileger immuno the applistaining for Ocation ofRM (arro the same Ows, B, C).R M concentration with the antibody
was capable to form a few alone to endothelial cellsation of anti-PAI-1The applic

while application of ual addition of ORM, tubes (Tab. 20, column4) but not the individ

ORM at 100 ng/ml plus VEGF enhanced tnduced endothelial he length of VEGF-i cantly (Tab. 20, column5).itubes signif







Figure 49: Localization of ORM on endothelial tubes treated with ORM in high concentration
wleadith ans to a weti-PAIake-1 anr stainitibodyng . Interefor ORM ostingly, applin endothcelial tation ubes of ORM compatare 3d00 ng/ml to the samwiteh combi anti PAI-1 nation with low antibody
constroncegenstrationt stai of Oning for ORM atR 1M00 ng in endothelia/ml (arrows, A,B). l tubes is seTenhe control se by treatmection isnt of endothe negative lial cellfor ORs with M (C). VEGF, The
i PAI-1 antibody (arrows, D). 0 ng/ml and antORM at 30

bes was achieved when VEGFlength of endothelial cells tuAdditional increase of the

re applied to endotheliaplus ORM (100 ng/ml) plus anti-PAI-1 weollagen l cells on c

othelial tubes wasease of VEGF-induced endgel (Tab. 20, column9). The most incr

I-1 and ORM in 300 ng/ml AVEGF, anti-Pseen by the combined application of

concentration (Tab. 20, column8). Also tof ORM (300 ng/ml) application he combined

s sultRe

with anti-PAI-1 was capable to induccolumn10).

m)m)mm((ngthngth Le LeubeubeTT

e endothelial tube formation (Tab. 20,

1 1 2 2 3 3 4 4 5 5 6 6


1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 1 100
Table 20: Graphical representation of determination of tube lengths. Representation of tube
lengths formed by endothelial cells treated only with basal medium (1), with VEGF-A alone (2), with
100 ng/ml of ORM alone (3), with anti-PAI-1 (15 ng/ml) antibody alone (column4), with 100 ng/ml of
ORM and VEGF-A simultaneously (5), with 15 ng/ml of anti-PAI-1 antibody and VEGF-A
simultaneously (6), with ORM at 300 ng/ml and VEGF-A simultaneously (7), the application of ORM at
300 ng/ml and anti-PAI-1 antibody in combination with VEGF-A (8), with 100 ng/ml of ORM, anti-PAI-1
antibody and VEGF-A simultaneously (9), with 300 ng/ml of ORM and anti-PAI-1 antibody
y (10). sleousimultan

sion sDiscu

5. Discussion


e urine lycoproteins in thification of new proteins/g provides the identThis studysample of patients with cancer of urinary bladder by combined application of
th subsequent mass spectrometric analyses, proteomic technology such as 2-DE wi chniques such as immunoblotting,conventional protein research te of bladder tissue, geneticsectionsochemical studies on paraffin immunohist in-vitro silencing via siRNA technique and finallyapproaches including gene ons assay on collagen gel. In tube formatimechanistic studies such as endothelial established for appropriate separation ofthis study, the optimum conditions were obtained y on urine samplesication of 2-DE technologurinary proteins for the applcancer of different stage as well as s with bladder from healthy persons and patientamong y, two urinary proteins, ORM and ZAG,patients on follow-up. Going this waother proteins found in the urine samples, which were not known to have a functional
relation to the stages of bladder cancer and particularly to angiogenesis until now,
focussed on the functions of ORM in rlywere identified. This work was particulaangiogenesis, genetic and he potential role of ORM in endothelial cells. To explore tapillary formation assay were studies such as endothelilal cin-vitromechanistical performed.

5.1 The impact of 2-DE for characterization of urinary proteins
fluids including in urine. In the last few years, new technologies Among them a widely used metwere developed to identhod is the technique of theify proteins in body
coupled with MS/MS (Spahr entire protein followed by LCproteolytic digestion of the et al., 2001). Surface-enhanced laser desorpsion/ionization (SELDI)-MS has been
utilized to profile urinary proteins as an alternative high-throughput approach (Rogers
Among these techniques 2-DE with subsequent et al., 2003; Schaub et al., 2004).MS analysis is the more effective method for generating reproducible results in
human proteome pattern and protein identification in body fluids including urine. This
method has an advantage over the technique of proteolysis with subsequent MS/MS
analysis due to a 2-DE which can show the relative protein expression profiles with
molecular weights and pI.

sion sDiscu


5.2 powerful tool for identiOptimized separation of urinary prfication of urine proteins oteins for performing of 2-DE as a
was to view complete protein pattern of At the beginning of this study the first aim address this aim this studyurine samples and to predict the function of proteins whicfocused on developing an effective sample preparation h are present in urine. To
strategy for 2-DE. Additional samDE images, but each additional step can resuple preparation stepslts in the selectiv can improve the quality of 2-e loss of protein
Malasit, 2005).species, which is disc To avussed in seoid loss of proteins, a sampleveral studies (Oh et al., 2004; preparation strategy was Thongboonkerd and chosen
e this preparation methods and of the 2-DE arples of ias simple as possible. The princdescribed in section of material and methods. e been of protein spots havlarge numberUntil now, through extensive studies, a specific breakdown products ofof them are charge-isoforms oridentified, but many abundant proteins swhether the pattern of these breakdowuch as albumin, fibrinogen, immunoglobulin, n products shows a diseasetce specific. However,
stion but needs future analyses. In the composition or not is an interesting queliterature there are,abundance of proteins and the co however, limited proteomic datmplex nature of urine. Ta on human urine due to low he majority of proteins
ed from cytoplasm and have functional urine are originatwhich are present in normal 2002; Oh et al., nderson and Anderson, oteins (Aproperties similar to plasma pr of the urine samples are:ations of proteomic analysis 2004). The current certain limit(i) the total protein amount is low, (ii) few proteins present in urine samples in a
urinary proteinss in identification ofrelatively high amount may lead to difficultie(iii) urine cpresent only in very low concentrationsontains high amounts of body filtra and the locations of tes such as water, salts, electrolytes expressed proteins and
ich disturb 2-DE process. and nitrogenous waste products whdes and techniques to achieve a higher There are several studies describing metho ch as dye- or solvent-precipitation,protein concentration in urine samples suultracentrifugation, lyophilisation and the removal of the proteins abundantly present
nkerd et 1998; Pang et al., 2002; Thongbood Williams, mples (Marshall anin urine saultracentrifugation technique was applied al., 2004). To overcome these difficulties the salt, electrolytes, and small moleculesusing a centrifugal filter column to removete urine samples. Furthermore, the ra(<10 kDa) and as well as to concent mples prior to theirar prepare the urine sdevelopment of novel methodologies to bette

sion sDiscu


able studies were Although, consider was a necessary step.esuse in 2-DE analysoaches st of these appran urinary proteome, mopublished on mapping the hum r to its use for 2-DE analyses such asrequire many steps for urine preparation prioprecipitation methods, ultracentrifugation, removal of gylcosaminoglycans, dialysis
the most abundanten immunosubtraction ofsteps, lyophilisation, gel filtration, evoonkerd and Malasit, 2005). e-fractionation tools (Thongbproteins and/or other pr Recently, after publishing the data presented in this work, a novel method of solidphase bas ty ligands under controlledon of proteins for affinied, selective adsorptioncentration of low-abundance ease the ccrsaturation conditions was described to inon of high-abundance proteins from same proteins and to decrease the concentratiher improvement in protein hulasiraman et al., 2005). A furtep (Tmixture in a single st recently described using beads coated concentration/equalization technique was alsoastagna et al., 2005) and in that study with hexameric peptide ligand libraries (Cproteins has been reported. additional 251 hidden urinary ed in literature at that methodological knowledge reportUnder consideration of the several techniques were performed at the time of the study presented in this work, ion methods (TCA, tsuch as different protein precipitabeginning of the experiments rehydration buffers. We applied by testing the use of different acetone, etc.) followedultracentrifugation as a simple method to avoid loss of proteins and alteration in
for urine samples this sample preparation method usednature of urine. Efficiency ofnd high-resolution human urine proteome was enough to obtain a reproducible apattern in 2-D gels. However, for further extended studies on urinary proteins,
ques mentioned above tohe novel technimbined with tultracentrifugation can be coimprove the protein separation in 2-D gel. 5.3 bladder cancer Urine proteomics enables the identification of proteins related to
ive and opic evaluation, which is expens cystoscBladder cancer is diagnosed byinvasive. Urine cytology is the current non-invasive gold standard but it has low
tzenberg, 2001; Murphy et al., 1984). The search for sensitivity (Konety and Ge e cystoscopy as a diagnosticentially replacbladder cancer biomarkers that could poterogeneity of this by the molecular hetand surveillance tool has been complicated on urine proteomics as a source for a e focused disease. Numerous studies havd SELDI-TOF-MS to urine lieVlahou (Vlahou et al., 2001) apppotential biomarker.

sion sDiscu


e several carcinoma (TCC). Up to datll bladderesamples to detect transitional c related to bladder cancer, are described h are supposed to beurinary proteins, whic urinary calreticulin (Kageyama et al.,arker for bladder cancer such asas a biomear matrix protein lis et al., 2004), nucl2004), calcium-binding protein psoriasin (Ce tumor antigen, and telomerase (Ramakumar et al., 1999). 22, bladder 2-D gel ation and of protein preparAfter optimization of the techniquesses on a we performed extensive analyesis on human urine samples electrophor er, andpersons, patient with bladder canclarge number of urine samples of healthy oteomic map of normal human urine with on of pr Comparispatients on follow-up.w and patients on follow-up revealed fe cancer h bladderurine of patients witThe major differences were seen especially but significant differences. similaritiesients and healthy persons. The similarities concerned between urinary pattern of patsome protein spots at 55 kDa, which were later identified by mass spectrometric
a which were present in all urine analyses as human serum albumin, and 66 kDsamples. 5.4 using 2-DE mayNovel protein spots in urine sampl be of diagnostic relevance es of patients with bladder cancer
this study with already the proteins identified inhe list ofThe comparison of t et al., 1996; Grover lises (Castagna et al., 2005; Cepublished urinary protein databasal., 2004; Pang et al., 2002; Pieper et al., 2004; and Resnick, 1993; Oh et garding erevealed similarities r Rasmussen et al., 1996; Thongboonkerd et al., 2002)in the urine abundantly and knowingly being present the detection of few proteins etc. Among a high min, immunolglobulin,deriving from blood plasma such as albu of the 2-DE gel only a small ilver stainingbeing identified in snumber of protein spots ning. These protein spots which have been part was also detectable in the CBB staiesent in the urine samplesentially up-regulated or only pridentified to be either differ of patients with bladder cancer were cut out from the gel and analyzed in mass
ation and ible identifice analyses led to the reproducspectrometric analyses. Thesns such as ORM and ZAG. characterization of protei 5.5 ORM is increased in urine samples of patients with bladder cancer
Prior to the discussion of the impact of ORM in bladder cancer it is necessary to give
scribed in 1950 by Schmid (Schmid, 1950;a brief survey about ORM. It was firstly de

sion sDiscu


e phase proteins playing a role in the Schmid, 1953) and belongs to a group of acut to stress, along with many othermodulation of the immune response The relatively high . id, 1980; Schmid, 1975)functions(Bennett and Schmmans is known to rise two- to concentration of ORM in the serum of healthy hu such as acute infection, inflammatory andfivefold in response to different conditions .s and cancer (Schmid, 1975)lymphoproliferative disordera polypeptide chain ed and is composed of The structure of ORM is well characterizacid as mentioned above. containing about 45 % carbohydratThus, its proposed immunomodulate including a large amount of fucosylicory activities have been and sialic
e the strongly fucosylated and sialylatedattributed to its glycosylation pattern, sincE-selectin and to inhibit complement ORM glycoforms have the ability to bind activation (De Graaf et al., 1993). An induced expression of sialyl Lewis X (sLeX) on
might influence the E- or ORM during acute inflammation has been rP-selectin-mediated influx oeported, leading tof sLeX-expressing leukocytes the speculation that it
expressiinto inflamed ORM could have a feedback inhibi It has been suggested that an increastory effect on the extravased level of sLeX-ation of
ORM is thought to modify the pleukocytes, by competition for thee E-selermeability ctin adhesion moleculesof the vascular endot (Lasky, 19helium, possibly by 92). So
interacting with the endotheliabeen shown that ORM binds to the vasculal glycocalyx (Curry and Michel, 1980). Thus far it has r endothelial cell surface and then causes
intercellular junction (Predescu et al., e cell without passing the transcytosis across th1998). is elevated in different types of ORMIt has been shown that the serum concentration al., 2000) leading to the lung and ovary cancer (Duche etof cancers such as breast, cancer cells themselves. The present assumption that ORM might be produced by tumors, sion in urinary bladderM expresdata here show a significantly increased ORy, the detection of er cancer. Consequentl in the invasive stages of bladdparticularlyadder cancer patients, as eins in the urine of bllarge amount of ORM protcreased production and secretion of ORM. tes an inademonstrated in this work, indicnumber of studies report about ORM glycoform determination In the literature, a high such as colorectal (Hansen et al., 1987)in serum diagnosis of different cancer types, However, only a few studies are and ovarian cancer (Dobryszycka W., 1992). available concerning the changes of ORM levels in the urine of patients. It has been

sion sDiscu


ute inflammation (Pang et al.,nts with acobserved to be increased in urine of patie .betic patients (Narita et al., 2004)2002) and of normoalbuminuric type2 diaAfter being identified in 2-DE of the urine analyses performed in this study also
vel of ORM in the urine samples ofed leimmunoblotting studies revealed an increaspatients with bladder cancer and patients on follow-up. To my knowledge this is the
first report showing that ORM is samples of patientsantly increased in urine significwith urinary bladder cancer, particularly in those with invasive tumor stages. The
patients with acute ved to be increased in urine of level of ORM has been obser patients 2 diabetic inflammation (Pang et al., 2002) and of normoalbuminuric typehe results presented in 2-DE and t(Narita et al., 2004). Correspondinglyimmunoblotting studies here, the present immunohistochemical results suggest that,
ent inflammatory cells and, interestingly,in addition to cancer cells, some tissue residendothelial cells of angiogenicly activated blood vessels which are to find in close
increase of ORM in the urine ofas source for thisssue serve association to tumor tit increase in the mean protein would explain the highesbladder cancer patients. This1, which marks the switch from a non-amount of ORM at tumor stage pTa to pT invading the lamina proria where the tumor to a tumor typevascularized superficial tumor with activation of to blood vesselsget direct accestumor cells son et al. the previous results presented by Soerensneovascularization. Confirmingngs show that human vascular endothelial (Sorensson et al., 1999), the present findie of ORM in endothelial cells esencly. Thus, the prcells produce ORM endogenous obably forming new tumor vessels asactivated by tumor angiogenesis or prthe endothelial-bound ORM but also ORM demonstrated here does not only reflect haracteristic phenomenons of clls. One of the essential originating from endothelial ce is the abnormal vascular leakage orlly activated blood vessels aangiogenic ORM has been reported to modify the permeability (Hashizume et al., 2000). Indeed,x (Curry ing with the endothelial glycocaly by interactendothelial permeability, possibly al., 1998). Serum level of ORM is increased in and Michel, 1980; Predescu etinflammatory and lymphoproliferative disorders and cancer, such as breast, lung and
lved the id, 1975) which might be invoovary cancer (Duche et al., 2000; Schmmodulation of vascular leakage. ajor carrier of sialyl Lewis X a mructure is that ORM is A further aspect of ORM st acute inflammation. This finding led to the speculation that particularly during(sLeX), via this carbohydrate moiety ORM might be able to bind to E- or P-selectin and might

sion sDiscu


influence the binding and rolling as well as extravasation of leukocytes into inflamed
ed that CEACAM1, as recently demonstratky, 1992). Similar to ORM, it waareas (Las carrying sLeX and Lewis X residues andosylated cell adhesion moleculehighly glyc ndothelial cells of new immature blood is up-regulated in eacting pro-angiogenic, eira-Ferrer et al., 2004). Thisr stages (Olivly tumovessels of bladder cancer in earicly les, which are present in angiogenstructural similarity between both molecumilar to CEACAM1 also ORM ition that sactivated blood vessels, leads to the assumption. To aegulation of angiogenesis and tumor vascularizmight be involved in the rogy and angiogenesis, ORM ORM in endothelial cell bioladdress the potential role of a siRNA in HDMECs were performed. sion and ORM gene silencing vioverexpres ental manipulations, genetically modifiedimAfter testing the efficiency of these exper (endothelial tube formation). rmation assayHDMECs were used in in vitro capillary fo 5.6 ORM support the VEEndothelial overexpression of ORM as GF-induced endothelial tube formation well as endothelial stimulation by
Until now it has been shown that ORM binds to endothelial cell surface (Boncela et
M plays a role in capillary ether ORal., 2001) but it is so far unclear whcells. The results presented here morphogenesis provided by endothelial the overexpressed ORM in HDMECs isdemonstrate clearly, that the majority ofing suggests that probably ORM produced secreted into the supernatant. This find as might also be mainly secreted into the extrendogenously by endothelial cells and in endothelial d in cultured HDMECcellular space and thus, the ORM detectein ORM r blood vessels might mainly result activated tumocells of angiogeniclybinding on endothelial cell surface. Also data obtained by endothelial tube assay as
While the use of HDMECs transfected for presented here support this interpretation. significant differences in comparison toORM in endothelial tube assay did not lead to the simultaneous,stimulated by VEGFthe wild type HDMECs when they were stimulation of wild type HDMECs by the supernatant of ORM overexpressing
and network of endothelial gnificantly increased length i and VEGF led to a sHDMECstubes in comparison to the stimulation of HDMECs by VEGF alone. This effect was
partly reversed when endothelial cells on collagen gel were stimulated
simultaneously with supernatant of ORM silenced HDMECs and VEGF indicating that
the ORM-siRNA used here is to some extent effective in the in-vitro mechanistic
ECs transfected for ORM and ORM siRNAstudies. Since the supernatant of HDM

sion sDiscu


by application ofdothelial tubes induced simultaneously was not able to block enVEGF completely one can assume that either the ORM-siRNA was not effective
enough to block the endogenous production of ORM totally or there are other factors
ORM in endothelial tube formation. replacing the functional effects of 5.7 ORM interaction with PAI-1 influences the VEGF-induced endothelial tube
formation To gain more insight into the function of ORM in capillary morphogenesis, further in
vitro endothelial tube formation studies were performed to investigate the role of the
M and PAI-ly it has been shown that ORinteraction between ORM and PAI-1. Recent ly influences thel cells which obvious1 form a complex on the surface of endotheliamigration of endothelial cells (Boncela et al., 2001). These studies show that it is the
r-1 (PAI-1) which constitutes the complex otor inhibitaactive form of plasminogen activtive form of PAI-1 suggesting complex stabilizes the acwith ORM. The building of thisy effect of PAI-1. The ORM-he inhibitorthat ORM-PAI-1 complex would increase tPAI-1 co-localization was found in thymosin β4 (Tβ4)-activated but not in quiescent
nce the helial cells). ORM may influebilical cord vein endothuman umHUVECs (ll as their ability to generate plasmin e properties of endothelial cells as weadhesivinase type plasminogen activator (uPA) in al., 2001). The role of the urok(Boncela et rature (Andreasen et al., 2000; Binder etangiogenesis is widely described in the litesapillai, 2003; Chorostowska-Nade al., 2001; Choong and al., 2007; Boncela etko et al., 2004b). While the generation Wynimko et al., 2004a; Chorostowska-Wyniml for the regulation of ssentia action of uPA or tPA is eof plasmin from plasminogen byented that uPA plays a crucial stem, it is well documion sythe activity of the coagulatuding the vascular basement the extra cellular matrix inclrole in the remodelling of membrane normally underlying the endothelial cell layer and enclosing the pericytes
of the basementA well construction in capillaries (Andreasen et al., 1997). integrity of the vascular wall and also formembrane is of importance for the structural ep during the new tich is an essential smigration activity of endothelial cells whformation of blood vessels by angiogenesis (Folkman and D'Amore, 1996). The pro-
cked by PAI-1 (Andreasen et al., 2000; effects mediated by uPA are bloangiogenicn to interact with uPA and uPAR howBinder et al., 2007) which has been sharacter This interaction shows a dual c(Andreasen et al., 1997; Binder et al., 2007).as demonstrated in figure 50: (i) The interaction of PAI-1 with uPA and uPAR leads

102sion sDiscuto the internalization of uPAR and its subsequent recycling and translocation to the
e of endothelial migration and in an increasmembrane at the cellular tips which resultangiogenic activity. (ii) The presence of ORM results in the building of a complex with
PAI-1 at the endothelial cells surface which stabilizes the activity of PAI-1 leading to
cycling which consequently result in the ation and rethe blockage of uPAR internalizand angiogenesis (Andreasen et al., 1997). suppression of endothelial migration

AAnormal procnormal proceessssBB+ORM+ORMCC+O+ORMRM
vvlevileviaaeell LDL LDLooff P P re reAAccII--ee1 1 ptptoror
dependedependennttStabStabililiizzaattioionnooff P PAAI-I-111-IAP-itn+a1-IAP-itn+a
uPuPAARRinterninternalalizatizationionFrFree ORMee ORM
uPuPAARRrereccyyclingclingtutuMiMibegbegrraattforforioiommnnat aat aininooddnn



Figure 50: Important interactions within the uPAR-uPA-PAI-1 systems and the role of ORM.
Normally uPresulting in acA andtivation of endotheli uPAR interaaction ll migration aeads to ind antengiogenernalization of uPAR sis (A). In the presenand then recyce of ORcling of M there is auPAR
recycomplecling. Thx of ORM ais results nd PinAI-1 whic inhibition of endothh stabilizes PAI-1 and elial migratiosuppn anressed ansgioge then unesis (B). PAR internThealiz additionation anald
applicof free ORM acation ofting anti-PAI-1 which blowith VEGF-A togetcks the comher proangipleogenicx building of PAI-1 and O and increases the RM increaseendothelial migration s the amount and
capillary formation, thereby, promotes angiogenesis (C).

sion sDiscu


antibody as used here in ication of the anti-PAI-1 r, the additional appleHowevbuilding of ORM-PAI-1 complex with he endothelial tube formation assay blocks te the angiogenic activity of endothelial probably two consequences which increasM and PAI-1 results in lex building between OR) The blockage of compicells: () the inhibition of ORM-iion as described above, and (atienhanced uPAR internalizly act pro- free ORM which obviousPAI-1-complex increases the amount ofangiogenicly when applied in combination with VEGF and enhances the VEGF-
. Thus, it is conceivable to postulate a role ofin-vitro induced capillary morphogenesis-1 in the vascular morphogenesis. the interaction between ORM and PAI 5.8 Zinc-alpha-2-glycoprbladder cancer particularlyotein is incr in the invasive stages eased in urine samples of patients with
that ZAG is detectable in an increased demonstrates for the first timeThis study e tumors ofwell as invasiv patients with superficial as amount in the urine samples of demonstrate that unohistochemical studiesurinary bladder. Interestingly, our immpresence of ZAG at the luminal surface of normal transitional epithelium of urinary
the stage pTa ise a papillary tumor ofbladder switches to the basal side onc est immunostaining for ZAG was found inobserved. Since in our studies the strongtumor cells invading the lamina propria of the bladder wall, we suggest a relationship
rther studies. In contrast to ORM, between tumor invasion and ZAG, which needs fu the superficial tumor stage pT1 to thee of ZAG is seen from the strongest increasassumption that the ZAG enhancement is tumor stage pT2, confirming our einvasiv ent of an invasive tumor and not with angiogenesis,associated with the developmwhereas ORM increase seems to be related to angiogenic activ tion as discussedae need further studies on a higher ed her involvabove. The exact mechanismse protein present in the lbine samples. ZAG is a solunumber of tumor tissues and urserum, and has also been found in the cytoplasm of normal secretory epithelial cells,
, as well as in salivary, bronchial, east, prostate, and liverincluding those of br gi and Schmid, 1961; Tada et al., 1991).gastrointestinal, and sweat glands (Burral types of malignant tumors (Brysk et Increased expression of ZAG occurs in seve vel of ZAG has been used as a canceral., 1997; ez-Itza et al., 1993) and the serum lemarker. Although the biological functions of ZAG remain largely unclarified, a
noma, MAC16, leading to purified from murine adenocarciing factorlipidmobilizcachexia, and from the urine of patients with cancer cachexia has been shown to be

sion sDiscu

identical to ZAG (Todorov et al., 1998).

sion of ZAG in adipocytes enhancesoverexpres

(Bing et al., 2004).

More recently, it was shown that



cachexia in mice

Consion luc

6 Conclusion In summary, the present results demonstrat


e that the combined use of proteomics

ology approach and mechanistic with molecular cell bitechnology assays in-vitro

der as actors related to disoreins and fool to detect new protserve as a powerful t

er and to clarify their role in the bladder cancshown here for human urinary

M plays aSpecifically, the findings here show that ORpathogenesis of the disorders.

morphogenesis which might be of relevanceclear role in the VEGF-induced capillary

for tumor vascularization and anti-angiogenic tumor therapy.


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8 Appendi



Map of 3.1(-) vector


tae ulum VicCurri


Vitae 8.2 Curriculum Irmak Surname : First Name: Ster 25.02.1978 : Birthdayrkey uBirthplace : Mardin/ TSingle : FamilyTurkish : lityNationa: Raumerstr. 1 Adress 45144 Essen, Deutschland Education Background:chool in Konya, Turkey : Primary S1982-1990 a, Turkey : High School in Kony1990-1993 epartment, Selcuk University, Turkey : Undergraduate in Chemistry D1994-1998

itute of ochemistry Division, Inst: Graduate (MSc.) in Chem. Dept., Bi1999-2001 ersity, Turkey Pure and Applied Science, Hacettepe Univy istry Division, Hacettepe Universit: PhD in Chem. Dept., Biochem2001-2002 and Applied Science, Turkey (not University, Institute of Pure completed)

: Scientific research in Department2003-2006 of Urology, University Hospital of Hamburg-Eppendorf (for doctoral thesis)

ent of Anatomy, University Hospital of 2006-present : Scientific research in DepartmEssen (for doctoral thesis) Employment: 2006-present of Essen : Research Scientist in Department of Anatomy, University Hospital
2002-2006 : Research Scientist in Department of Urology, University Hospital of

tment of Chemistry, Biochemistry : Research Scientist in Depar 1999-2002 Division, Hacettepe University, Turkey

tae ulum VicCurri



1. Tilki D, IrHammerer P, Friedricmak S, Oh MG, Schuch G, Galaliveira-Ferrer L, Hauschildlae R, Stief CG, K J, Miethe K,ilic Atakaya H, E, Huland H,
switch in prostate cancerErgun S. CEA-related cell adhesion mo, Oncogene, lecule-1 is25(36) (2006),4965-74. involved in angiogenic
2. Ster IrmakFriedrich, Hartwig Huland, Suleyman , Derya Tilki, Jochen Heukeshoven, Ergun, Stage-DependenLeticia Oliveira-Ferrer, t Increase of Martin
Proteomics 5(16)Orosomucoid and Zi (2005),4296- 304. nc alpha-2 Glycoprotein in Urinary Bladder Cancer,
Hauschild, Sonja Gudrun Ziegeler, JessicaL.Oliveira-Ferrer, Derya Tilki, 3. Loges, Ster IrmakErgun, Dual Role of CEACAM1 in A, Ergin Kilic, Hartwig Huland, ngiogenesis and Invasion of HumanMartin Friedrich, Suleyman
Reseach 64 (2004), 8932-8938. er, CancerUrinary Bladder Canc

4. Eroğlu M., Irmak S., Acar A., Denkbaş E.B., Design and Evaluation of a
Chemotherapy in SuperficiMucoadhesive Therapeutic al Bladder Cancer, InteAgent Delivery System for Postoperative rnational Journal of
Pharmaceutics, 235 (2002) 5159. Presentations (Abstracts Available):1. Irmak S, Tilki D, Olivorosomucoid (ORM) in vascular endoteira-Ferrer L, Ergün S,helial cells and Urinary proteomics and the role oangiogenesis.102nd Annual f
ft, 30 March-2 April, 2007, Giessen, Meeting of the Anatomische Gesellscharal) OGermany. (2. S. Irmakdetection of orosomucoid in , D. Tilki, J. Heukeshoven, L. Oliveiraurinary bladder cancer, 17-Ferrer, , M. Friedrich, S. Ergün., The th Symposium
h 2006, Essen, Germany.(Poster) Experimentelle Urologie, 17-18 Marc3. S. Irmak, L. Oliveira-Ferrer, J. Heukeshoven, M. Friedrich, H. Huland, S. Ergün.,
Determination of urinary protein patternanalyses., European Association of Urology in bladder(EAU), XIXth Congres carcinoma by proteomic s, 24-27 March
2004, Vienna, Austria.(Poster) 4. Sevinc, E. Kilic, M. FriM.Fernando, L. Oliveira-Ferrer, D.edrich, C. Wagener, S. Ergün, Tilki, G. ZeigelerDual role, J. Hauschild, of CEACAM1 in S. Irmak, S.
inary bladder cancer., European angiogenesis and invasion of human ur h Congress, 24-27 March 2004, Vienna,Association of Urology(EAU), XIXtAustria.(Poster) 5. S. Irmak, L. Oliveira-Determination of urinary protein patternFerrer, J. Heukeshoven, in bladderM. Friedrich, H. Huland, S. E carcinoma by proteomic rgün.,
th analyses., 16Germany.(Poster) Experimental Urology Congress, 11-13 March 2004, Lübeck,
6. Denkbaş E.B., Irmak S.Mucoadhesive Polymeric Carriers to Use , Özdemir N., Eroğin Postlu M., Acar M., Mitomycin-C Loaded operative Chemotherapy in
Studies, IX. International Symposium on In-vitro Superficial Bladder Cancer: chnology, Antalya, Turkey, 19-22 Eylül 2002. (Poster) Biomedical Science and Te

cCurritae ulum Vi


7. Denkbaş E. B.., Kõlõçay E., Birlikseven C., Irmak S., Öztürk E., Preparation of
pheres and determination of their magnetical magnetic chitosan microsndproperties, 2June, 2001, Kõrõkkale. (Poster) National Congress of Chromatography, Kõrõkkale University, 6-8

8. Kõlõçay E., Irmak S., Baysal M.Y., Denkbaş E.B., Preparation and
CharacterizImmobilization, 3ardtion of Magnetic Ch Mediterranean Basin Conferencitosan Microspheres for Enzyme e on Analytical Chemistry,
a, Turkey. (Poster) June 4-9, 2000, Antaly Courses, Congress and Summer Schools:1. .102nd2007; Giessen, Germany. Annual Meeting of the Anatomische Gesellschaft, 30 March-2 April,
2. 17th Symposium Experimentelle Urologie, 17-18 March 2006, Essen,
Germany. 3. 15th Annual International CEA Symposium, 21-24 July 2005, Berlin,
Germany. 4. Vienna, Austria. European Association of Urology(EAU), XIXth Congress, 24-27 March 2004,
5. 16th Experimental Urology Congress, 11-13 March 2004, Lübeck, Germany.
6. 2nd2001, Kõrõkkale, Turkey. National Congress of Chromatography, Kõrõkkale University, 6-8 June,
7. Controlled Turkey. Release Systems, Marmara University, 11 May, 2001, Istanbul,
8. 7th National Symposium on Biomedical Science and Technology, Hacettepe
University, 25-27 September, 2000, Ankara, Turkey. 9. Modern and ClassiUniversity, 23 August - 2 September 2000, Kucal Biochemistşadasõ, İry Methods, Summer School, Agean zmir, Turkey.
10. 3rdJune, 2000, Antalya, Turkey. Mediterranean Basin Conference on Analytical Chemistry, MBCAC III, 4-9
11 ."1st National Chromatography Congress, 16-18 June, 1999, Kõrõkkale
kkale, Turkey. õrõUniversity, K12 Selçuklu, Konya, Turkey. .Certificate of Education, Selçuk University, Education Faculty, 1997,

sity, Education Faculty, 1997, rUnive

g unrErklä

Ergün leyman üSDr.


Erklärung eiche 6 ionsordnung der FachberAbs. 2, Nr. 8 der PromotHiermit erkläre ich, gem § 6 ss ich das Arbeitsgebiet, dem das Thema bis 9 zur Erlangung des Dr. rer. nat., da oid (ORM) in vascularization of bladderUrinary proteomics and the role of orosomucLehre vertrete und den Antrag von Ster cancer zuzuordnen ist, in Forschung und worte. rIrmak befü Essen, 13. April 2007 Prof. Dr. Süleyman Ergün
Erklärung g der Fachbereiche . 8 der PromotionsordnunHiermit erkläre ich, gem. § 6 Abs. 2, Nr6 bis 9 zur Erlangung des Dr. rer. nat., dass ich die vorliegende Dissertation
ebenen Hilfsmittel anderen als der angegselbständig verfasst und mich keinerbedient habe. Essen, 13. April 2007 Ster Irmak
Erklärung g der Fachbereiche. 8 der PromotionsordnunHiermit erkläre ich, gem. § 6 Abs. 2, Nrdass ich keine anderen Promotionen bzw. r Erlangung des Dr. rer. nat., u6 bis 9 z durchgeführt habe und dass diese ArbeitPromotionsversuche in der Vergangenheit von keiner anderen Fakultät abgelehnt worden ist. Essen, 13. April 2007 Ster Irmak

ter Irmak

ter Irmak