Characterization of leukotriene D_1tn4-induced gene signatures in human endothelial cells [Elektronische Ressource] / von Barbara Uzonyi
154 pages

Characterization of leukotriene D_1tn4-induced gene signatures in human endothelial cells [Elektronische Ressource] / von Barbara Uzonyi

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Characterization of leukotriene D -induced gene s ignatures in 4human endot helial cel lsDissertationzur Erlangung de s akadem ischen Grades doctor rerum naturalium(Dr. rer. nat.)vorgelegt dem Rat der Biologi sch-P harm azeutischen Fakultätder Friedrich-Schiller-U niversität Jenavon Barbara Uzonyigeboren am 23.01.1979 i n Buda pestGutachter:1.P rofessor Dr. A ndreas J. R. H abenicht (Jena)2.P rofessor Dr. Re inhard Wetzker (J ena)3.P rofessor Dr. H artm ut Kühn (Berlin)Tag de r Doktorprüfung: 09.03.2007Tag de r öffentlichen Verteidigung: 21.05.2007Table of contentsIntroduction.................................................................................................................................61.1. L eukotrienes and t heir receptors........................................................................................61.1.1. L T biosynthesis...........................................................................................................61.1.2. P hysiologi cal and pathologi cal rol e of t he LTs..........................................................91.1.3. L T receptors................................................................................................................91.1.3.1. BL T-Rs..............................................................................................................101.1.3.2. Cys LT-Rs101.1.3.3. F unctional studies on c ysLT-Rs...............................................................

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Publié le 01 janvier 2007
Nombre de lectures 22
Poids de l'ouvrage 6 Mo

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Characterization of leukotriene D -induced gene s ignatures in 4
human endot helial cel ls
Dissertation
zur Erlangung de s akadem ischen Grades doctor rerum naturalium
(Dr. rer. nat.)
vorgelegt dem Rat der Biologi sch-P harm azeutischen Fakultät
der Friedrich-Schiller-U niversität Jena
von Barbara Uzonyi
geboren am 23.01.1979 i n Buda pestGutachter:
1.P rofessor Dr. A ndreas J. R. H abenicht (Jena)
2.P rofessor Dr. Re inhard Wetzker (J ena)
3.P rofessor Dr. H artm ut Kühn (Berlin)
Tag de r Doktorprüfung: 09.03.2007
Tag de r öffentlichen Verteidigung: 21.05.2007Table of contents
Introduction.................................................................................................................................6
1.1. L eukotrienes and t heir receptors........................................................................................6
1.1.1. L T biosynthesis...........................................................................................................6
1.1.2. P hysiologi cal and pathologi cal rol e of t he LTs..........................................................9
1.1.3. L T receptors................................................................................................................9
1.1.3.1. BL T-Rs..............................................................................................................10
1.1.3.2. Cys LT-Rs10
1.1.3.3. F unctional studies on c ysLT-Rs.........................................................................12
1.1.3.4. Cys LT-Rs in the cardiovascular system ............................................................13
1.1.4. L T effects on t he endothelium ..................................................................................14
1.2. T hrombin and i ts receptors..............................................................................................16
1.2.1. T hrombin ge neration and i nactivation......................................................................17
1.2.2. T hrombin receptors...................................................................................................18
1.2.2.1. M echanism of PAR activation and signaling.....................................................18
1.2.2.2. P ARs in the cardiovascular system ....................................................................19
1.2.3. T hrombin effects on t he endothelium .......................................................................20
Rationale....................................................................................................................................21
Aims............................................................................................................................................21
Materials an d me thods.............................................................................................................22
2.1. M aterials..........................................................................................................................22
2.1.1. Re agents....................................................................................................................22
2.1.2. K its............................................................................................................................22
2.1.3. Buffe rs and solutions................................................................................................23
2.2. M ethods24
2.2.1. E ndothelial cell isolation and culture ........................................................................24
2+2.2.2. M easurem ent of [Ca] ............................................................................................25ic
2.2.3. RN A extraction.........................................................................................................26
2.2.4. M icroarray.................................................................................................................27
2.2.4.1. T erms27
2.2.4.2. Ra w data: signal, call, detection p va lue ............................................................27
2.2.4.3. G enechip quality control....................................................................................28
2.2.4.4. A nalysis..............................................................................................................292.2.5. Q ua ntitative reverse transcription pol ym erase chain reaction (qRT-P CR) ..............30
2.2.5.1. Re rverse transcription........................................................................................30
2.2.5.2. P rim er design.....................................................................................................30
2.2.5.3. S em i-qua ntitative PCR .......................................................................................31
2.2.5.4. Re am plification and clean-up of t he standard fragment....................................31
2.2.5.5. S eque ncing.........................................................................................................32
2.2.5.6. qP CR ..................................................................................................................32
2.2.6. P rotein extraction and concentration measure m ent..................................................34
2.2.6.1. L ysis in Laem m li buffer.....................................................................................34
2.2.6.2. L ysis in Tris-Triton buffer.................................................................................34
2.2.6.3. P rotein concentration measure m ent...................................................................34
2.2.7. S DS-polyacrylam ide ge l electrophoresis and Western bl ot......................................34
2.2.7.1. P onceau Red staining.........................................................................................35
2.2.7.2. Im m unostaining.................................................................................................35
2.2.8. IL -8 ELISA...............................................................................................................36
2.2.9. T F proc oagulant activity assay36
2.2.10. F low cytom etry.......................................................................................................37
2.2.11. Im m unofluore scence...............................................................................................37
2.2.12. S tatistics..................................................................................................................38
Results........................................................................................................................................39
3.1. L TD -induced ge ne signatures.........................................................................................394
3.1.1. L TD -induced early ge nes resem ble those activated by t hrombin............................414
3.1.2. L TD and t hrombin added t ogether augment ge ne expression.................................414
3.1.3. V alidation of t he microarray results by qRT-P CR ...................................................43
3.1.4. L TD stim ulates im m ediate-early ge nes...................................................................464
3.1.5. L TD -induced ge ne signatures are the result of c ysLT -R stim ulation....................464 2
3.2. Cha racterization of L TD -induced early ge nes................................................................474
3.2.1. L TD induces the EGR and NR4A transcription factors..........................................474
3.2.2. L TD up-regulates inflam m atory ge nes in HU V ECs ................................................504
3.2.3. L TD -m ediated i nduction of DSCR1........................................................................524
3.3.3.1. Invol vem ent of N FAT in LTD -induced ge ne expression.................................524
3.2.4. L TD trigge rs TF expression and cell-associated proc oagulant activity..................554
3.3. L ate ge nes regulated by L TD ..........................................................................................564
3.3.1. Induc tion of t he com plem ent regulator CD55 by L TD ...........................................5643.3.2. D own-regulation of t he cysLT -R mRNA by L TD .................................................582 4
Discussion...................................................................................................................................59
4.1. P otential rol e of L TD -induced t ranscription factors in cardiovascular pathobiology....594
4.2. L TD -induced ge nes in leukocyte recruitm ent and extracellular matrix rearrangem ent.634
4.3. P rocoagulant EC phenotype evoked by L TD .................................................................654
4.4. L TD -induced CD 55 may prot ect ECs against enhanced com plem ent activation..........674
4.5. N egative feedback mechanism s of c ysLT -R activation may involve DSCR1...............682
4.6. L TD stim ulation leads to cysLT -R down-regulation in ECs .........................................704 2
4.7. Comparison of L TD - and t hrombin-induced ge ne expression patterns..........................704
4.8. Conc lusions......................................................................................................................73
Summary...................................................................................................................................

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