Thrombin-induced VEGF expression in human retinal pigment epithelial cells.
OBJECTIVE
The purpose of the present study was to investigate the effects of thrombin and thrombin in combination with other proangiogenic factors on VEGF expression in hRPE cells.


METHODS
hRPE cells were stimulated with thrombin TNF-alpha, monocytes, and TGF-beta2. After stimulation, conditioned medium and lysed cells were subjected to ELISA, Western blot analysis, immunocytochemistry, and RT-PCR analyses. Inhibitors specific for various signal transduction pathways were used to determine the signaling pathways involved.


RESULTS
Treatment of RPE cells with thrombin resulted in dose- and time-dependent increases in VEGF mRNA levels and protein production. hRPE VEGF expression is predominantly protease-activated receptor (PAR)-1 dependent. Approximately 80% of thrombin-induced VEGF secretion was abrogated by inhibitors of MAPK/ERK kinase (MEK), p38, c-Jun NH2-terminal kinase (JNK), protein tyrosine kinase (PTK), phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), nuclear factor-kappaB (NF-kappaB), and reactive oxygen species (ROS). Analyses of VEGF protein production and mRNA synthesis revealed that VEGF induction by thrombin plus TNF-alpha or coculture with monocytes was additive, whereas that by co-incubation with TGF-beta2 was synergistic. The costimulated VEGF production by TGF-beta2 plus thrombin was an average of three times higher than the sum of that induced by each agent alone. Furthermore, BAPTA [bis-(o-aminophenoxy)ethane-N,N',N'-tetraacetic acid], a calcium chelator, blocked the VEGF secretion induced by thrombin and thrombin plus TGF-beta2 by 65% and 20%, respectively, but had no effect on that induced by TGF-beta2 alone.


CONCLUSIONS
Thrombin alone and in combination with TNF-alpha, monocytes, and TGF-beta2 potently stimulated VEGF expression in hRPE cells via multiple signaling pathways. The thrombin-induced calcium mobilization may play an important permissive role in maximizing TGF-beta2-induced VEGF expression in RPE cells.
Bian
Zong-Mei
ZM
Department of Ophthalmology, University of Michigan, Ann Arbor, Michigan 48105, USA.
Elner
Susan G
SG
Elner
Victor M
VM
D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004359:Drug Therapy, Combination; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D012333:RNA, Messenger; D053781:Transforming Growth Factor beta2; D014409:Tumor Necrosis Factor-alpha; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013917:Thrombin