The induction of an angiogenic response in corneal myofibroblasts by platelet-activating factor (PAF).
OBJECTIVE
Although the exact mechanisms underlying corneal neovascularization remain unclear, cytokines and growth factors play an important role in their development. We have shown previously that the inflammatory mediator platelet-activating factor (PAF) is a potent inducer of corneal neovascularization in vivo. In this study, we investigate the role of stromal myofibroblasts in neovascularization and the effect of PAF on this process.


METHODS
Myofibroblasts were obtained from rabbit corneal keratocytes and identified with anti-α-SMA antibody. Cells were treated with PAF (100 nM) for 24 hr. In some experiments, cells were pre-treated with the PAF antagonist LAU-0901 (150 nM). Expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) was examined by immunofluorescence and immunoblotting. To study the effect of myofibroblasts on vessel formation in vitro, Vybrant(®) CM-DiI labeled human umbilical vein endothelial cells (HUVECs) were cultured on myofibroblasts in a thin layer of collagen gel. CD31 was used as the cell marker of HUVEC.


RESULTS
VEGF and TSP-1 were not detectable in keratocytes, but they were positively stained in myofibroblasts. PAF induced a significant increase in VEGF expression and a decrease in TSP-1 expression. These changes were inhibited in the presence of LAU-0901. HUVECs co-cultured with corneal myofibroblasts formed a typical structure of vessel-like tubes within 1 week. The addition of PAF to the medium increased HUVEC-induced vessel-like tube formation, which was abolished by LAU-0901. Addition of anti-VEGF antibody to the medium completely prevented the formation of vessel-like tubes.


CONCLUSIONS
We provide evidence for the role of stromal myofibroblasts in the corneal neovascularization process. By enhancing VEGF production and decreasing TSP-1 production in myofibroblasts, PAF augments the angiogenic response. The PAF antagonist LAU-0901 could represent a new therapeutic venue for inhibiting corneal neovascularization.
He
Jiucheng
J
Neuroscience Center of Excellence and the Department of Ophthalmology, Louisiana State University Health Sciences Center, School of Medicine, New Orleans, Louisiana, USA.
Eastlack
Jason P
JP
Bazan
Haydee E P
HE
D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D003315:Cornea / Q000166:cytology*; D016510:Corneal Neovascularization / Q000139:chemically induced*; D004095:Dihydropyridines / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D058628:Myofibroblasts / Q000187:drug effects*; D010972:Platelet Activating Factor / Q000009:adverse effects* / Q000037:antagonists & inhibitors; D011817:Rabbits; D019700:Thrombospondin 1 / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D004095:Dihydropyridines; C487837:LAU 0901; D010972:Platelet Activating Factor; D019700:Thrombospondin 1; D042461:Vascular Endothelial Growth Factor A