The role of PDGF receptor inhibitors and PI3-kinase signaling in the pathogenesis of corneal neovascularization.
OBJECTIVE
Corneal neovascularization remains an unsolved therapeutic problem. Platelet-derived growth factor (PDGF) is directly linked to vessel formation and stabilization. This study was undertaken to elucidate the mechanisms by which PDGF exerts its effects on corneal angiogenesis.


METHODS
Corneal neovascularization was induced in C57 mice by removal of the limbal epithelium. When mature vessels appeared after 7 days, mice were treated with the PDGF receptor-beta inhibitor AG 1296 or the phosphatidylinositol 3-kinase (PI3-K)-inhibitors wortmannin and LY294002, respectively, using an intraperitoneally implanted miniosmotic pump. At day 14 after scraping, corneas of treated and untreated (control) mice were dissected and immunostained with FITC-CD31 antibody for endothelial cells and with Cy3-SMA (smooth muscle actin) for pericytes. VEGF (vascular endothelial growth factor), ang1/2 (angiopoietin 1 and 2), and PDGF mRNA levels of treated and untreated corneas were determined by real-time RT-PCR.


RESULTS
Mice treated with the PDGF inhibitor AG 1296 showed an inhibition of corneal neovascularization of 21.1% and a reduction of pericytes of 52% in the newly formed vessels compared with untreated animals. VEGF, ang1, ang2, and PDGF mRNA expression was reduced in the corneas of AG 1296-treated mice compared with the respective control. Treatment with the PI3-K inhibitors wortmannin and LY29002 had similar effects, inducing a decrease in corneal neovascularization and a reduction of VEGF, ang1, ang2, and PDGF mRNA levels.


CONCLUSIONS
Inhibition of the PDGF signal pathway results in loss of pericytes and a reduction in vessel density in the neovascularized cornea that correlates with reduced expression of PDGF, ang1/2, and VEGF mRNA. Furthermore, PI3-K was shown to be involved in the regulation of VEGF, ang1, and PDGF, as the PI3-K inhibitors wortmannin or LY294002 had similar effects. Because PDGF is a known stimulus for PI3-K activation, it can be postulated that the observed decrease in VEGF, ang1/2, and PDGF mRNA levels on administration of the PDGF inhibitor is caused by the decreased activation of the PI3-K signaling cascade.
Dell
Susanne
S
Department of Vitreoretinal Surgery, Center for Ophthalmology, University of Cologne, Cologne, Germany.
Peters
Swaantje
S
Müther
Philipp
P
Kociok
Norbert
N
Joussen
Antonia M
AM
D000730:Androstadienes / Q000494:pharmacology; D042683:Angiopoietin-1 / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D042702:Angiopoietin-2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002867:Chromones / Q000494:pharmacology; D003315:Cornea / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D016510:Corneal Neovascularization / Q000209:etiology* / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009025:Morpholines / Q000494:pharmacology; D020286:Pericytes / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D020032:Tyrphostins / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D020032:Tyrphostins; D042461:Vascular Endothelial Growth Factor A; D000077191:Wortmannin; C467628:Angpt1 protein, mouse; C467480:vascular endothelial growth factor A, mouse; C106250:6,7-dimethoxy-3-phenylquinoxaline; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one