The Therapeutic Effect Of VEGF And PDGFB Receptor Tyrosine Kinases Inhibitors In Mice Corneal Angiogenesis

Physiologically avascular cornea is transparent with conjunctiva vessels ending at the limbus.But in numerous pathological processes,such as infection,trauma, immunologic diseases,corneal neovascularization,the formation of new blood vessels from limbus can occur.Although corneal neovasculariztion(CNV) has some advances in reducing inflammation,fasting wound healing,preventing corneal melt,it may significantly alter vision acuity,and worsen the prognosis of subsequent penetrating keratoplasty,which is caused by the loss of the immunologic privilege of the cornea. The rejection risk can reach to 90%.In China,only the CNV caused by trauma occupied 10%of all corneal diseases.In Shanghai area,more than 2/3 of total 500 patients received PKP in Eye & ENT hospital of Fudan University annually suffered from CNV.And some patients even lose the chance to receive the surgery because of severe CNV.The treatment of corneal neovascularizaion remains largely unsuccessful.Endothelial cells,pericytes and basement are the main components of new vessels. Angiogenesis is a complex processes,including endothelial cells proliferation, migration and remodelling.The major steps are:a) endothelial cells and pericytes activated;b) original basement membrane degradation;c) proliferation and migration of activated endothelial cells and pericytes;d) new vascular tube formation e) recruitment of pericytes;f) new basement deposition;g) vascular netwok formation. Previous studies have disclosed that although multiple stimuli may be involved in the development of new vessels,vascular endothelial growth factor plays a major role in angiogenesis.In pathologic processes,VEGF mRNA expression is up regulated, which stimulates endothelial cells.Active endothelial cells can excrete matrix metalloproteinase(MMPs) to breakdown basement membrane and extracellular matrix(ECM).Following proteolytic degradation of the ECM,"leader"endothelial cells start to migrate through the degraded matrix.They are followed by proliferating endothelial cells to form blood sprouts.So VEGF has become a therapeutic target.And VEGF blockade has been recently validated as an effective strategy for inhibition of new blood vessel growth.However,several studies have also shown that anti-VEGF therapy may not be so sufficient to cause vessels regression in advanced stage. Because of the limited ability to impact established vessels,the role of pericytes in new vessels formation has been realized gradually.According to the clinicopathological study on 196 corneal buttons by Cursiefen,2 weeks after onset of CNV,more than 80%of new vessels were covered by pericytes. Basic researches show that PDGF-B is expressed by the sprouting endothelium and PDGFR-βis expressed by the pericyte.An endothelial PDGF-B signal controls pericyte recruitment to angiogenic vessels.Moreover,pericytes release VEGF,which in turn affect endotelial cells survival.The pericyte recruited to vessels means the stability and matuation of new vessels.At this stage,the endothelial cells become less dependent on VEGE This is the reason why interference with VEGF/VEGFR pathway is not effective.Besides the paracrine manner,direct cell-cell contact also enhances the endothelial cells survival.Based on previous studies,it is reasonable to inhibit PDGFB signaling pathway to cause disruption of pericytes recruitment.Then endothelial cells without pericytes support undergo apoptosis after VEGF withdrawal.This antiangiogenic therapy has been validated in stumor-associated angiogeness vessels.VEGF and PDGFB proteins bind to their receptors,VEGFR-2 and PDGFR-βrespectively,to stimulate endothelial cells and pericytes proliferation.These are transmembrane receptors with intracellular tyrosine kinase domain(RTK).Recently, some small molecular RTK inhibitors(TKI) have been developed which demonstrate potential therapeutic efficient.These drugs interrupt VEGF/VEGFR or PDGF/PDGFR-βsignaling through inhibition of receptor phosphorylation.These drugs have been used to treat tumor-associated angiogenesis,but seldom applied in corneal neovascularization.In our study,two RTK inhibitors,targeting VEGF/VEGFR and PDGF/PDGFR-β,were used alone or in combination for corneal new vessels. PartⅠPericyte Recruitment in Mice Corneal AngiogenessPurpose:To observe the occurrence and morphologic characteristics of pericytes recruitment to corneal new vessels in mice model.Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/l) to the central cornea.At 3,7,10,14,21 and 28 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.Result:3 days after scraping,pericytes only coated to the base of angiogenic sprout. 10 days after scraping,pericytes moved toward the tip direction.14-21 days after scraping,all vascular loops were covered by pericytes.Pericyte density was higher in main trunk than in branch vessel.The pericytes in main trunk exhibited a rounded morphology and closely associated with each other.Others in branch appeared flat shape with long processes wrapping the endothelium.28 days after scraping,the coverage of pericyte decreased,and some pericytes detached from endothelium. VEGF,PDGFB and ANG1 mRNA expression was up-regulated after scraping.Conclusion:The coverage of corneal new vessels by pericyte lags behined the blood sprout formation.Pericytes demonstrate morphology and density difference in main trunk and branch vessels.PartⅡThe Therapeutic Effect of VEGF and PDGFB Receptor Tyrosine Kinases inhibitors in Growing Stage of Mice Corneal AngiogenesisPurpose:To compare using VEGF and PDGFB receptor tyrosine kinase inhibitors alone and in combination for the treatment of growing stage mice corneal new vessels.Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/L) to the central cornea.Either VEGF receptor inhibitorⅡor PDGFB receptor inhibitor or both were administrated by intraperitoneal injection once a day for 10 days.Phosphate-buffered saline(PBS) was used as control.At 10 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.Result:Mice treated with the VEGF,PDGFB receptor inhibitor or both showed an inhibition of corneal neovascularization of 15.4%,12.18%and 17.19%respectively, compared with untreated animals.VEGF,PDGFB and ANG1 mRNA expression was not impaired in the corneas treated with VEGF or PDGFB receptor inhibitor alone. But ANG1 mRNA expression was decreased in corneas treated with both.Conclusion:Inhibition of VEGF and PDGFB signaling alone was effective to early stage corneal neovascularization.Combination therapy showed more significant efficiency.PartⅢThe Therapeutic Effect of VEGF and PDGFB Receptor Tyrosine Kinases inhibitors in Maturated Stage of Mice Corneal AngiogenesisPurpose:To compare using VEGF and PDGFB receptor tyrosine kinase inhibitors alone and in combination for the treatment of maturated stage mice corneal new vessels.Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/L) to the central cornea.At 10 days after scraping,Either VEGF receptor inhibitorⅡor PDGFB receptor inhibitor or both was administrated by intraperitoneal injection once a day for 10 days.Phosphate-buffered saline(PBS) was used as control.At 21 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.Result:Mice treated with the VEGF,PDGFB inhibitor or both showed an inhibition of corneal neovascularization of 3.04%(P＞0.05),12.59%and 20.85%respectively, compared with untreated animals.VEGF,PDGFB and ANG1 mRNA expression was not impaired in the corneas treated with VEGF alone.But VEGF,PDGFB and ANG1 mRNA expression was decreased in corneas treated with PDGF inhibitor alone or both.Conclusion:Inhibition of VEGF signaling was not effective to maturated stage corneal neovascularization.Inhibition of PDGFB signaling resulted in blood regression.Combination therapy showed more significant efficiency.