Keratocytes Promote Corneal Neovascularization Through VEGFr3 Induced By PPAR?-inhibition

Background:The cornea,with the characteristic of transparency and no blood vessels,is the significant part of visual system.However,the cornealneovascularization will invade into the corneal stroma as the cornea lost the feature of transparency in the certain pathological conditions.The causes of cornealneovascularization are quite complicated,as the same time,the pathogenesises are also obscure.Our group has researched that the keratocyte,with the secretion of MMP13 degrading the extracellular matrix to provide the space,played the important roles in the formation of corneal neovascularization.But the mechanism of how the synthesis and secretion of the MMP13 is induced by the keratocytes has never been researched clearly.Purpose:Discuss the mechanism that keratocytes upregulate the expression of VEGFr3 and MMP13,promote the corneal neovascularization(CNV).Method:Corneal neovascularization model is established by corneal alkali burns and the mouse are divided into three groups such as normal control group,PPARa agonist(200uM Fenofibrate)group and vehicle control(0.1%DMSO)group.1)examinated the CNV length and area with slit lamp microscope at 1?3?5?7 days after alkali burns;2)examinated VEGFr3?MMP13?PPAR??ACSM1?ACADM?ADH7 mRNA and protein expression with qRT-PCR and Western Bolt method;3)identified the relation between PPARa expression and corneal neovascularization formation in suit hybrization(ISH)and immunohistochemistry(IHC)staining detection method;4)examinated the TG accumulation in oil red O staining detection method.The primery mouse keratocytes are treated by 20uM Fenofibrate or 0.01%DMSO at 24 hours and then are examinated the VEGFr3?MMP13?PPARa?ACSMI?ACADM?ADH7 mRNA and protein expression with qRT-PCR and Western Bolt method.At the same time,PPAR?-/-mouse corneal alkali burn models and primary keratocytes models are established to testify that PPARa gene can promote the ACSM1?ACADM?ADH7 and inhibite the VEGFr3 and MMP13 mRNA and protein expression.Result:After the corneal alkali burns 1-5 days,CNV length and area are increasing continually,but CNV begin to decrease at the 7 days through the slit lamp examination;the progress of CNV is positive correlation with the expression of MMP13?VEGFr3,however,negative correlation with the expression of PPARa?ACSM1?ACADM?ADH7;TG is accumulated in the corneal stroma in Oil red O staining detecting method.PPARa mRNA is mainly expressed in corneal epithelial cell?stroma cells?endothelial cells in the normal cornea;PPARa expression is markedly decreased in cornea,especially stroma cells after corneal alkali burns through the ISH and ICH,which indicates the CNV is related to the inhibition of PPARa expression.The local application of Fenofibrate can promote PPARa expression,inhibite the expression of VEGFr3 and MMP13 and the CNV progress.PPARa gene knockout can improve the CNV progress through which promotes VEGFr3 and MMP13 expression in mice cornea.Primary keratocytes models indicate that actived PPARa can downregulate the VEGFr3?MMP13 expression in the keratocytes.PPARa gene knockout can upregulate the VEGFr3?MMP13 exppression in the keratocytes.Conclusion:Keratocytes can promote the expression of VEGFr3 and MMP13 and promote the formation of CNV through PPARa downregulation.