Study Of VEGF-C And VEGFR-3 Expression Changes With Intervention By Fenofibrate In Human Retinal Pigment Epithelial Cells And BN Rat Choroidal Neovascularization In Vitro/in Vivo

ObjectiveThrough in vitro experiments, this research studies and probes into the impacts of fenofibrate on vitality of the human retinal pigment epithelium (RPE) and the human umbilical vein endothelial cell (HUVEC) as well as the possible mechanism. Through in vivo experiments, it discusses the changing rules of VEGFC and VEGFR-3 of choroid and retina during the formation process of choroidal neovascularization in the laser-induced BN rats and the influences of fenofibrate on the formation process of choroidal neovascularization so as to provide new clues for the treatment of choroidal neovascularization disease.Methods1. RPE and HUVEC cells are divided into RPE normal group, RPE+fenofibrate group, RPE hypoxia group, and RPE hypoxia+fenofibrate group; HUVEC normal group and HUVEC+hypoxia supernate group. The healthy BN adult rats are randomly divided into blank control group and experimental group which is further divided into fenofibrate intervention group (drug group) and non-intervention group, and each group takes 1 w,2w,3w and 4w as the observation points.2. The superoxide anion probe is applied to detect the RPE hypoxic conditions; MTT is utilized to detect the cell viability; and ELISA is used to detect the expression of VEGFC and VEGFR-3 in the RPE cell culture supernatant. Cell wound healing assay is implemented to detect the cell migration ability; cell lumen formation experiment is conducted to detect the vascularization ability of HUVEC cells; and qRT-PCR test and Western-blot test are adopted to detect the expression levels of mRNA and protein in VEGFC and VEGFR-3 in RPE cells. In addition, FFA is used to detect the leakage area and strength of choroidal neovascularization at laser points for different time nodes in fenofibrate intervention group and non-intervention group. Isolectin B4-FITC dyed flat mounts of choroidal neovascularization is utilized to observe the staining degree of neovascular endothelial cells at the laser points during different time nodes. The BN rat eyes are processed for frozen section and immunofluorescence method is applied to detect the distribution situation of VEGFC and VEGFR-3 in choroid and retina at laser points. Moreover, qRT-PCR technique is utilized to quantitatively detect the expression changes of VEGFC and VEGFR-3 in choroid and retina of different groups at different time nodes; meanwhile Western Blot technique is also used to quantitatively detect the expression changes of VEGFC and VEGFR-3 in choroid and retina of different groups at different time nodes.Results1. Fenofibrate can restrain the increase of VEGFC and VEGFR-3 expression in RPE cells and cell culture supernatants caused by hypoxia.2. RPE cells in hypoxia culture supernatants can promote the viability of HUVEC cells, the migration of cells and the formation of lumens, while fenofibrate can restrain this process.3. After the formation of choroidal neovascularization in laser-induced BN rats, the expression of VEGFC and VEGFR-3 in choroid and retina can be observed, of which VEGFC is mainly expressed in retinal ganglion cell layers above the laser points and VEGFR-3 is mainly expressed in the neovascularization areas of choroid laser points. However, the expression of the above mentioned factors will not be observed in normal choroid and retina.4. After the formation of choroidal neovascularization in laser-induced BN rats, the expression of VEGFC and VEGFR-3 in choroid and retina presents certain regularities. For instance, with the passage of time, the expression of VEGFC in choroid and retina shows a slow declining trend while the expression of VEGFR-3 in choroid presents a gradual rise and maintains the high expression level, but the expression of VEGFR-3 in retina is basically none or a little.5. Fenofibrate can obviously slow the formation and development process of choroidal neovascularization in laser-induced BN rats and significantly reduce the expression of VEGFC in choroid and retina. The expression decline of VEGFR-3 in choroid is related with the expression reduction of VEGFC caused by Fenofibrate.Conclusions1. Due to hypoxia, the expression of VEGFC and VEGFR-3 rises in CoCl2-induced culture RPE cells, but fenofibrate can restrain the expression increase of VEGFC and VEGFR-3 in RPE cells under hypoxic condition.2. When RPE is hypoxic, the concentration of VEGFC and VEGFR-3 cell culture supernatants will rise, which promotes proliferation, migration and neovascularization vitality of blood capillary in HUVEC cells. This indicates that RPE cells play an important role in the CNV formation process caused by hypoxia. As fenofibrate can restrain the expression increase of VEGFC and VEGFR-3 in RPE cells under hypoxic condition, it may indirectly influence the vitality of HUVEC cells through adjusting RPE functional activities so as to achieve the formation of neovascularization.3. VEGFC is mainly expressed in retinal ganglion cell layers above the laser damage area of BN rats and VEGFR-3 is mainly expressed in the choroid damage area of laser points.4. Fenofibrate can reduce the VEGFC expression in choroid and retina, and its role in decreasing VEGFR-3 in choroid may be indirectly affected through VEGFC.