Abstract：Objective: To investigate the functions and underlying mechanisms of melanoma cell adhesion molecule cluster of differentiation 146 (CD146) in retinal vascular endothelial cells. Methods: In this experimental study, oxygen induced retinopathy (OIR) mouse model (n=16) was constructed from January to June 2021, and the age-matched mice were used as the control (n=16). Retinal vascular endothelia were isolated by the magnetic beads-based method. Real-time quantitative PCR was performed to detect the expression level mRNAin retinas and cells in the two groups. Immunofuorescent staining identifed the expression and location of CD146 in retinas. Western blot assay determined the protein expressions in human retinal microvascular endothelial cell (HRMEC) and retinas. CD146 small interfering RNA(siCD146) transfected HRMEC was used as the transfection experimental group, while nonsense small interfering RNA transfection was used as the negative transfection control group (NC). MTS, EDU and fow cytometry were used to evaluate cell viability, cell proliferation, and cell cycle, respectively. Transwell and scratch assay were used to measure cell migration. Matrigel angiogenesis assay examined the ability of tube formation. And siRNAs were delivered into the retinas by intravitreal injection. Flat-mount of retina stained for isolectin B4 (Ib4) analyzed the neovascularization and avascular region. Independent sample t-test was used for data analysis. Results: Immunofuorescent staining showed that CD146 was specifcally expressed on retinal vessels. Compared to the normal mice, the level of CD146 mRNAin vascular retinal endothelial cells of OIR model mice were twice as high as those of normal mice (t=6.57, P=0.003). The protein expression of CD146 was also increased in hypoxia (1% O2) compared to the normoxia (21% O2) (t=5.79, P=0.010). CD146 inhibition by siRNAtransfection inhibited HRMEC migration signifcantly (Transwell assay: siCD146-1: t=6.15, P=0.003; siCD146-2: t=3.88, P=0.005. Scratch assay: siCD146-1: t=2.73, P=0.041; siCD146-2: t=4.10, P=0.006). The matrigel angiogenesis assay showed that siCD146 transfection also inhibited the ability of tube formation (siCD146-1: t=2.81, P=0.048; siCD146-2: t=3.10, P=0.036). Western blot analysis showed that the protein expression levels of RHOA (t=3.60, P=0.023; t=4.12, P=0.015) and p-P38 (t=2.89, P=0.044; t=3.07, P=0.038) were decreased. Retinal fat-mount showed that siCD146 injection group obviously relieved the neovascularization (t=8.94, P=0.001) and avascular region (t=11.24, P=0.001). Western blot analysis showed that the expression levels of CD146 (t=5.62, P=0.030), P-p38 (t=6.87, P=0.021), and RHOA (t=5.06, P=0.037) were downregulated significantly in siCD146 groups compared to the NC groups. Conclusions: Knockdown of CD146 significantly inhibited retinal vascular endothelial cell migration and angiogenesis by downregulating the expression of p38 and RHOA.
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