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Objective To investigate the role of bone morphogenetic protein (BMP) 7 peptidomimetics THR123 in the regulation of proliferative vitreoretinopathy (PVR) and epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells.
Methods Primary human RPE cells were isolated from donor eyes.The RPE cells was cultured for 48 hours with conventional medium, medium containing 10 ng/ml transforming growth factor-β2 (TGF-β2), medium containing 10 ng/ml TGF-β2 and 5 μg/ml THR123, respectively, serving as negative control group, TGF-β2 stimulation group and THR123-treated group.The relative protein expression levels of E-cadherin, α-smooth muscle actin (α-SMA), fibronectin (FN) activin receptor-like kinase-2 (ALK2) and Snail family zinc finger transcription factor 1, Snail1) were detected by Western blot analysis in each group.RPE cell contractile function was detected by collagen gel contraction assay.RPE cell migration function was detected by transwell assay.RPE cell polarity was detected by transepithelial resistance.For the in vivo model, the rabbit PVR model was established by intravitreal injection of RPE cells and cytokine platelet-derived growth factor.The PVR rabbits were randomly divide into PVR group, 0.5 μg/ml THR123 group and 5 μg/ml THR123 group, and were intravitreally injected with corresponding concentration of THR123 on the day of modelling and 7 days after modelling.The fundus conditions of rabbits were observed every 7 days.Retinal detachment was checked by B-scan ultrasonography every 7 days, and PVR grading was performed on 14 and 28 days after modelling.On day 28 after modelling, the rabbit was sacrificed by air embolism and the eyeball was enucleated.Hematoxylin-eosin (HE) staining and α-SMA immunofluorescence staining were performed to compare the differences in PVR progression among groups.In the LDN inhibition group, BMP receptor inhibitor LDN193189 was added to primary RPE cells and the expression of EMT markers and cell functional differences of RPE cells were compared between negative control group and LDN inhibition group.Small interfering RNA of control and Snail1 was transfected into RPE cells of siNC+ TGF-β2 group and siSnail1+ TGF-β2 group, respectively, and the differences in EMT markers and function of RPE cells were compares.The protocol involving human specimens, cells and animals in this study was opproved by the Medical Ethics Committee of Shanghai General Hospital (No.2021SQ057).
Results There were statistically significant differences in gel contraction ratio, transepithelial resistance and relative expressions of E-Cadherin, FN and α-SMA proteins among the negative control group, TGF-β2 stimulation group and THR123-treated group ( F=28.38, 136.30, 38.50, 2.53, 53.54; all P<0.01).Compared with the negative control group and THR123-treated group, the gel contraction ratio, transepithelial resistance, and relative expression of E-cadherin in the TGF-β2 stimulation group decreased, and the relative expression of α-SMA and FN increased, with statistically significant differences (all P<0.01).On day 28 after modeling, the PVR grades in the PVR group, 5 μg/ml THR123 group and 5 μg/ml THR123 group were 4.00±0.45, 2.80±0.37, 1.80±0.37, respectively, with a statistically significant difference among groups ( F=7.583, P=0.007).The PVR grade of rabbit eyes was significantly lower in the 5 μg/ml THR123 group than in the PVR group ( P<0.05).HE staining showed that the number of preretinal proliferative membranes was less in the 5 μg/ml THR123 group than in the PVR group and the 0.5 μg/ml THR123 group, and no retinal detachment was found.Immunofluorescence staining showed that the expression of α-SMA in vitreous cavity and retina was significantly lower in the 5 μg/ml THR123 group than in the PVR group and the 0.5 μg/ml THR123 group.Compared with the negative control group and THR123-treated group, the relative expression of ALK2 protein in the TGF-β2 stimulation group was significantly decreased, and the relative expression of Snail1 protein was significantly increased (all P<0.01).Compared with the negative control group, the gel contraction ratio, transepithelial resistance and relative expression of E-cadherin protein in the LDN inhibition group were significantly reduced, and the relative expression of FN, α-SMA and Snail1 proteins were significantly increased (all P<0.01).Compared with the siNC+ TGF-β2 group, the gel contraction ratio and the relative expression of E-cadherin protein in the siSnail1+ TGF-β2 group were significantly increased, and the relative expression of FN, α-SMA and Snail1 proteins were significantly decreased (all P<0.05).
Conclusions THR123 can activate the BMP pathway to inhibit Snail1, thereby inhibiting the EMT process in RPE cells and PVR occurrence.It is expected to provide potential targets for PVR drug therapy.