The Effects And Mechanisms Of CD44 In Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy(PVR)is a blinding disease occurring secondary to rhegmatogenous retinal detachment(RRD),ocular trauma,or ocular surgical procedures.PVR has been considered as an exaggerated wound-healing process,and is characterized by the formation of contractile fibrocellular membranes within the vitreous cavity and on the preretinal and subretinal surfaces.At present,surgery remains the mainstay of treatment in PVR.Although there have been tremendous advancements in surgical techniques and management,PVR cannot be effectively treated and is still the primary cause of surgical failure when treating retinal detachment or ocular trauma.Therefore,in order to improve postoperative visual function,it is particularly important to explore new prophylactic and therapeutic approaches based on a deeper understanding of the pathogenesis of PVR.The mechanisms of PVR are orchestrated by multiple elements such as growth factors,cytokines,extracellular matrix proteins and various cells.So far,a growing body of evidence has been indicated that the retinal pigment epithelial(RPE)cell plays a crucial role in PVR.The terminally differentiated RPE cell is mitotically quiescent under physiological conditions.The RPE,which is located between the neural retina and the choroid,plays various roles indispensable for function of the neural retina and the choroid.However,when the eye suffers from a retinal break or trauma,multiple factors lead to the disruption of junctional complexes in RPE cells.Subsequently,RPE cells become detached from Bruch's membrane,migrate through breaks in the neural retina,proliferate,and transform into myofibroblasts.These cells participate in the growth of fibrotic membranes,which contract and lead to further retinal detachment and blurring vision.In the process,RPE cells undergo epithelial-mesenchymal transition(EMT),which plays a pivotal role in the development of PVR.During EMT,RPE cells transdifferentiate into mesenchymal cells that are characterized by enhanced proliferative and migratory capacity,and resistance to apoptosis.They produce extracellular matrix proteins and lead to PVR.CD44 is a ubiquitous transmembrane glycoprotein which is expressed on a variety of cell types and is implicated in many physiological and pathological processes including development,inflammation,immune response,wound healing and cancer progression.CD44 participates in signal-transduction processes by establishing specific transmembrane complexes with extracellular ligands and by organizing signaling cascades through association with the actin cytoskeleton,thereby regulating many cellular processes,such as cell growth,differentiation and migration.CD44 has not been identified on normal RPE cells which remain mitotically quiescent.Under pathologic conditions,such as RRD,CD44 is expressed on proliferating and migrating RPE cells.The upregulation of CD44 in RPE cells has been correlated with the pathological progression of RRD.CD44 has been proposed to promote tissue fibrosis in lung,liver,kidney and so on.But the role of CD44 in PVR needs to be further studied.4-methylumbelliferone(4MU)is a derivative of coumarin that has choleretic and antispasmodic effects.4MU has anti-tumor,anti-inflammatory and anti-fibrotic effects by inhibiting the synthesis of hyaluronic acid.And 4MU abrogates cell proliferation and migration via downregulation of CD44 expression and its mechanisms of effects involve the inhibition of intracellular signal-transduction pathways.Therefore,we speculate that 4MU may prevent the formation of fibrocellular membranes in PVR by inhibiting the proliferation and migration of RPE cells.Therefore,in this study,we collected PVR membranes and detected the expression of CD44 in the membranes.Then the in vitro model of ARPE-19 cell EMT was established by using TGF-?2,and the effects and mechanisms of CD44 and 4MU in RPE cell EMT were investigated.This study further clarified the pathogenesis of PVR,and provided a theoretical basis for the prevention and treatment of PVR.ObjectiveThis study aimed to detect the expression of CD44 in the PVR membrane,and to explore the effects and mechanisms of CD44 in the epithelial-mesenchymal transition of ARPE-19 cells.We further investigated the effects and mechanisms of 4MU in ARPE-19 cell EMT.Methods1.PVR membranes were obtained from patients during routine surgical procedures and were evaluated by indirect immunofluorescence for CD44 and cytokeratin-18(CK-18).2.In vitro model of ARPE-19 cell EMT was created by incubating with TGF-?2.The morphological changes of RPE cells were observed.Cell proliferation was analyzed by Cell Counting Kit-8(CCK-8)assays at 24 h after the treatment of TGF-?2.The migratory capacity of RPE cells was evaluated by cell scratch assay and Transwell cell migration assay,and the expression of ?-SMA,Fibronectin(FN),N-cadherin and Vimentin in ARPE-19 cells were detected by Western Blot.In addition,the expression of CD44 and mesenchymal proteins were detected at different time points of TGF-?2treatment.3.ARPE-19 cells were transfected with CD44 si RNA.After transfection with si RNA,ARPE-19 cells were incubated with TGF-?2,and cell proliferation and migration in each group were detected.Western blot was used to measure the expression of ?-SMA,FN,N-cadherin,Vimentin,Smad2/Smad3 and phospho-Smad2/phosphoSmad3 at 48 h after TGF-?2 treatment.4.CD44 antibody was used to inhibit the effect of CD44.ARPE-19 cells were preincubated with 20?g/m L antibody to Ig G or CD44 and then treated with TGF-?2.Western Blot was used to detect the expression of ?-SMA,FN,N-cadherin,Vimentin,Smad2/Smad3 and phospho-Smad2/phospho-Smad3.5.ARPE-19 cells were incubated with different concentrations of 4MU,and the proliferation and migration of RPE cells were analyzed.Western Blot was used to detect the expression of CD44,?-SMA,FN,N-cadherin,and Vimentin.To screen for the suitable concentration of 4MU.6.We chose 0.8m M as the appropriate concentration of 4MU,and divided the experiment into three groups,including control group,TGF-?2 group and TGF-?2+4MU group.The control group was normally cultured ARPE-19 cells.The TGF-?2group was ARPE-19 cells treated with 10ng/m L TGF-?2.ARPE-19 cells were treated with 10ng/m L TGF-?2 in the presence of 0.8m M 4MU in the TGF-?2+4MU group.The proliferation ability of RPE cells in each group was measured by CCK-8 kit.RPE cell migration was evaluated by cell scratch assay and Transwell cell migration assay.Western Blot was used to detect the expression of CD44,?-SMA,FN,N-cadherin,Vimentin,Smad2/Smad3 and phospho-Smad2/phospho-Smad3.Results1.The PVR membrane samples were obtained at vitrectomy from 6 patients with PVR.All 6 samples contained cells positive for CD44.And there are identifiable cells positive for both CD44 and CK-18.2.ARPE-19 cells treated with TGF-?2 changed from cobblestone-like epithelial appearance to long-spindle fibroblast-like shape.The results of CCK-8 assay showed that compared with the control group,TGF-?2 promoted RPE cell proliferation(P<0.0001).The results of scratch assay and Transwell migration assay showed that TGF-?2 induced RPE cell migration(P<0.05).Compared to the control group,TGF-?2 significantly stimulated the upregulation of ?-SMA,FN,N-cadherin and Vimentin in RPE cells,and the difference was statistically significant(P<0.05).TGF-?2increased the expression of CD44,and the expression of CD44 was the highest at 24 h of TGF-?2 induction,and there was a downward trend of CD44 at 48 h,but there was no significant difference between 24 h and 48h(P=0.3919).?-SMA,FN,N-cadherin and Vimentin were significantly up-regulated in a time-dependent manner and were the highest at 48 h after TGF-?2 treatment(P<0.05).3.CD44 si RNA was used to knock down CD44 in ARPE-19 cells.Compared with the TGF-?2 group and the NCsi RNA+TGF-?2 group,RPE cells proliferation was reduced in the CD44 si RNA+TGF-?2 group(P<0.05).The scratched healing area of the CD44 si RNA+TGF-?2 group was reduced at 24 h and 48h(P<0.05).The migrated RPE cells were also significantly decreased in the CD44 si RNA+TGF-?2 group(P<0.05).At48 h,the expression of ?-SMA,FN,N-cadherin and Vimentin decreased in CD44 si RNA+TGF-?2 group(P<0.05),and the ratio of phospho-Smad2/phosphoSmad3 to total Smad2/Smad3 was also reduced.4.Compared with the TGF-?2 group and the Ig Gm Ab+TGF-?2 group,the X expression of ?-SMA,FN,N-cadherin,Vimentin and the ratio of phosphoSmad2/phospho-Smad3 to total Smad2/Smad3 decreased in the CD44 m Ab+TGF-?2group,and the difference was statistically significant(P<0.05).5.After 24 h of 4MU treatment,compared with the control group,0.2 m M 4MU had no significant inhibitory effect on the proliferation of ARPE-19 cells(P=0.1237),and the other four concentrations of 4MU significantly inhibited RPE cells proliferation(P<0.05).The proliferation of ARPE-19 cells in different concentrations of 4MU groups was reduced compared with the control group at 48h(P<0.05).The results of the cell scratch assay showed that compared with the control group,4MU significantly hampered ARPE-19 cells migration in a time and dose-dependent manner(P<0.05).Compared with the control group,4MU reduced the expression of CD44,?-SMA,FN,N-cadherin and Vimentin in ARPE-19 cells,and the inhibitory effect of 4MU at a concentration of 0.8 m M was the most obvious.6.The results of CCK-8 showed that RPE cells proliferation in the TGF-?2+4MU group was reduced compared to the TGF-?2 group,and the difference was statistically significant(P<0.05).The results of cell scratch assay showed that the scratched healing area in the TGF-?2+4MU group was smaller than that in the TGF-?2 group at 24 h and48h(P<0.05).The results of Transwell cell migration assay showed that compared with TGF-?2 group,RPE cells migration was reduced in TGF-?2+4MU group(P<0.05).The results of Western blotting showed the expression of ?-SMA,FN,N-cadherin and Vimentin and the ratio between phospho-Smad2/phospho-Smad3 and total Smad2/Smad3 in ARPE-19 cells in TGF-?2+4MU group were lower than that of the TGF-?2 group(P<0.05).Conclusions1.The RPE cells in the PVR membrane have the expression of CD44.2.TGF-?2 promoted EMT in ARPE-19 cells in vitro,and ARPE-19 cells were accompanied by up-regulation of CD44 during EMT.3.CD44 si RNA and CD44 antibody may inhibit RPE cell EMT by inhibiting the phosphorylation of Smad2 and Smad3.4.4MU may inhibit TGF-?2-induced EMT in RPE cells through the SMAD pathway.