Integrin-mediated Signaling Pathway Influences Lens Posterior Capsular Opacification

IntroductionAfter cataract is the most common disorder to affect visual acuity after the operation. Posterior capsular opacification ( PCO) is its pathogenesis basis, caused by the adhesion, proliferation and migration of the remained human lens epithelial cells (hLECs). It also correlates with the transdifferentiation and extracellular matrix (ECM) deposition. Hence, the etiology of cellular adhesion and proliferation becomes the key point to PCO prevention. Basic and clinical research on PCO has been carried on for many years before the suitable solution can be found. Great number of reports demonstrate that cellular proliferation acts on PCO, but few has dwelled on the ECM influence in cell destine. The interaction between hLECs and ECM, and its downstream adhesion - dependent signaling pathway may throw light on PCO research.Integrins, acting as the cellular adhesive receptors described by Hynes in 1984, mediate the cell - ECM adhesion to form the integrity. As the plasmas membrane receptor family, integrins mediate transmembrane communication as well as cell adhesion, join efforts in the ECM - integrins - cytoskelleton pathway, activate signaling transduction and take great part in physiologic process. Ras - Raf - MAPK and PI3K/PKB are the major downstream pathways of inte-grin - mediated signaling. Phosphatidylinositol - 3 -kinase (PI3K) / protein kinase B ( PKB) is activated by growth factors and correlate with them. PKB activity is regulated by PI3K and makes important effects in cellular metabolism.In our research, the experiments are well organized to form a whole thesis. That's to say, two components of ECM (laminin and fibronectin) , integrins, focal adhesion kinase (FAK) and downstream signals (PDK/PKB) make the theme to go through the total research. Effective blockage of this signaling trans-duction may inhibit hLECs proliferation as well as adhesion. It can also reduce sac wrinkle and fibrogenesis, which decreases the loss of visual acuity caused by PCO.Immortalized hLECs are applied to research PCO, which can't perfectly mimic the pathogenesis of PCO. In our experiments, hLECs in vitro culture condition is improved, and the primarily or secondarily cells grow out fluently and exuberantly, similar to the progress of PCO. The interaction between hLECS and its extracellular matrix, as the theme of our research, bring forth the brand new idea for PCO prevention and treatment.ObjectiveTo investigate the effects of Laminin ( Ln) and Fibronectin ( Fn) on the hLECs primarily cultured in vitro, the possible mechanisms of retinoic acid ( RA) in alleviating PCO, and the effects of Laminin on the activation of protein -kinase -B (PKB) and the activating pathway in hLECs.MethodsTwenty - eight pieces of lens anterior capsule were cut into scraps and seeded on dishes coated with Ln, Fn or BSA. The morphological characters of the hLECs were detected with inversed microscopy. And the dates when epithelial or mesenchymal cells grew out were recorded. The expressions of Cx43, ANp63 and integrin^! were demonstrated with indirect irnrnunofluorescent microscopy. Reverse transcriptase - polymerase chain reaction ( RT - PCR) was used to show the mRNA level of ANp63 and integrinfjj. The 3rd passage subcultured cells were involved in these experiments. The morphological features of the hLECs were recorded with inverse microscopy and digital camera. The effects of RA on hLECs proliferation were evaluated by MTT assay, and the inhibited ratios were calculated. The expressions of integrin{3j and vimentin were detected with immunofluorescent microscopy. Laminin was added into the nutrient medium of subcultured hLECs with the end concentration of 5 fig/ml. The cells werecollected 0, 10, 20, 40 and 60 minutes after Laminin treatment, [-y -32P] -ATP incorporation assay was used to detect the PKB kinase activity. Wortman-nin, the Specific inhibitor of phosphatidylinosital -3 -kinase (PI3K), was applied to pretreat the hLECs for 1 hour at the concentrition of lOOnM. Then Laminin was added and PKB activity was detected.ResultsThe hLECs in the Ln and Fn groups grew out much earlier [(2.4±0.5) d ] with more viable cells than the control group, but the emergence of fibroblas-tic cells in the Fn group [(7.6±1.0)d] were also earlier than that of the Ln group [ ( 12. 7 ± 1. 4) d] ( P < 0. 05 ) . The positive ratio of Cx43 in the Ln group (23/28) was the highest (P <0. 05) , and that of ANp63 (24/28) and integrin|3i( 25/28) in the Fn group was also the highest among the three groups (P <0. 05). The analyses of RT - PCR products were in accordance with the immunofluorescent results. 10"8M ~10~6M RA - treated group showed more epithelial cells and less fibroblastic ones, while 10~ M group did the decrease in both the two kinds of cells by inverse microscopy. MTT assay demonstrated that 10" ~10"M RA inhibited the proliferation of the hLECs. The inhibition was concentration - dependent and time - dependent ( P <0.05). The inhibition ratios were between 10. 8% and 32. 0%. In the 10~6M ~ 10 "5M RA - treated group, the positive expression of integrinBi was higher ( P <0. 05), and vimen-tin fibers spread well all over the cells in 10 "7 ~10 "5M groups. PKB activity of the membrane and cytosol reached the peak value 40minutes after Laminin treatment , as 2. 72 and 2.00 times as much that of the control one. There was significant difference in PKB activity between the Laminin treated group and the control one. Both membrane and cytosol activity of PKB decreased significantly after wortmannin pretreatment.ConclusionsLn can promote persistently the proliferation of hLECs primarily cultured in...