Effects Of Mechanical Stress On Human Retinal Pigment Epithelial Cells In Vitro

Background and AimProliferative vitreoretinopathy (PVR) is an inflammative common complication of retinal detachment and the leading cause of anatomical failure in surgery. PVR is an excessively wound healing response occurring after RPE cell wounding. RPEs are major proliferative cells in PVR. In the pathological state, such as rhegmatogenous retinal detachment (RRD) etc, The activated RPE cells migrate from their normal, sessile site to vitreous and surface of the retina, in which they begin to dedifferentiate, migrate, proliferate, change phenotype and secrete many kinds of inflammatory factors and cytokines. Every one plays its role according to temporal situation, thus forms a chain of reaction. But it is not completely clear the mechanism of modulation on RPE cells growth in the pathogenesis of PVR. Wounding and repair of RPEs is the main stage at the beginning process. The past research mostly focused on the cell culture. Less study aimed at the biological force and wounding to RPEs during retinal detachment and PVR formation.The RPE lies between the retina and the Bruch membrane. As an important barrier between blood and retina, the RPEs play a vital role in ocular metabolism. Many ocular diseases are related to RPE cells. Mechanical force is a common phenomenon that exists in organism. In physiological orpathological process, the RPE cells are exposed to force coming from different sources. Vitreous and hyperplastic membrane are main origins of stretch. When RRD happened, the RPEs were exposed directly. The stretch force from the neural retina and liquefied vitreous would affect the RPEs. When the eye moved, the force endured by the RPEs would much stronger. The contraction of hyperplastic membrane can also stretch the RPEs. When it was bleeded around the RPEs, which often happened in choroids break, choroidal neovascularization (CNV) occurred in diseases like age-related macular degeneration (AMD). The contraction and the following scar formation is also an stress factors to RPEs. Furthermore, some exterior factors, such as trauma and medical-derived factors would stretch the RPEs.Cells were in the complex circumstance with force factor. Force affected RPEs significantly on growth, metabolism, phenotype change, gene expression, autocrine and paracrine. As an important kind of cells in the eye, it was necessary for us to study the biological effects on the RPEs produced by force. It would be helpful for us to detect the pathogenesis.Our work aimed at this principal course. After incubation, RPEs adsorbed beads and were stretched in the magnetic field. The model simulated that RPEs were stretched by the vitreous during retinal detachment. At the beginning stage of retinal detachment, we tried to study the change of migration, proliferation, signaling pathways and extracellular matrix (ECM) metabolism. Stretch that had ever been, ignored became an important factor. We thought much of it to complete our research, which broadened thoughts.The aim of our experiments is: (1)to establish and observe the experimental mechanical stress model of retinal pigment epithelial cells (RPEs) in vitro,(2)to observe the migration and proliferation of human RPEs under mechanical stress in vitro and the effects of a long effective corticosteroid (triamcinolone acetonide) on it. (3)to observe the MMPs expression of human RPEs under mechanical stress in vitro and the effects ofSB203580 (inhibitor of p38) on it. ?to observe the calcium flux and the MAPK signaling pathways of RPEs during mechanical stress in vitro. Methods(1)Ferric oxide microparticles, coated with collagen, were added to dishes containing substrate-attached RPEs. After incubation, the cell layer was washed to remove unbound beads and loosely attached beads. Combination of beads and cells were observed by optical microscope and scan electronic microscopy. We also monitored fluorescence changes of cells by confocal microscopy prior to and after vertical magnetic force application.(2)The cell layer bound with beads was laid in an invariable magnetic field. At each time point, confluent monolayers of human RPE cells were denuded, the migration of RPE was documented respectively. The RPE proliferation was evaluated with MTT assay and 3H-TdR absorbtion. The effects of triamcinolone acetonide (TA) on the migration and proliferation of RPE under the mechanical stress were also studied.(3)At each time point, the mRNA and protein levels of MMP-2, MMP-9, TIMP-2 and FN were analysed with RT-PCR and WESTERN BLOT. Secretion of MMP-2 was evaluated by ELISA. The effects of SB203580(inhibitor of p38) on the MMPs expression of RPE under the mechanical stress were also studied.(4)Cultured human RPE cells were analyzed by using the fluorescence Ca²⁺ dye fluo-3/AM and laser scanning confocal microscope (LSCM) before and after force stimulation. The activation state of the MAP kinases extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in RPEs was determined over a time course from 3 minutes to 30 minutes with western-blot analysis. To examine the role of p38 kinase in the response to force stimulation, cells were grown for 30 minutes in the presence or absence of inhibitor of p38 (SB203580). The expression change of active p38 kinase was observed with fluorescence staining.Results(1)Distribution of beads on the surface of RPEs showed separated or clumped pattern, localized in central area rather than in the peripheral. Integration rate could be achieved as 38.3%. Preincubating cells with collagen would limit the combination of beads with cells to much extent. The basic fluorescence quantity is 20.2±4.7U. A sudden increase can be seen after magnetic force application. At 5th minute the quantity is over 4 times as the basic. Then it climbs gradually and peaks at 15th minute, followed by a gradual reversal. Without force for 10 minutes, it reaches to the basic level by and large.(2)The mechanical stress facilitated the migration and proliferation of RPEs in the acute stage. TA restrained the process in a concentration-dependent manner. Compared with the control group, the facilitative migration rate was 23.67±2.53%(P=0.031 )30min under the stress. The facilitative proliferation rate was 15.72±0.46% (P=0.037 ) and 24.8±0.23% (P=0.003) 15min and 30min after exposure of the stress, respectively.(3)MMP-2, -9; TIMP-2; and FN were expressed in cultured RPE cells. mRNA of FN and MMP-2 increased at 15min and 4h after stress, respectively. Protein of MMP-2 began to increase at 12h, but not FN. SB203580 blocked the upregulation stimulated by stress. The expressions of MMP-9 and TIMP-2 did not change during 12h.(4)Normal fluorescence in RPE cells was strong and distributed throughout the cells. The nucleus appeared to be more fluorescent than the cytoplasm. After force stimulation, a rapid increase in fluorescence intensity was observed, followed by a rapid decrease by adding MnCl₂. There was no obvious difference in increased curve in cells preincubated with EGTA. By adding CaCl₂, the fluorescence intensity increased again. Moreover, afterforce stimulation, the fluorescence intensity in cells preincubated with cytochalasin D increased over two bold than in normal cells. Total ERK, JNK, and p38 were detected in RPEs. Active ERK, active p38, and barely no active JNK were detected. Activation of ERK was unchanged during the stess. In contrast, although p38 activation was barely detected in the normal cells, this stress-activated protein kinase exhibited a robust activation after 5 minutes. SB203580 blocked the p38 activation during force stimulation. The force stimulation also increased the fluorescence quantity of active p38 with fluorescence staining. Conclusions(1)The mechanical force can be applied to the RPE cells by this model, which can imitate the state of RPEs before retinal detachment.(2)The mechanical stress can stimulate migration and proliferation of RPEs. TA restrained the process in a concentration-dependent manner. Thus, it might potentially contribute to the development of retinal detachment or fundus hyperplastic diseases.(3)The mechanical stress may be play an important role in the upregulation of expression of MMP- 2 and FN in RPE cells and account for a directional shift in the balance between MMPs and TIMPs, which may be important for the integrity of ECM components.(4)The mechanical stress may increase the calcium influx of RPEs and initiate the release of intracellular calcium store. The calcium influx may be a signal before injury of RPEs. Mechanical stress can affect RPEs, which may take effects partially via p38 signalling pathway.In conclusion, the mechanical stress may potentially contribute to the development of retinal detachment or fundus hyperplastic diseases. Avoiding being stretched and the following pathological process may be a new way of prevention and cure to such kind of diseases, which needs further study for the future.