Effects Of The Electric Field On Human Retinal Pigment Epithelial Cells

BackgroundWound healing is important for maintaining the normal structure and function of tissues. During this process, an endogenous electric field might play a role. When the epithelium is wounded, the transepithelial potential will drive current out of the disrupted area, and create the endogenous electric field in turn. It has been proven that many types of cells respond to direct current electric fields (EFs) by changing their biologic characters, especially the migration capacity. Studies have shown that cells from multiple species and tissues display directed migration to the EFs, known as galvanotaxis or electrotaxis. Further more, the wound healing of the skin and the cornea can be accelerated by EFs. With the accumulated experimental evidence, it can be suggested electric signals play an important role in the directed cell migration of wound healing.Up to now, the mechanisms that guide both cell migration to electric cues and EF-induced wound healing have not been illustrated thoroughly. However, some assumptions have been introduced, including the asymmetries of cytoskeletal molecules and growth factors receptors, and the activation of signalling pathways. In addition, cell attachment to the extracellular matrix (ECM) is critical for migration and is mediated by members of the integrin family. Integrins are heterodimeric transmembrane receptors and play a central role in regulating cell adhesion, migration, proliferation and differentiation by mediating interactions between the extracellular matrix and the cell. Integrins exist in several activation states on the cell surface and activation induces integrin clustering. This leads to recruitment of multiple signaling molecules and the regulation of different signaling pathways. Furthermore, cell-to-substratum linkage sites mediated by integrin binding to matrix proteins must be regulated in order for cells to generate traction forces and to initiate directional movement. Focal adhesion kinase (FAK) plays an important role during this process. FAK is a nonreceptor protein-tyrosine kinase that localizes to focal contact sites. It associates with integrin receptors and recruits other molecules to the site of this interaction, thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton. The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylates FAK and is a key negative regulator of FAK signalling. Taken together, to investigate the effects of EFs on integrins and integrin-mediated signaling might be helpful to further understand the mechanisms of EFs on cells.Retinal pigment epithelium (RPE) is essential for the integrity and function of neural retina. Dysfunction or injury to RPE has been linked to many devastating eye disorders. However, RPE cells are under the influence from both neural retina and choroid, and treatment targeting the RPE cells usually has poor effect.Among the diseases which relate to the RPE disfunction, age-related macular degeneration (AMD) is a common cause for legal blindness. AMD is a complex and multifactorial disease and prevalence will further increase given demographic developments in ageing populations. A complex interaction of metabolic, functional, genetic and environmental factors seems to create a stage for chronically developing changes in ocular structures of the macular region (choriocapillaries, Bruch's membrane, retinal pigment epithelium, photoreceptors) which may contribute to varying degrees to the onset and final picture of AMD. Current therapeutic options are limited for this disease so far. According to the histopathology of AMD, recovery of the normal structure and function of RPE cells seems to be the key point for proper treatment. Based on the effects of EFs on wound healing, we suppose that applying EFs on the RPE cells may be helpful in controlling RPE cell movements and more importantly, for AMD treatment.AimsTo establish the experiment model of RPE cells exposed to EFs, and investigate the effects of EFs on the viability, migration and proliferation of human RPE (hRPE) cells and the possible mechanisms.Methods1) A RPE cell exposed to EF model was established. Cultured hRPE cells were exposed to EFs within the range of 0~10 V/cm and images of the cells were obtained. The viability of the cells was determined by means of staining with trypan blue and AgNORs during and after exposure to EFs. Flow cytometry was applied to assess the apoptosis of the cells.2) To observe the effect of EFs on hRPE cell migration, the hRPE cultured in the medium with and without serum and EGF (epidermal growth factor) were exposed to EFs at 2, 4, 6, 8, 10 V/cm for 3h. Images of the cells were obtained every 15 min and directedness of movement were measured by tracing the position of cell nuclei before and after EF application, the directionality of migration was described by cosineФ, whereФwas the angle between the field axis and the vector drawn by the net cell translocation path. The parameters used to quantify cell migration in this study include the average cosineФ, the average distance, the average velocity and the migration index. The cell number and cell density at different time point were calculated, and the cell circle was analyzed by flow cytometry. The expression cyclin E was analyzed by Western blot.3) In the hRPE cells pretreated with or without cytochalasin B and EFs, the distribution of F-actin andβ1 integrin was measured by immunohistochemistry. The expression ofβ1 integrin was determined by PCR and Western blotting and the expression of FAK and pFAK was determined by Western blotting.4) Using liposome mediated method, PTEN were transfected into hRPE cells. The transfected cells were selected with 400 mg/L G418 and clones were picked and expanded. The transfected cells were exposed to the EFs as indicated above, and the directedness of the cell movement was measured. Flow cytometry was used to assess the cell circle, and Western blot was used to detect the expression of FAK and pFAK in the cells.Results1) The EF-exposure model for the experiment was stable and reliable. EFs below 2 V/cm did not affect the hRPE cell shape. After exposed to the EFs of 2~10 V/cm for 3 h, hRPE cells were elongated and oriented with their long axes perpendicular to the vector of the field. After stopped the EF exposure and cultured for an additional 12 h, the cells regained their normal shape and randomly distributed in the culture medium. Trypan blue and AgNORs staining showed no effect of EFs on the viability of RPE cells during 3 h exposure (P>0.05). The result of flow cytometry showed no obvious apoptosis in hRPE cells before and after EF exposure.2) Without exposure to EFs, RPE cells in the culture were randomly distributed with no obvious changes to their motility during the course of the experiment, and the cosineФwas 0.02±0.10. After exposed to EFs, hRPE cells were migrated to the cathode, and this directed translocation was more conspicuous with the value of cosineФclose to 1 when the field strength increased. Cells cultured in serum free medium showed slight polarization and the cosineФwas 0.30±0.12. Cultured in the medium with serum or serum plus added EGF, cells showed obvious cathodal migration in EFs. Increased electric field strength could enhance this cathodal directedness. When the polarity of the electric field was reversed, the cells reversed their direction of migration accordingly. In cultures with EFs, hRPE cell density and cell growth rate were increased higher than that of the control cultures (P<0.05). Flow cytometry showed the percent of the cell population in the G0/G1 phase in EF-exposed cells decreased, whereas the percentage of the cell population in the S and G2/M phases increased significantly. Western blot analysis of hRPE cells showed that EF exposure induced a significant increase in the expression of cyclin E.3) Immunofluorescence staining revealed that F-actin formed a stress fiber network across the cytoplasm in the normal RPE cells. After exposure to an EF for 3 h, the actin bundles accumulated at the lateral borders of RPE cells, especially towards the cathode side.β1 integrin was weakly expressed in normal RPE cells. Three hours after exposure to EFs, the staining ofβ1 integrin had increased in hRPE cells, with the stain density accumulating at the side facing cathode. After treated with cytochalasin B, a disruption of F-actin stress fibers in the cells was observed and random distributed deposits ofβ1 integrin staining were also detected. Exposure to EFs did not reverse these phenomena. The results of RT-PCR and Western blot showed an increase inβ1 integrin mRNA and protein expression of hRPE cells after exposure to EFs. This up-regulation ofβ1 integrin was blocked by cytochalasin B. FAK was steadily detected in normal RPE cells or in the cells exposed to EFs. The level of pFAK was low in RPE cells without exposure to EFs. After the cells were exposed to EFs, the relative intensity between the band density of pFAK and that ofβ-actin significantly increased compared with the controls (P<0.05).4) PTEN expressing vector was successfully transfected into hRPE cells and dramatically inhibited the polarization and directed migration of hRPE cells in EFs. Flow cytometry showed the percentage of the cell population in the S and G2/M phases in the transfected cells decreased whereas the percent of the cell population in the G1 phase increased significantly (P<0.01). EF exposure did not reverse this cell circle inhibition. Western blot analysis revealed that the expression of FAK in hRPE cells was not affected by PTEN transfection or EF exposure. However, the increased expression of pFAK in hRPE cells after EF exposure was blocked by PTEN transfection.Conclusions1) Limited period of exposure to EFs under 10 V/cm does not affect the normal viability of hRPE cells.2) EFs induce directed migration of hRPE cells, which can be enhanced by serum or EGF. EFs promote the proliferation of hRPE cells in certain exposure period by up-regulating the expression of cyclin E.3) Exposure to EFs induces the polymerization of cytoskeleton and up-regulate the expression of integrin. Activation of FAK signaling pathway may also involved in this interation. 4) FAK and PTEN mediate directional sensing of cell migration in response to electric signals.Similar research has not been reported at home and abroad.