| AMD is the leading cause of blindness in people over 50 years old in the Western world. It is a degenerative eye disease that affects the macula. The macula is the central part of the retina at the back of the eye and responsible for the clear central vision needed for daily activities like reading or driving. AMD causes the progressive loss of central vision, leaving only peripheral vision intact. There are two types of macular degeneration: dry AMD which accounts for 85-90% of all cases and wet AMD which is the most serious form and accounts the other 10~15% cases. Dry macular degeneration rarely causes severe visual impairment. The reason for the AMD is not very clear now, so there is currently no effective treatment for dry AMD. However, there are some clinically-proven treatments for some forms of wet AMD.Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is one subtype of phospholipase A2s which are a growing family of enzymes. It was purified from mouse macrophage cell line and several investigated tissues and cells contain iPLA2-VIA activites. iPLA2-VIA catalyse the hydrolysis of the sn-2 acyl-ester bonds in phospholipids, leading to the release of arachidonic acid and lysophospholipids. AA can be converted into eicosanoids through the action of a variety of prostaglandin synthases, lipoxygenases. Eicosanoids are potent inflammatory mediator in the human body and play a role in a wide range of physiological and pathological processes such as sleep regulation, immune responses, and pain perception. The lysophospholipid play a role in cell signals and can serve as a precursor for lipid mediators such as lysophosphatidic acid or platelet activating factor (PAF) which is another inflammatory factor. In addition of the key role in homeostatic membrane phospholipid, iPLA2-VIA is involved in several cell signal transduction in many physiopathological processes such as proliferation, secretion and apoptosis. However, there is no report about the function of iPLA2-VIA in the human eye until now.The aim of our study was to investigate the role of iPLA2-VIA on phagocytosis of RPE cells. We utilized biochemistry, cytobiology, immunology, molecular biology and other theories like bioinformation; used many methods which include immunohistochemical staining, immunofluorescent staining, western blotting, activity assay and transfection method as well to investigate the change of iPLA2-VIA protein expression level and the activity of this enzymen after phagocytosis by ARPE-19 cell line. This study establishs a basis for further investigation of iPLA2 in Age related macular degeneration and possible intervention therapy methods. For this purpose, we did the following experiments:Chapter 1 Expression of iPLA2-VIA in the normal human eye and ARPE-19 cell line In order to explore the expression of iPLA2 in the normal human eye, a specific antibody of iPLA2-VIA from Cayman Company was used for the paraffin-embedded human eye sections. In the cornea, we found strong to moderate detectable expression of iPLA2-VIA in the epithelial and endothelium cells. Weak to moderate iPLA2-VIA expression was found in the dilator and sphincter muscle. Pigmented and non-pigmented ciliary epithelial cells as well as the ciliary muscle revealed moderate iPLA2-VIA expression. Strong expression of iPLA2-VIA was observed in various layers of the retina. Most abundant expression was detected in the RPE cells, the ganglion cells and the nerve fiber layer. Moderate iPLA2-VIA expression was found in the photoreceptor inner segment, the inner and outer plexiform layers, whereas no expression appeared in the nuclear layers and the photoreceptor outer segments. Moderate iPLA2-VIA expression appeared in both the anterior and equatorial lens epithelium.Western blotting analysis, using specific antibody against human iPLA2-VIA, revealed the protein expression in ARPE-19 cells. iPLA2-VIA identified a ~88 kDa band, and a ~70 kDa band. Immunohistochemical staining of ARPE-19 cells confirmed the protein expression. The authenticity of the 70 kDa protein to which the Cayman antibody react, was evaluated by immunoprecipitation. An iPLA2-VIA directed antibody produced by Santa Cruz (sc-14463) was used to immunoprecipitate iPLA2-VIA from ARPE-19 cells. In this precipitate, both the ~88 kDa protein as well as the ~70 kDa protein was recognized by Western blot analysis using the Cayman antibody, strongly indicating that this protein is an iPLA2-VIA protein product. Confocal fluorescence staining was used and iPLA2-VIA was found in the cytosol only, not the nuclear of ARPE-19 cells. This result was also confirmed by the immunohistochemical staining of human eye sections.Our study demonstrates for the first time the presence of iPLA2-VIA in human eye and ARPE-19 cells. We suggest that iPLA2-VIA play an important role in the different functions of RPE cells.Chaper 2 Involvement of iPLA2-VIA in the phagocytosis of ARPE-19 cellsThe photoreceptor cells and retinal pigment epithelium cells (RPE) are the two outer layers in the retina. RPE cells support photoreceptor cell function by shuttling nutrients, oxygen and various survival factors to the photoreceptor cell layer. They provide crucial functions for the maintenance of the retinal environment and the renewal of photoreceptor cells. Abnormal phagocytosis by RPE cells will induce different retinal disease such as age related macular degeneration (AMD).In previous study we have explored the high expression level of iPLA2-VIA in the human RPE cells, so in this part we will futher to detect the role of iPLA2-VIA in RPE phagocytosis. Bovine POS was isolated and labeled by biotin-streptividine method, and it was used to establish the phagocytosis model with ARPE-19 cells. The peak of ARPE-19 cells phagocytosis is after 12 hours after fed with POS. The results from Western blotting and activity assay showed that the expression and activity of iPLA2-VIA were both upregulated after phagocytosis and mean while BEL-the specific inhibitor can inhibit the upregulated activity. Further we chose 10μM BEL which does not have significant cytotoxicity to ARPE-19 cells to detect the inhibition of BEL at different time points and found the phagocytosis decreased after BEL treatment, not time-dependent. The inhibition of ARPE-19 phagocytosis of POS by different BEL concentrations was explored as well, and the result showed that BEL concentrations between 5 and 20μM revealed equal inhibitory effect on ARPE-19 phagocytosis of POS.iPLA2-VIA is proved by using different experimental methods that it is involved and upregulated during the RPE phagocytosis process. AMD is a genetically complex disorder of the photoreceptor-RPE-Bruch's membrane-choriocapillaris complex, meaning the abnormal RPE phagocytosis might be the main reason of AMD. Our results shed new light on understanding the mechanistic pathway and exploring new medicine treatment for AMD.Chapter 3 Role of iPLA2-VIA in the phagocytosis of mouse primary RPE cellsThe purpose of the experiments in this part was to explore the role of iPLA2-VIA in the phagocytosis of mouse primary RPE cells by repeating most of the experiments of iPLA2-VIA on mouse primary cultured RPE cells instead of ARPE-19 cells. Mouse RPE cells were isolated from C57BL/6 mice. They were harvest and grow well with lot of melanin granules inside. The nuclei could not be seen under the light microscope. The cells grew on the bottom of the culture plates and spontaneous pseudopod formation starts from 4 to 5 days. After 8 to 10 days mouse RPE cells can be confluent. We used anti-cytokeratin antibody to stain the cultured cells and found all the cells got stained which comfirmed that the cells we used for experiments were RPE cells. Phagocytosis model was successfully setup then by using mouse primary RPE cells and labeled bovine POS. iPLA2-VIA specific inhibitor BEL decreased 33.6% of the phagocytosis. Further more, the transfection with murine iPLA2-VIA vector upregulated 15.58% of phagocytosis while SiRNA vector transfected cells phagocytosis level downregulated 15.63%. We suggest that iPLA2-VIA plays an important role on the regulation of RPE phagocytosis, and it may also be involved in the regulation of photoreceptor cell renewal.The primary culture of mouse RPE cells was successful and was used to establish a new phagocytosis model. iPLA2-VIA was revealed to upregulate the phagocytosis by using specific inhibitor and transfection method. This result confirms our previous results which from ARPE-19 phagocytosis model. In conclusion, iPLA2-VIA is a regulator of RPE phagocytosis process and it may also contribute to the serious retina diseases like AMD. It may give a hint towards possible treatment using the iPLA2 related drugs for dry AMD.The expression of iPLA2-VIA in the RPE cells as well as its involvement in the phagcotysosis of RPE cells is a novel finding in the world. However, there are still some shortages in our experiments. First, ARPE-19 as well as mouse primary RPE cells are not human RPE cells, and not aged RPE cells in real AMD condition. Second, bovine POS was not human POS or murine POS. There may have some immuno rejection since the different species. The best choice should be use murine POS with mouse RPE cells or human POS with ARPE-19 cells. So we need to consider all these aspects and to do the further experiments. |
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