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Mausumi Bandyopadhyay, Baerbel Rohrer; Effect Of Complement Activation On Human Embryonic RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1226.
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Complement deposition in the aging eye is one of the pathological outcomes, a growing problem related to malfunction of vision and thereby blindness. Considering the fact that, chronic tissue inflammation is intimately related with activation of complement cascades associated with age, we attempted to evaluate biological consequences of complement deposition in primary RPE cells to better understand the possible role of complement activation in the pathology of AMD.
Human embryonic RPE cells are isolated from aborted fetus as per IRB guidelines. The primary RPE cells were cultured untill they form monolayers with stable transepithelial resistance (TER). The cells were treated exogenously with 25% normal human serum as source of complement and 0.5mM hydrogen peroxide to induce oxidative stress. In this experiment, our aim is to determine mechanisms of RPE cell damage. After each experiment cells and supernatant were collected for study.
The observations in this set of experiments show that the treatment of oxidatively stressed embryonic RPE cells with NHS resulted in a rapid loss of barrier function as measured by TER. This effect was due to sublytic complement activation triggered by the alternative pathway, since no cell lysis could be observed; TER reduction could be prevented using C7-depleted serum; and treatment with the alternative pathway inhibitor CR2-fH prevented the effect of NHS. Treatment of RPE monolayers with H2O2 and NHS resulted in an increase in ROS production above the levels generated by H2O2 alone, although short-term, mitochondrial function, as assessed by ATP content, was unaffected. Finally, H2O2 and NHS resulted in increased expression and secretion of matrix metalloproteases MMP2 and MMP9. The active forms of the enzymes are elevated in the basal compartment.
Taken together, these results confirmed our previous findings in ARPE-19 cells that oxidative stress sensitizes RPE cells to complement attack. In addition, these findings for the first time, link oxidative stress, complement activation, and basal release of activated MMP2/9, which have all been associated with the pathogenesis of AMD.
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