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21 December 2009

Complement 5b-9 complex-induced alterations in human RPE cell physiology

Wenbo LiABCDEF, Song ChenABCDEFG, Minwang MaDEF, Jiangyuan QianBC, Xiubin MaBF

Med Sci Monit 2010; 16(1): BR17-23 :: ID: 878311

Abstract

Background
Recent evidence shows that complements are closely related to the occurrence of choroidal neovascularization (CNV). We studied the effect of complement 5b-9 complex (C5b-9) on membrane permeability and molecular biological behavior in cultured human retinal pigment epithelium (RPE) cells and considered the role of C5b-9 in CNV.
Material and Method
Human RPE cells were exposed to different concentrations of C5b-9 for 24 hours, then observed through light and electron microscopy. The dynamics of calcium ion change in cells exposed to sublysis C5b-9 were analyzed by confocal laser scanning microscope, and the amount of VEGF and TGF-beta2 mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR) RESULTS: RPE cells were destroyed when exposed to 80 microg/ml and 40 microg/ml C5b-9. The structure of RPE cells was not obviously changed when exposed to 20 microg/ml or less C5b-9; however, pigment granules are released from the cell membrane when observed using electron microscopy. In most of the cells, calcium fluorescence intensity increased rapidly after the deposition of C5b-9, to a peak at 4 min, lasted for about 6 min, and then began to decrease. The expression of VEGF and TGF- beta2 mRNA in RPE cells with C5b-9 was increased at 4 h and decreased at 24 h, but they were higher than in the control group.
Results

Conclusions
These observations suggest C5b-9 can induce a change in membrane permeability, an increase in cytoplasmic calcium ion concentration, and significant up-regulation of angiogenic factors in cultured RPE cells, which may be one of many potential mechanisms of CNV formation.

Keywords: Transforming Growth Factor beta2 - metabolism, Reverse Transcriptase Polymerase Chain Reaction, Retinal Pigment Epithelium - ultrastructure, Multiprotein Complexes - toxicity, Microscopy, Electron, Microscopy, Confocal, DNA Primers - genetics, Complement C9 - toxicity, Complement C5b - toxicity, Choroidal Neovascularization - metabolism, Cells, Cultured, Cell Membrane Permeability - drug effects, Calcium - metabolism, Vascular Endothelial Growth Factor A - metabolism

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