April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Sublytic Activation of the Complement Cascade in Human Embryonic RPE Cell Monolayers Creates a Permissive Environment for Amd Pathology
Author Affiliations & Notes
  • M. Bandyopadhyay
    Neuroscience, MUSC, Charleston, South Carolina
  • B. Rohrer
    Ophthalmology, Med Univ of South Carolina, Charleston, South Carolina
  • Footnotes
    Commercial Relationships  M. Bandyopadhyay, None; B. Rohrer, WO/2007/149567, P.
  • Footnotes
    Support  Foundation Fighting Blindness, Research to Prevent Bindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6177. doi:
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      M. Bandyopadhyay, B. Rohrer; Sublytic Activation of the Complement Cascade in Human Embryonic RPE Cell Monolayers Creates a Permissive Environment for Amd Pathology. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6177.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Polymorphisms in different complement proteins each increase the risk for developing AMD; and membrane attack complex (MAC) deposition density is correlated with severity of AMD and the loss of RPE cells. Previously we have shown that oxidative stress sensitizes RPE monolayers (ARPE-19) to complement attack resulting in sublyic MAC activation and VEGF secretion. We extended this analysis two fold by testing human embryonic RPE cells, and by examining whether sublytic complement activation is directly involved in pathology by either impairing RPE cell function or by the production and release of compounds from RPE cells that could contribute to AMD pathology.

Methods: : Human embryonic RPE cells were isolated from aborted fetus as per IRB guidelines and grown in MEM media and 5% FBS. RPE monolayers with stable transepithelial resistance (TER) were established in transwells plates; FBS was eliminated 24 hours prior to the experiments. Monolayers were treated with H2O2 to induce oxidative stress and complement-sufficient serum CSS; followed by biochemical or histological analysis of cells and supernatants.

Results: : As in the ARPE-19 cells, in human embryonic RPE monolayers, combined treatment with H2O2 and CSS disrupted barrier function as determined by TER measurements, whereas either treatment alone had no effect. Likewise, inhibitor studies and ELISA measurements confirmed that this effect was dependent upon the activation of the alternative pathway and VEGF secretion. As expected, H2O2 treatment resulted in the generation of reactive oxygen species and superoxide, an effect that was significantly augmented by the addition of CSS. Short-term treatment with H2O2 and CSS did not affect structural integrity (ZO1 and occludin staining), rod outer segment uptake or mitochondrial metabolism. However, H2O2 and CSS, but not either treatment alone, resulted in 4-10-fold increased expression of matrix metalloproteases MMP2 and MMP9.

Conclusions: : Taken together, these studies confirmed in human embryonic RPE cells that oxidative stress and sublytic complement activation on the surface of RPE cells results in VEGF release, which mediates disruption of the cell monolayer. Furthermore, H2O2 and CSS increased expression of matrix metalloproteases, which are thought to play an important role in remodeling of extracellular matrix such as Bruch’s membrane. These findings link the following readouts that have all been associated with the pathogenesis of AMD; oxidative stress, complement activation, apical VEGF release, and MMPs.

Keywords: age-related macular degeneration • retinal pigment epithelium • extracellular matrix 
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