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Kelly Mulfaul, Nathaniel Kevin Mullin, Joseph C Giacalone, Arwin Shrestha, Heather T Daggett, Edwin M Stone, Budd A. Tucker, Robert F Mullins; Local CFH synthesis protects choroidal endothelial cells from membrane attack complex formation.. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2927.
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Early in age-related macular degeneration (AMD), choroidal endothelial cells (CECs) in the choriocapillaris degenerate concurrently with the choroidal accumulation of the membrane attack complex (MAC), a multiprotein pore which causes cell lysis. MAC formation is controlled by negative regulators such as complement factor H (CFH). A polymorphism in CFH (Y402H) increases risk of AMD by 4-7-fold and results in reduced affinity for binding of this inhibitor to the choroidal extracellular matrix. We hypothesize that changes in local CFH protein levels lead to increased MAC formation on CECs early in AMD.
To assess the role of CEC-derived CFH, an immortalized CEC line generated from a healthy donor eye was transfected with a pool of 3 CFH siRNAs or a scrambled control for 48 h prior to treatment with 25% normal human serum (NHS) or 25% heat-inactivated NHS. Levels of CFH mRNA and protein were determined and MAC formation was quantified by immunofluorescence. In addition, we generated iPSC-derived CECs from a patient homozygous for the Y402H polymorphism and harboring a stop mutation in CFH. We divided differentiated cells into two populations one un-transduced and one transduced with a lentiviral vector to drive overexpression of wildtype CFH. iPSC derived CECs were treated with 25% NHS or 25% C5 depleted NHS. MAC formation was visualized by immunofluorescence and quantified by FACs analysis.
siRNA delivery resulted in 60% knockdown of CFH mRNA (p< 0.001) and 50% knockdown of secreted CFH protein. In the absence of CEC-synthesized CFH, we observed an increase in the abundance of MAC immunofluorescence on CECs (2-fold change, p < 0.001). AMD iPSC-derived CECs produced negligible levels of secreted CFH protein by ELISA. Lentiviral overexpression resulted in an 18-fold increase of CFH protein, which was accompanied by decreased MAC immunofluorescence (Figure 1).
We have demonstrated that a reduction in local synthesis of CFH protein by CECs leads to increased MAC deposition in cells exposed to complement-intact serum and that overexpression of CFH locally can protect CECs from MAC. Thus, increased expression of local wildtype CFH protein to CECs may protect against choriocapillaris degeneration and AMD progression.
This is a 2021 ARVO Annual Meeting abstract.
Figure 1. AMD iPSC derived CECs are susceptible to MAC deposition (Red) whereas overexpression of CFH protects against MAC formation.
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