Sublytic Membrane-Attack-Complex (MAC) Activation Alters Regulated Rather than Constitutive Vascular Endothelial Growth Factor (VEGF) Secretion in Retinal Pigment Epithelium Monolayers

Abstract

Uncontrolled activation of the alternative complement pathway and secretion of vascular endothelial growth factor (VEGF) are thought to be associated with age-related macular degeneration (AMD). Previously, we have shown that in RPE monolayers, oxidative-stress reduced complement inhibition on the cell surface. The resulting increased level of sublytic complement activation resulted in VEGF release, which disrupted the barrier facility of these cells as determined by transepithelial resistance (TER) measurements. Induced rather than basal VEGF release in RPE is thought to be controlled by different mechanisms, including voltage-dependent calcium channel (VDCC) activation and mitogenactivated protein kinases. Here we examined the potential intracellular links between sublytic complement activation and VEGF release in RPE cells challenged with H 2 O 2 and complement-sufficient normal human serum (NHS). Disruption of barrier function by H 2 O 2 ؉ NHS rapidly increased Ras expression and Erk and Src phosphorylation, but had no effect on P38 phosphorylation. Either treatment alone had little effect. TER reduction could be attenuated by inhibiting Ras, Erk and Src activation, or blocking VDCC or VEGF-R2 activation, but not by inhibiting P38. Combinatorial analysis of inhibitor effects demonstrated that sublytic complement activation triggers VEGF secretion via two pathways, Src and Ras-Erk, with the latter being amplified by VEGF-R2 activation, but has no effect on constitutive VEGF secretion mediated via P38. Finally, effects on TER were directly correlated with release of VEGF; and sublytic MAC activation decreased levels of zfp36, a negative modulator of VEGF transcription, resulting in increased VEGF expression. Taken together, identifying how sublytic MAC induces VEGF expression and secretion might offer opportunities to selectively inhibit pathological VEGF release only.

Protective responses to sublytic complement in the retinal pigment epithelium

“…Polarized RPE were exposed to either 10% NHS or 60 μg/mL purified C9 reconstituted in 10% C9-depleted serum (Quidel) for 10 min (11,12). Heat-inactivated NHS (56°C for 30 min) and C9-depleted sera were used as negative controls.…”

“…The complement cascade is activated by insults that disturb RPE homeostasis, including defective clearance of phagocytosed photoreceptor outer segments (6) and abnormal accumulation of vitamin A dimers (7-9) or oxidized lipids (10) in the RPE. Studies also show that exposure to sublytic levels of MAC alters RPE barrier integrity, causes mitochondrial stress, and induces secretion of VEGF and proinflammatory cytokines (11,12). Thus, RPE dysfunction activates the complement cascade, which can promote further RPE damage and establish a vicious cycle of chronic inflammation within the retina (13).…”

Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease

“…These proteins can deposit on the plasma membrane of RPE cells to create a sublytic complex that degrades RPE function without killing the cell (45,46). CD46 is expressed in the basolateral membranes of human RPE cells, where it is exposed to the choroidal circulation.…”

Complement Stimulates Retinal Pigment Epithelial Cells to Undergo Pro-Inflammatory Changes

“…Others have found an influence of complement on the regulation of the vascular endothelial growth factor production of RPE cells and discovered a decreased epithelial resistance when normal human serum was combined with oxidative stress [21]. The induction of C5b-9 on the basal side of primary porcine RPE cells was also accompanied by an increased expression of CD59 [22].…”