Cellular senescence recapitulates many features of aging.
25,26 Thus, to develop an in vitro model of CC aging, RF/6A ECs were cultured to high passages (>P60) to achieve replicative senescence and compared with lower passage (<P40) cells for expression levels of senescence-associated β-galactosidase (SA-β-gal), a well-known senescence marker.
24 Staining with X-gal, which yields a blue color when cleaved by SA-β-gal, revealed a 9-fold greater (
P < 0.001) expression of SA-β-gal in higher passage cells than in the lower passage counterparts (
Fig. 1A). To further confirm the senescent phenotype, we compared the expression levels of p21 in the low and high passage cells. p21 is an inhibitor of cyclin-dependent kinase that promotes cell growth arrest and, thereby, senescence.
27 Our qPCR measurements revealed that high passage ECs exhibit 1.7-fold higher levels (
P < 0.01) of p21 mRNA than low passage ECs (
Fig. 1B). Consistent with the typical senescence phenotype, we also observed a markedly greater percentage of “enlarged” cells (projected area >4000 μm
2) in the high passage ECs than in the lower passage cells (
Supplementary Fig. S1). The low and high passage cells were subsequently termed “normal” and “senescent” ECs, respectively. Notably, the SA-β-gal–expressing senescent ECs did not exhibit any loss of endothelial phenotype, as judged by similar expression levels of classical endothelial-specific markers CD31 (
Fig. 1C) and CD146 (
Fig. 1D) in normal and senescent ECs.
To test their sensitivity to complement activation associated with dry AMD, we exposed these cells to complement-competent NHS. By activating the alternative complement pathway, NHS treatment leads to surface MAC deposition similar to that observed in the CC of human dry AMD eyes.
28,29 Flow cytometry analysis of NHS-treated normal and senescent ECs labeled with anti-C5b-9 (anti-MAC) revealed similar degrees of surface MAC deposition in these cells (
Fig. 2A). However, when compared with normal ECs, the senescent cells underwent a 3-fold increase (
P < 0.001) in complement-induced lysis, as judged by greater trypan blue incorporation within the senescent ECs (
Fig. 2B,
Supplementary Fig. S2). Notably, this increase in complement-induced lysis of senescent ECs did not result from reduced expression of endogenous cell-surface MAC inhibitory factor CD59, whose surface levels were comparable in both normal and senescent ECs (
Supplementary Fig. S3). Predictably, the lysed (trypan blue-positive) cells exhibited a rounded morphology (
Fig. 2B; inset), which is indicative of impaired cell viability and death.
30,31