Increased cell stiffness contributes to complement-mediated injury of choroidal endothelial cells in a monkey model of early age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet no therapies exist for ~85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigmented epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of the small GTPases Rac and Rho, because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility, while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential. © 2022 The Pathological Society of Great Britain and Ireland.

Keywords: Rac; Rho; actin; age-related macular degeneration; choroid; complement activation; endothelial cells; mechanotransduction; stiffness.

Figure 1.. Rhesus monkeys with macular drusen exhibit increased MAC deposition and choriocapillaris atrophy

(A) Retinal fundus images were used to categorize rhesus monkey eyes as young normal (YN; 6–9 years old), old normal (ON; 14–21 years old), or old with moderate-to-severe macular drusen (AMD; 14–30 years old). Bottom panel shows magnified view of the macular region outlined in the top panel, with arrows indicating drusen accumulation in AMD eyes. (B) Confocal images of macular sections from YN, ON, and AMD eyes labeled with anti-C5b-9 (yellow) and anti-collagen IV (green) reveal MAC immunoreactivity within drusen, and at the level of sub-RPE and choriocapillaris. Notably, MAC immunoreactivity was not observed in RPE. Acellular capillaries (asterisks) were identified by the absence of DAPI-labeled (purple) cell nucleus on the luminal side of collagen IV-positive capillaries. Scale bar, 25 μm. RPE, Retinal pigmented epithelium; CC, choriocapillaris. (C) Fluorescence intensity analysis showed that AMD eyes exhibit significantly higher (~1.5-fold; p<0.001) MAC immunoreactivity in the sub-RPE and choriocapillaris regions than YN eyes. (D) Counting of acellular capillaries revealed that AMD eyes have a significantly (p<0.001) higher degree of choriocapillaris atrophy in the macula than YN and ON eyes.

Figure 2.. Phenotypic characterization of isolated monkey choroidal ECs

(A) Phase contrast images of macular choroidal ECs isolated from YN, ON, and AMD monkeys reveal colony formation (arrows) characteristic of EC cultures. Scale bar: 100 μm. (B, C) RT-qPCR analysis of the isolated choroidal ECs indicate comparable mRNA levels of VE-cadherin (CDH5) and VEGFR2, key endothelial-specific markers. mRNA levels were normalized to GAPDH and expressed as mean ± sem. (D, E) Monkey choroidal ECs were labeled with anti-CD146 or anti-CD31 (empty histograms) antibody, or isotype-matched control antibody (solid gray histogram) and subjected to flow cytometry analysis. Histograms of cell counts versus fluorescence intensity indicate notable expression of the endothelial cell surface markers CD146 and CD31 in these cells.

Figure 3.. Choroidal ECs from drusen-laden eyes exhibit increased susceptibility to complement injury

(A) Choroidal ECs were treated with complement-competent normal human serum (NHS, 5% v/v; 3 h, 37 °C) prior to phalloidin labeling of F-actin cytoskeletal filaments. Representative fluorescent images and quantitative analysis of F-actin integrity revealed that AMD ECs undergo significantly greater cytoskeletal damage when compared with YN and ON cells (p<0.01; n≥300 cells/condition). Scale bar, 25 μm. (B) NHS-treated choroidal ECs were labeled with anti-C5b-9 to detect surface membrane attack complex (MAC) deposition. Representative confocal fluorescence images and quantitative image analysis (bar graph; n≥75 cells/condition) reveal no significant differences (p>0.05) in MAC deposition between these cells after 3 h of NHS treatment.

Figure 4.. Choroidal ECs from drusen-laden eyes exhibit higher stiffness

(A) EC stiffness was measured using a biological-grade AFM fitted with a silicon nitride cantilever tip that was modified with a 5 μm-diameter glass bead. Quantitative analysis of multiple (n≥60) force indentation measurements revealed that the stiffness of choroidal ECs isolated from AMD eyes was 2-fold higher than that of their young normal (YN) counterparts (p<0.05). (B) Normal, untreated choroidal ECs were stained with fluorescently-labeled phalloidin to visualize the constitutive organization of F-actin cytoskeletal filaments. Representative fluorescent images revealed robust longitudinal actin stress fibers in YN and ON ECs but a peripheral actin arrangement in AMD ECs, as indicated by the white arrowheads. Scale bar, 50 μm.

Figure 5.. Inhibiting Rac-mediated cell stiffness reduces the susceptibility of AMD ECs to complement injury

(A) Rac activity in choroidal ECs was measured using Rac G-LISA activation assay, which revealed a 3.6-fold increase (p<0.01) in Rac activity in AMD ECs when compared with YN cells. Bars indicate mean ± sem. (B) Phalloidin staining of AMD ECs treated with pharmacological Rac inhibitor (NSC23776; 1 mM) shows disruption of peripheral actin by Rac inhibition. (C) AFM was used to measure the stiffness of AMD ECs treated with or without NSC23776. Quantitative analysis of multiple (n≥60) force indentation measurements indicated that Rac inhibition significantly reduced cell stiffness. (D) Representative fluorescence images of phalloidin-labeled AMD ECs and quantitative analysis of F-actin integrity (box plot; n>100 cells/condition) show that inhibiting Rac-mediated cell stiffness significantly prevented complement injury in AMD ECs. Scale bar, 25 μm.

Figure 6.. RhoA confers YN ECs protection against complement injury

(A) RhoA activity in choroidal ECs was measured using RhoA G-LISA activation assay, which revealed a ~60% decrease in Rho activity in AMD ECs when compared with YN cells (p<0.05). Bars indicate mean ± sem. (B) Representative western blot bands (18 kDa) for phosphorylated MLC (pMLC) and their densitometric analysis (mean ± SD) normalized to total MLC shows that MLC phosphorylation is reduced by ~50% in AMD ECs (p<0.001). (C) Phalloidin staining of YN ECs treated with pharmacological ROCK inhibitor (Y27632; 100 μM) shows disruption of longitudinal actin stress fibers by RhoA/ROCK inhibition. (D) YN ECs were co-treated with NHS and Rho/ROCK inhibitor Y27632 (100 μM). Representative fluorescence images of phalloidin-labeled cells and quantitative analysis of F-actin integrity (box plot; n>100 cells/condition) show that inhibiting RhoA/ROCK-mediated contractility caused an ~40% increase in complement injury in YN ECs (p<0.001). Scale bar, 25 μm.