Abstract
Purpose :
Early age-related macular degeneration (AMD) is marked by a significant loss of endothelial cells (ECs) in the choroidal vessels (CVs). Yet, little is known about the mechanisms underlying this CV degeneration. Based on our recent report that choroidal EC stiffening contributes to cytoskeletal injury in a monkey model of early AMD, here we investigated in detail the role and regulation of endothelial mechanobiology in choroidal EC loss associated with early AMD.
Methods :
Choroidal ECs were isolated from the macula of ‘old healthy’ (OH; 20 yrs old) and old ‘drusen-laden early AMD’ (AMD; 19 yrs old) rhesus monkeys. ECs were compared for their susceptibility to death by AMD stressors (oxidative stress and complement activation) as well as transcriptome- and proteome-wide levels of mechanobiology regulators. Subendothelial matrix stiffness and composition were assessed using atomic force microscopy and immunofluorescence. The causal role of subendothelial matrix stiffness in OH and AMD EC death was assessed by growing them on synthetic matrices mimicking OH or AMD matrix stiffness and treating with AMD stressors. Finally, we assessed the ability of AMD-secreted matrix to activate complement (using immunolabeling) and any potential effects of the complement-activated matrix on choroidal EC death by AMD stressors.
Results :
We found that choroidal ECs from AMD monkeys are more necrotic than the OH ECs. Transcriptomic and proteomics analysis of AMD vs OH choroidal ECs revealed that the top-ranked biological pathway based on the differentially expressed genes relates to extracellular matrix. Indeed, AMD ECs secreted greater levels of matrix crosslinking enzyme tissue transglutaminase 2 and structural proteins collagen IV and fibronectin, which correlated with a significantly stiffer subendothelial matrix. Importantly, AMD and OH ECs grown on synthetic matrices revealed that matrix stiffening alone increases choroidal EC susceptibility to AMD stressors. Finally, we found the AMD-secreted matrix exhibits substantial complement deposition that, in turn, exacerbates AMD EC death in response to AMD stressors.
Conclusions :
Our findings implicate the stiffer subendothelial matrix and its complement-activating potential as previously-unrecognized risk factors for choroidal EC death in response to AMD stressors, thereby offering a new insight into CV loss associated with early AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.