Abstract
Purpose :
Although many studies demonstrate higher prevalence of primary open angle glaucoma (POAG) in those of African descent, no physiologic findings have been identified to explain these phenotypic differences. We tested the hypothesis that there are fundamental differences in mitochondrial function and antioxidant protection between self-reported African American (AA) and White (W) subjects in an in vitro model of trabecular meshwork (TM) from POAG patients and healthy donors (controls).
Methods :
TM specimens collected from POAG patients undergoing glaucoma surgery were analyzed by RNA-sequencing (RNA-seq). Control donor and POAG TM tissues, divided by race, were cultured following standard methods and confirmed with specific cell markers. Intracellular reactive oxygen species (ROS) production was measured in live TM cells by CellROXTM Orange after hydrogen peroxide (H2O2) challenge. Major antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT), and their transcriptional factor, nuclear factor erythroid 2-related factor 2 (NRF2), were measured at RNA (qPCR) and protein levels (Western blot). Select genes of interest from RNA-seq analysis were also evaluated with qPCR. Student T-Test and Tukey's multiple comparisons test were applied for data analysis.
Results :
The most significant expression differences from RNA-seq data were identified in nuclear genes encoding protein components of mitochondrial Complex I, III and IV. POAG TM tissue from AA (n=3) demonstrated higher expression of these genes (p<0.01) vs. W (n=4). qPCR assessment of these genes in TM tissues from control donors (6AA, 6W) and POAG patient tissue (4AA, 5W) did not show any racial differential. For the primary cultured TM cells, ROS production in AA TM cells was significantly higher than W (p=0.004) following H2O2 challenge. qPCR showed NRF2 and SOD2 were significantly lower in AA than W (p=0.02, 0.04), with no difference in CAT. Western blot confirmed protein levels of NRF2 and SOD2 were significantly lower in AA TM cells than W (p=0.003, 0.04).
Conclusions :
Fundamental differences in oxidative stress and antioxidant protection may be important risk factors for racial disparities in POAG risk and severity. The primary TM cell culture model derived from POAG patient tissue and healthy donor controls provides powerful tools to study distinct differences of TM function between AA and W.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.