Abstract
Purpose :
Mitochondrial dysfunction of retinal pigment epithelial (RPE) cells plays a key role in age-related macular degeneration (AMD) pathogenesis. We have previously shown that RPE cells derived from AMD donors have a distinct mitochondrial gene expression profile as well as distinct phenotype when compared to age-matched controls. Here, we analyze the mitochondrial gene expression and function of these lines cultured on nitrite-modified extracellular matrix (ECM), an in vitro aged Bruch’s membrane model, to investigate whether a nitrite-modified ECM alters mitochondrial function and gene expression in AMD-derived RPE.
Methods :
iPSCs were generated from fibroblasts isolated from four AMD patients (2 atrophic and 2 exudative). RPE derived from iPSCs were generated using established protocols and analyzed by morphology, cell type specific marker expression, transepithelial resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cells were cultured on nitrite ECM for two weeks and subject to cell viability and mitochondrial function analysis using the Seahorse platform. DNA microarray with 23,000 well annotated genes, including mitochondrial structure and function-related genes.
Results :
iPSCs and differentiated RPE displayed cell-type-specific morphology, markers, TER, and phagocytic capacity. Cell viability and mitochondrial respiration were reduced in AMD-derived RPE cells on nitrite ECM, compared to cells cultured on unmodified ECM; mitochondrial related genes such as ATPase Cu++ transporting beta polypeptide (ATP7B), cytochrome P450 family 24 subfamily A polypeptide 1 (CYP24A1) and glutaminase 2 (GLS2) display a distinct expression pattern in AMD-derived RPE when cultured on nitrite-modified ECM compared to AMD-derived lines cultured on unmodified ECM.
Conclusions :
Here, we provide evidence that nitrite modification of the ECM affect cell viability and mitochondrial function in iPSC-derived from AMD patients. Further exploration of the mitochondrial genes affected in this model may pave the way for novel treatments for AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.