Heterogeneous membrane cholesterol in large and small rat mesenteric endothelial cells

Abstract

Introduction: Endothelial dysfunction is a common event in cardiovascular disease, characterized by damage to the endothelium and impaired local blood flow regulation, including the regulation by the gasotransmitters, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S). We and others have shown that H 2 S is a vasodilator; however, not all arterial segments respond similarly to this gasotransmitter. We have shown H 2 S-sensitivity in large arteries (>200 μm) is dependent on the membrane cholesterol content of endothelial cells (EC), while smaller arteries (<200 μm) are inherently highly sensitive to H 2 S. In addition, we have demonstrated ECs from large arteries contain more cholesterol in the plasma membrane than ECs from smaller arteries. Taken together, these results suggest that EC signaling is dependent on the level of membrane cholesterol and that the cholesterol content of the plasma membrane is differentially regulated in arterial segments within the same organ. This study investigated the mechanism mediating differences in endothelial membrane cholesterol between large and small arteries. Our single-cell RNAseq data suggests that the differences in cholesterol content between small and large arteries may result from increased EC expression of ATP-binding cassette family a1 (Abca1) in small arteries. Therefore, we hypothesized that augmented cholesterol efflux by Abca1 mediates the lower membrane cholesterol in ECs of resistance arteries facilitating vasodilatory signaling pathways. Methods: Endothelial tubes were generated by enzymatic digestion followed by titration of mesenteric arteries from male Sprague-Dawley rats (Envigo 175 – 225g, N=5 for all experiments). Cholesterol in endothelial tubes was measured using filipin-lll (50 μg/ml), while Abca1 was measured using immunofluorescence staining and confocal microscopy. We performed in-vitro studies manipulating membrane cholesterol by treating rat mesenteric ECs with Abca1 inhibitor, probucol (10μM), or Abca1 knockdown (KD) through RNA interference. Results: Our data reveal an increase in filipin-lll fluorescence in large artery ECs compared to ECs from small arteries and that Abca1 expression is greater in small artery ECs. Inhibition and KD of Abca1 using probucol or siRNA, respectively, ­ in vitro increased EC membrane cholesterol. Conclusions: Abca1 is an important regulator of EC membrane cholesterol and mediates the lower EC membrane cholesterol in small arteries compared to large arteries. Future investigations will explore whether hypercholesterolemia causes an increase in EC membrane cholesterol in the microcirculation resulting in endothelial dysfunction and impaired organ blood flow control. Funding: 1) NIH 1R01HL160606 (JSN), 2) T32HL007736 (JRA), and 3) UNM Comprehensive Cancer Center Support Grant NCI P30CA118100 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.