Cholesterol depletion following chronic hypoxia unmasks EGFR-signaling in pulmonary vascular smooth muscle through disinhibition of Rac1
Authors: Ahmadian Rosstin , Anderson Jacob , Naik Jay , Walker Benjimen , Jernigan Nikki , Resta Thomas 
Published: May 2023

Introduction: Chronic hypoxia (CH) leads to increased pulmonary vascular resistance and resultant pulmonary hypertension (PH) in patients with chronic obstructive pulmonary disease and sleep apnea. We recently demonstrated that CH augments vasoconstrictor reactivity through a switch in pulmonary arterial smooth muscle cell (PASMC) signaling from Ca2+-dependent mechanisms towards a novel epidermal growth factor receptor (EGFR)-NADPH oxidase 2 (NOX2)-Rho kinase Ca2+ sensitization pathway. The overall objective of this study is to understand the mechanism by which CH mediates this switch in signaling. We have recently demonstrated that CH decreases membrane cholesterol, a lipid membrane domain component that can regulate both EGFR and NOX2 activity in other cell types. Since Rac1 is an important regulator of NOX2, we hypothesized that cholesterol depletion following CH unmasks EGFR-signaling through disinhibition of Rac1. Methods: We validated our ability to manipulate membrane cholesterol levels by treating isolated pulmonary arteries from control and CH (4 wk @ PB = 380 mmHg) Sprague Dawley rats with either cholesterol (2 mM), epicholesterol (cholesterol epimer; 2 mM), MβCD (cholesterol chelator; 10 mM), or vehicle. PASMC membrane cholesterol content was quantified by filipin (50 μg/ml) fluorescence using confocal microscopy. NOX2 activity was assessed via epidermal growth factor (EGF)-induced superoxide production +/- gp91ds-tat (selective NOX2 inhibitor; 50 μM) in primary cultures of PASMCs from control and CH rats. Superoxide levels were measured by dihydroethidium fluorescence (5 μM). We additionally assessed Rac1 activity by G-LISA (Cytoskeleton) in these cells. Subsequently, we examined vasoconstrictor responses to increasing concentrations of EGF (10-10 – 10-6 M) in pressurized, endothelium-disrupted pulmonary arteries (~150 μm diameter) from control rats. A subset of arteries was treated with gp91ds-tat (50 μM), fasudil (Rho kinase inhibitor; 10 μM), or their respective vehicles. Results: Cholesterol supplementation significantly (P<0.05) increased PASMC membrane cholesterol in both control and CH groups. Conversely, treatment with either MβCD or epicholesterol significantly decreased membrane cholesterol in arteries from control animals only. Additionally, epicholesterol increased NOX2-dependent superoxide production and Rac1 activity in PASMCs from control but not CH rats. In agreement with our hypothesis, the epicholesterol-dependent substitution of membrane cholesterol revealed concentration-dependent vasoconstriction to EGF in pulmonary arteries from control rats. Treatment with either fasudil or gp91ds-tat attenuated this effect. Conclusions: These results suggest decreased PASMC membrane cholesterol following CH augments EGFR-mediated vasoconstriction through disinhibition of NOX2 and Rac1. This work was supported by NIH R01 HL132883 to T. Resta, NIH T32 HL007736 to R. Ahmadian, and NIH F31 HL162502-01 to R. Ahmadian. 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.