Eicosapentaenoic acid reduces pulmonary endothelial caveolae protein and restores nitric oxide

ABSTRACT

INTRODUCTION: Loss of pulmonary endothelial cell (EC) nitric oxide (NO) bioavailability following infection by SARS-CoV-2 or influenza leads to increased viral uptake, thrombosis and enhanced inflammation. EC production of NO by its synthase (eNOS) is downregulated by proteins in membrane caveolae, including caveolae-associated protein 2. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its bioactive lipid metabolites reduce inflammation and improve EC function in various tissues. These benefits of EPA may contribute to the pronounced cardiovascular event reduction reported in REDUCE-IT. As a result, EPA is now being tested in patients at risk for COVID-19. This study tested the effects of EPA on expression of caveolae-associated protein 2 and NO bioavailability in pulmonary ECs under conditions of inflammation caused by the cytokine interleukin-6 (IL-6). METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Cells (including untreated controls) were stimulated with calcium ionophore to measure maximum production of NO and peroxynitrite (ONOO-) using tandem porphyrinic nanosensors. Proteomic analysis was performed using LC/ MS to capture relative expression levels >1,000 proteins. Only significant (p<0.05) changes in protein expression between treatment groups >2-fold were further analyzed. RESULTS: HMVEC-L challenged with IL-6 showed a pronounced loss of NO bioavailability. EPA treatment increased NO release (17%, p<0.05) and decreased ONOO- release (16%, p<0.05) compared with IL-6 treatment. The [NO]/[ONOO-] ratio, a marker of eNOS coupling efficiency, decreased by 35% (p<0.001) following exposure to IL-6 but EPA treatment increased the ratio by 39% (p<0.001). Improved NO bioavailability correlated with >3-fold reduction in caveolae-associated protein 2 (p<0.05). CONCLUSIONS: EPA restored NO bioavailability and reduced expression of caveolae-associated protein 2 in pulmonary ECs following IL-6 treatment. The ability of EPA to inhibit endothelial inflammatory changes and restore NO bioavailability has therapeutic implications for patients at risk for SARS-CoV-2 infection and other inflammatory states.