Necessary Role of Ceramides in the Human Microvascular Endothelium During Health and Disease

Background: Elevated plasma ceramides and microvascular dysfunction both independently predict adverse cardiac events. Despite the known detrimental effects of ceramide on the microvasculature, evidence suggests that activation of the shear-sensitive, ceramide-forming enzyme NSmase (neutral sphingomyelinase) elicits formation of vasoprotective nitric oxide (NO). Here, we explore a novel hypothesis that acute ceramide formation through NSmase is necessary for maintaining NO signaling within the human microvascular endothelium. We further define the mechanism through which ceramide exerts beneficial effects and discern key mechanistic differences between arterioles from otherwise healthy adults (non–coronary artery disease [CAD]) and patients diagnosed with CAD. Methods: Human arterioles were dissected from discarded surgical adipose tissue (n=166), and vascular reactivity to flow and C2-ceramide was assessed. Shear-induced NO and mitochondrial hydrogen peroxide (H 2 O 2 ) production were measured in arterioles using fluorescence microscopy. H 2 O 2 fluorescence was assessed in isolated human umbilical vein endothelial cells. Results: Inhibition of NSmase in arterioles from otherwise healthy adults induced a switch from NO to NOX-2 (NADPH-oxidase 2)–dependent H 2 O 2 -mediated flow-induced dilation. Endothelial dysfunction was prevented by treatment with sphingosine-1-phosphate (S1P) and partially prevented by C2-ceramide and an agonist of S1P-receptor 1 (S1PR1); the inhibition of the S1P/S1PR1 signaling axis induced endothelial dysfunction via NOX-2. Ceramide increased NO production in arterioles from non-CAD adults, an effect that was diminished with inhibition of S1P/S1PR1/S1P-receptor 3 signaling. In arterioles from patients with CAD, inhibition of NSmase impaired the overall ability to induce mitochondrial H 2 O 2 production and subsequently dilate to flow, an effect not restored with exogenous S1P. Acute ceramide administration to arterioles from patients with CAD promoted H 2 O 2 as opposed to NO production, an effect dependent on S1P-receptor 3 signaling. Conclusion: These data suggest that despite differential downstream signaling between health and disease, NSmase-mediated ceramide formation is necessary for proper functioning of the human microvascular endothelium. Therapeutic strategies that aim to significantly lower ceramide formation may prove detrimental to the microvasculature.