Necessary Role of Acute Ceramide Formation in The Human Microvascular Endothelium During Health and Disease

Elevated plasma ceramides independently predict adverse cardiac events and we have previously shown that exposure to exogenous ceramide induces microvascular endothelial dysfunction in arterioles from otherwise healthy adults (0-1 risk factors for heart disease). However, evidence also suggests that activation of the shear-sensitive, ceramide forming enzyme neutral sphingomyelinase (NSmase) enhances vasoprotective nitric oxide (NO) production. 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 and patients with coronary artery disease (CAD).

Human arterioles were dissected from otherwise discarded surgical adipose tissue (n=123), and vascular reactivity to flow and C2-ceramide was assessed. Shear-induced NO production was measured in arterioles using fluorescence microscopy. Hydrogen peroxide (H₂O₂) fluorescence was assessed in isolated human umbilical vein endothelial cells.

Inhibition of NSmase in arterioles from otherwise healthy adults induced a switch from NO to H₂O₂-mediated flow-induced dilation within 30 minutes. In endothelial cells, NSmase inhibition acutely increased H₂O₂ production. Endothelial dysfunction in both models was prevented by treatment with C2-ceramide, S1P, and an agonist of S1P-receptor 1 (S1PR1), while the inhibition of S1P/S1PR1 signaling axis induced endothelial dysfunction. Ceramide increased NO production in arterioles from healthy adults, an effect that was diminished with inhibition of S1P/S1PR1/S1PR3 signaling. In arterioles from patients with CAD, inhibition of NSmase impaired dilation to flow. This effect was not restored with exogenous S1P. Although, inhibition of S1P/S1PR3 signaling impaired normal dilation to flow. Acute ceramide administration to arterioles from patients with CAD also promoted H₂O₂ as opposed to NO production, an effect dependent on S1PR3 signaling.

These data suggest that despite key differences in downstream signaling between health and disease, acute NSmase-mediated ceramide formation and its subsequent conversion to S1P is necessary for proper functioning of the human microvascular endothelium. As such, therapeutic strategies that aim to significantly lower ceramide formation may prove detrimental to the microvasculature.