Abstract 11818: The Beneficial Effects of Ceramide on Human Microvascular Endothelial Function is Dependent on S1P/S1PR1 Signaling Axis

Abstract

Despite the known damaging effects of elevated intracellular ceramides on human microvascular function, our recent work showed that loss of ceramide-formation through neutral sphingomyelinase (NSmase) promotes endothelial dysfunction in arterioles from healthy adults. This presents as a switch in the endothelial-derived mediator responsible for eliciting dilation to flow (flow-induced dilation; FID), from the vasoprotective nitric oxide (NO) to pro-inflammatory hydrogen peroxide (H 2 O 2 ). The mechanism through which ceramide exerts beneficial effects on the vasculature remains poorly understood. One of ceramide’s metabolites, sphingosine-1-phosphate (S1P), has been shown to stimulate NO production through the activation of S1P receptor 1 (S1PR1). Thus, we hypothesize that ceramide promotes NO-mediated FID through its metabolism to S1P and activation of S1PR1. Arterioles (100-250μm) from healthy adults were dissected from discarded surgical adipose tissue. Vessels were cannulated in organ chambers, pre-constricted with endothelin-1, and changes in internal diameter in response to increases in flow or C2-ceramide was measured via videomicroscopy. Difference between mean % dilation ±SE with two-way ANOVA is reported. Agonism of S1PR1 (CYM5442, 1μM, 30min) during NSmase inhibition (GW4869, 4μM, 30min) restored dependency on NO for FID, as L-NAME (100μM; NO synthase inhibitor) diminished FID (GW+CYM, n=4 vs GW+CYM+L-NAME, n=4: -70.3±20.0, p=0.01). A similar switch to NO-mediated FID was seen with C2-ceramide (10μM)+GW4869 treatment (GW+C2, n=5 vs GW+C2+L-NAME, n=4: -52.5±5.9, p<0.001). Inhibition of S1PR1 (W146, 10μM, 30min) induced a switch from NO to H 2 O 2 mediated FID, as catalase (500U/ml; W146, n=5 vs W146+Catalase, n=4: -34.0±7.9; p=0.01) but not L-NAME (W146, n=5 vs W146+L-NAME, n=4; -1.4±9.9, p=0.89) impaired FID. Acute ceramide-induced dilation (1min) was diminished in the presence of L-NAME (C2, n=5 vs C2+L-NAME, n=4: -33.40±10.06, p=0.02) and W146 (C2, n=5 vs C2+W146, n=4; -39.7±5.9, p=0.01). Together, these data suggest that ceramide induces NO-signaling through the activation of S1PR1 and provide mechanistic insight into how ceramide may exert beneficial effects within the human microvascular endothelium.