Endothelial Sphingolipid De Novo Synthesis Controls Blood Pressure by Regulating Signal Transduction and NO via Ceramide

Abstract

Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus-related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)-the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.

Keywords: blood pressure; ceramides; endothelial; sphingolipids; vascular resistance.

Figure 1.. Endothelial sphingolipid de novo biosynthesis is an important source of local and circulating ceramide and S1P.

(A) WB and densitometric analysis of SPTLC1 and SPTLC2 in ECKO-Sptlc2 and Sptlc2^f/f EC after 4-OHT (1μM, 72h) treatment. β-actin, loading control. (B) SPT activity in ECKO-Sptlc2 and Sptlc2^f/f EC. [³H]-serine and palmitoyl-CoA were used as substrates for SPT. Sphinganine, the reaction product, was separated in TLC and quantified. (C, D) LC-MS/MS quantification of total and specific ceramides in Sptlc2^f/f and ECKO-Sptlc2 after 4-OHT treatment. (A-D) N≥3 independent EC isolations/group; 4 mice/EC isolation. (E, F) LC-MS/MS analysis of ceramides, Sph and S1P in Sptlc2^f/f and ECKO-Sptlc2 plasma (n≥ 9/group). dhSph-1P=Dihydro-sphingosine-1-phosphate, dhSph=dihydro-sphingosine, Sph=sphingosine. Data are expressed as mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001 compared to Sptlc2^f/f. Statistical significance was determined by Unpaired t-test.

Figure 2.. Endothelial-derived SL control vascular tone by preserving signaling transduction to agonists and flow.

(A) Ach-mediated vasodilation of MA in absence (n≥12 mice/group, n≥22 MA/group) and presence of L-NIO (100 μM, 20 min; n≥5 mice/group n≥8 MA/group), and L-NIO-induced vasoconstriction in Sptlc2^f/f and ECKO-Sptlc2 MA at baseline (n≥5 mice/group n≥8 MA/group). (B) Plasma nitrite levels (n≥6/group). (C) WB analysis for P-VASP and VASP on ECKO-Sptlc2 and Sptlc2^f/f thoracic aortas (n=9 mice/group) and relative quantification. Vasodilation in response to: (D) Sphingosine-1-phosphate (S1P; n=9 mice/ group, n=9 MA/group); thrombin (n≥3 mice/group, n≥4 MA/group); prostacyclin (PGI₂; n≥4 mice/group, n≥6 MA/group); (E) VEGF (n≥5 mice/group, n≥5 MA/group) and (F) flow (n≥12 mice/group, n≥15 MA/group). All data represent mean ± SEM. ** p≤0.01 and *** p≤0.001 ECKO-Sptlc2 vs. Sptlc2^f/f. Statistical significance was determined by Unpaired t-test (A, B, C) or Two-way ANOVA (A, D-F).

Figure 3.. Radiotelemetry measurements of BP.

(A) Systolic, (B) diastolic and (C) mean BP, and (D) heart rate (HR) were measured by radiotelemetry for three consecutive days following the recovery from surgery (9 days) in ECKO-Sptlc2 (n=6) and Sptlc2^f/f (n=6). (E) Analysis of low to high frequency (LF/HF) ratios in the same groups of mice. All data represent mean ± SEM. *** p≤0.001 ECKO-Sptlc2 vs. Sptlc2^f/f mice. Statistical analysis was performed with Two-way ANOVA with Sidak post-test (A-D) and unpaired t-test (E).

Figure 4.. Prevailing role of C16:0-Cer vs. C24:0- and C24:1-Cer in restoring VEGF-mediated vasodilation.

ECKO-Sptlc2 mice were treated with C16:0-Cer, C24:0-Cer, C24:1-Cer at the doses of 3 mg/Kg/d (left panels) or 10 mg/Kg/d (right panels) i.p. for 2 consecutive days. VEGF-induced vasodilation in MA from ECKO-Sptlc2 mice treated with (A) C16:0-Cer (n≥4 mice/group); (B) C24:0-Cer (n≥ 4mice/group); (C) C24:1-Cer (n≥ 4mice/group), or vehicle. (D) Maximum VEGF-induced vasodilation (Emax) of MA from ECKO-Sptlc2 mice treated with two different doses of C16:0-Cer, C24:0-Cer or C24:1-Cer compared to vehicle (indicated as 0 on X-axis). (E) Schematic carton representing the different ceramide-specific effects on VEGF-induced vasodilation. All data represent mean ± SEM. *** p≤0.001 ECKO-Sptlc2 + corn oil vs. Sptlc2^f/f + corn oil; ° p≤0.05, °° p≤0.01 and °°° p≤0.001 ECKO-Sptlc2 + ceramide vs. ECKO-Sptlc2 + corn oil. Statistical significance was determined by Two-way ANOVA (A,B,C) or One-way ANOVA (D).

Figure 5.. C24:0- and C24:1-Cer prevail over C16:0-Cer in restoring flow-mediated vasodilation in ECKO-Sptlc2 MA.

ECKO-Sptlc2 mice treated with C16:0-Cer, C24:0-Cer, C24:1-Cer at the doses of 3 mg/Kg/d (left panels) or 10 mg/Kg/d (right panels) i.p. for 2 consecutive days. Flow-induced vasodilation in MA from ECKO-Sptlc2 mice treated with (A) C16:0-Cer (n≥ 4mice/group); (B) C24:0-Cer (n≥4 mice/group); (C) C24:1-Cer (n≥4 mice/group). (D) Flow-induced maximum vasodilation (Emax) of MA from ECKO-Sptlc2 mice treated with two different doses of C16:0-Cer, C24:0-Cer or C24:1-Cer compared to corn oil as vehicle. (E) Scheme representing specific effects of different ceramides on flow-induced vasodilation. Data are expressed as mean ± SEM. *** p≤0.001 ECKO-Sptlc2 + corn oil vs. Sptlc2^f/f + corn oil; ° p≤0.05 and °°° p≤0.001 ECKO-Sptlc2 + ceramide vs. ECKO-Sptlc2 + corn oil. Statistical significance was determined by Two-way ANOVA (A, B, C) or One-way ANOVA (D).

Figure 6.. C16:0-cer restores VEGFR2-mediated signaling in EC treated with myriocin.

(A) WB analysis was performed on HUVEC treated with myriocin, inhibitor of SPT, or vehicle, followed by VEGF stimulation (100 ng/ml, 2 min). Some HUVEC treated with myriocin, were incubated with C16:0-Cer to restore VEGF signaling. Membranes were incubated with antibodies against P-VEGFR2 (Y1175), VEGFR2, P-AKT (S473), AKT, P-eNOS (S1176) and eNOS. (B-C) Densitometric analysis of indicated phospho/total protein ratios (n=4 independent experiments). (E,F) Sphingolipid measurements by LC-MS/MS in Sptlc2^f/f and ECKO-Sptlc2 EC, treated with 300nM C16-Cer for the indicated times. (E) Total and (F) individual ceramides (3 independent EC isolations/group; 3 mice/EC isolation). Data are expressed as mean ± SEM. * p≤0.05; ** p≤0.01; *** p≤0.001. Statistical significance was determined by One-way ANOVA.