Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

BACKGROUND: Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. METHODS: We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. RESULTS: We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B (reticulon-4B), an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. CONCLUSIONS: These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

Sphingosine-1-phosphate controls endothelial sphingolipid homeostasis via ORMDL.

Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.

Involvement of 3',5'-cyclic inosine monophosphate in cystathionine γ-lyase-dependent regulation of the vascular tone.

BACKGROUND AND PURPOSE: l-cysteine or hydrogen sulfide (H2 S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 µM followed by vasodilatation at 30-100 µM. Here, we have investigated the signalling involved in the H2 S-induced contraction. EXPERIMENTAL APPROACH: Vascular response to NaHS or l-cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ-lyase (CSE-/- ), soluble guanylyl cyclase (sGCα1-/- ) and endothelial nitric oxide synthase (eNOS-/- ) knock-out mice. The cAMP, cGMP and inosine 3',5'-cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated. KEY RESULTS: CSE-derived H2 S-induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2 S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell-permeable analogue of cIMP elicits concentration-dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE-/- mice, confirms that H2 S-induced contraction involves cIMP. CONCLUSION AND IMPLICATIONS: The endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE-derived H2 S that is mediated by cIMP.

Anomalous Kv 7 channel activity in human malignant hyperthermia syndrome unmasks a key role for H2 S and persulfidation in skeletal muscle.

BACKGROUND AND PURPOSE: Human malignant hyperthermia (MH) syndrome is induced by volatile anaesthetics and involves increased levels of cystathionine ß-synthase (CBS)-derived H2 S within skeletal muscle. This increase contributes to skeletal muscle hypercontractility. Kv 7 channels, expressed in skeletal muscle, may be a molecular target for H2 S. Here, we have investigated the role of Kv 7 channels in MH. EXPERIMENTAL APPROACH: Skeletal muscle biopsies were obtained from MH-susceptible (MHS) and MH-negative (MHN) patients. Immunohistochemistry, RT-PCR, Western blot, and in vitro contracture test (IVCT) were carried out. Development and characterization of primary human skeletal muscle cells (PHSKMC) and evaluation of cell membrane potential were also performed. The persulfidation state of Kv 7 channels and polysulfide levels were measured. KEY RESULTS: Kv 7 channels were similarly expressed in MHN and MHS biopsies. The IVCT revealed an anomalous contractility of MHS biopsies following exposure to the Kv 7 channel opener retigabine. Incubation of negative biopsies with NaHS, prior to retigabine addition, led to an MHS-like positive response. MHS-derived PHSKMC challenged with retigabine showed a paradoxical depolarizing effect, compared with the canonical hyperpolarizing effect. CBS expression and activity were increased in MHS biopsies, resulting in a major polysulfide bioavailability. Persulfidation of Kv 7.4 channels was significantly higher in MHS than in MHN biopsies. CONCLUSIONS AND IMPLICATIONS: In skeletal muscle of MHS patients, CBS-derived H2 S induced persulfidation of Kv 7 channels. This post-translational modification switches the hyperpolarizing activity into depolarizing. This mechanism can contribute to the pathological skeletal muscle hypercontractility typical of MH syndrome. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.