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.

Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

AIMS: Growing evidence correlate the accrual of the sphingolipid ceramide in plasma and cardiac tissue with heart failure (HF). Regulation of sphingolipid metabolism in the heart and the pathological impact of its derangement remain poorly understood. Recently, we discovered that Nogo-B, a membrane protein of endoplasmic reticulum, abundant in the vascular wall, down-regulates the sphingolipid de novo biosynthesis via serine palmitoyltransferase (SPT), first and rate liming enzyme, to impact vascular functions and blood pressure. Nogo-A, a splice isoform of Nogo, is transiently expressed in cardiomyocyte (CM) following pressure overload. Cardiac Nogo is up-regulated in dilated and ischaemic cardiomyopathies in animals and humans. However, its biological function in the heart remains unknown. METHODS AND RESULTS: We discovered that Nogo-A is a negative regulator of SPT activity and refrains ceramide de novo biosynthesis in CM exposed to haemodynamic stress, hence limiting ceramide accrual. At 7 days following transverse aortic constriction (TAC), SPT activity was significantly up-regulated in CM lacking Nogo-A and correlated with ceramide accrual, particularly very long-chain ceramides, which are the most abundant in CM, resulting in the suppression of 'beneficial' autophagy. At 3 months post-TAC, mice lacking Nogo-A in CM showed worse pathological cardiac hypertrophy and dysfunction, with ca. 50% mortality rate. CONCLUSION: Mechanistically, Nogo-A refrains ceramides from accrual, therefore preserves the 'beneficial' autophagy, mitochondrial function, and metabolic gene expression, limiting the progression to HF under sustained stress.

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.

Sphingolipid Metabolism and Signaling in Endothelial Cell Functions.

The endothelium, inner layer of blood vessels, constitutes a metabolically active paracrine, endocrine, and autocrine organ, able to sense the neighboring environment and exert a variety of biological functions important to preserve the health of vasculature, tissues, and organs. Sphingolipids are both fundamental structural components of the eukaryotic membranes and signaling molecules regulating a variety of biological functions. Ceramide and sphingosine-1-phosphate (S1P), bioactive sphingolipids, have emerged as important regulators of cardiovascular functions in health and disease. In this review we discuss recent insights into the role of ceramide and S1P biosynthesis and signaling in regulating endothelial cell functions, in health and diseases. We also highlight advances into the mechanisms regulating serine palmitoyltransferase, the first and rate-limiting enzyme of de novo sphingolipid biosynthesis, with an emphasis on its inhibitors, ORMDL and NOGO-B. Understanding the molecular mechanisms regulating the sphingolipid de novo biosynthesis may provide the foundation for therapeutic modulation of this pathway in a variety of conditions, including cardiovascular diseases, associated with derangement of this pathway.

Endothelial Spns2 and ApoM Regulation of Vascular Tone and Hypertension Via Sphingosine-1-Phosphate.

Background Most of the circulating sphingosine-1-phosphate (S1P) is bound to ApoM (apolipoprotein M) of high-density lipoprotein (HDL) and mediates many beneficial effects of HDL on the vasculature via G protein-coupled S1P receptors. HDL-bound S1P is decreased in atherosclerosis, myocardial infarction, and diabetes mellitus. In addition to being the target, the endothelium is a source of S1P, which is transported outside of the cells by Spinster-2, contributing to circulating S1P as well as to local signaling. Mice lacking endothelial S1P receptor 1 are hypertensive, suggesting a vasculoprotective role of S1P signaling. This study investigates the role of endothelial-derived S1P and ApoM-bound S1P in regulating vascular tone and blood pressure. Methods and Results ApoM knockout (ApoM KO) mice and mice lacking endothelial Spinster-2 (ECKO-Spns2) were infused with angiotensin II for 28 days. Blood pressure, measured by telemetry and tail-cuff, was significantly increased in both ECKO-Spns2 and ApoM KO versus control mice, at baseline and following angiotensin II. Notably, ECKO-Spns2 presented an impaired vasodilation to flow and blood pressure dipping, which is clinically associated with increased risk for cardiovascular events. In hypertension, both groups presented reduced flow-mediated vasodilation and some degree of impairment in endothelial NO production, which was more evident in ECKO-Spns2. Increased hypertension in ECKO-Spns2 and ApoM KO mice correlated with worsened cardiac hypertrophy versus controls. Conclusions Our study identifies an important role for Spinster-2 and ApoM-HDL in blood pressure homeostasis via S1P-NO signaling and dissects the pathophysiological impact of endothelial-derived S1P and ApoM of HDL-bound S1P in hypertension and cardiac hypertrophy.

Early Morphofunctional Changes in AngII-Infused Mice Contribute to Regional Onset of Aortic Aneurysm and Dissection.

Aortic aneurysms and dissections are silent and lethal conditions, whose pathogenesis remains incompletely understood. Although angiotensin II (AngII)-infused ApoE-/- mice have been widely used to study aortic aneurysm and dissection, early morphofunctional alterations preceding the onset of these conditions remain unknown. The goal of this study was to unveil early morphofunctional changes underlying the onset of aneurysm and dissection. At 3 days post-AngII infusion, suprarenal abdominal aorta presented significant volumetric dilatation and microstructural damage. Ex vivo assessment of vascular reactivity of the suprarenal dissection-prone aorta and its side branches, showed an endothelial and contractile dysfunctions that were severe in the suprarenal aorta, moderate distally, and absent in the side branches, mirroring the susceptibility to dissection of these different vascular segments. Early and specific morphofunctional changes of the suprarenal aorta may contribute to the regional onset of aortic aneurysm and dissection by exacerbating the biomechanical burden arising from its side branches.

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

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.

Sphingolipid Signature of Human Feto-Placental Vasculature in Preeclampsia.

Bioactive sphingolipids are emerging as key regulators of vascular function and homeostasis. While most of the clinical studies have been devoted to profile circulating sphingolipids in maternal plasma, little is known about the role of the sphingolipid at the feto-placental vasculature, which is in direct contact with the offspring circulation. Our study aims to compare the sphingolipid profile of normal with preeclamptic (PE) placental chorionic arteries and isolated endothelial cells, with the goal of unveiling potential underlying pathomechanisms in the vasculature. Dihydrosphingosine and sphingomyelin (SM) concentrations (C16:0-, C18:0-, and C24:0- sphingomyelin) were significantly increased in chorionic arteries of preeclamptic placentas, whereas total ceramide, although showing a downward trend, were not statistically different. Moreover, RNA and immunofluorescence analysis showed impaired sphingosine-1-phosphate (S1P) synthesis and signaling in PE vessels. Our data reveal that the exposure to a deranged maternal intrauterine environment during PE alters the sphingolipid signature and gene expression on the fetal side of the placental vasculature. This pathological remodeling consists in increased serine palmitoyltransferase (SPT) activity and SM accrual in PE chorionic arteries, with concomitance impairment endothelial S1P signaling in the endothelium of these vessels. The increase of endothelial S1P phosphatase, lyase and S1PR2, and blunted S1PR1 expression support the onset of the pathological phenotype in chorionic arteries.

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases, but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

BiP/GRP78 Mediates ERAD Targeting of Proteins Produced by Membrane-Bound Ribosomes Stalled at the STOP-Codon.

Translational stalling of ribosome bound to endoplasmic reticulum (ER) membrane requires an accurate clearance of the associated polypeptides, which is not completely understood in mammals. We characterized in mammalian cells the model of ribosomal stalling at the STOP-codon based on proteins tagged at the C-terminus with the picornavirus 2A peptide followed by a termination codon instead of the Proline (2A*). We exploited the 2A* stalling model to characterize the pathway of degradation of ER-targeted polypeptides. We report that the ER chaperone BiP/GRP78 is a new main factor involved. Moreover, degradation of the ER-stalled polypeptides required the activities of the AAA-ATPase VCP/p97, its associated deubiquitinylase YOD1, the ribosome-associated ubiquitin ligase Listerin and the proteasome. In human proteome, we found two human C-terminal amino acid sequences that cause similar stalling at the STOP-codon. Our data suggest that translational stalling at the ER membrane activates protein degradation at the interface of ribosomal- and ER-associated quality control systems.

Sphingolipid De Novo Biosynthesis: A Rheostat of Cardiovascular Homeostasis.

Sphingolipids (SL) are both fundamental structural components of the eukaryotic membranes and signaling molecules that regulate a variety of biological functions. The highly-bioactive lipids, ceramide and sphingosine-1-phosphate, have emerged as important regulators of cardiovascular function in health and disease. In this review we discuss recent insights into the role of SLs, particularly ceramide and sphingosine-1-phosphate, in the pathophysiology of the cardiovascular system. We also highlight advances into the molecular mechanisms regulating serine palmitoyltransferase, the first and rate-limiting enzyme of de novo SL biosynthesis, with an emphasis on the recently discovered inhibitors of serine palmitoyltransferase, ORMDL and NOGO-B proteins. Understanding the molecular mechanisms regulating this biosynthetic pathway may lead to the development of novel therapeutic approaches for the treatment of cardiovascular diseases.

The VCP/p97 and YOD1 Proteins Have Different Substrate-dependent Activities in Endoplasmic Reticulum-associated Degradation (ERAD).

Endoplasmic reticulum-associated degradation (ERAD) is an essential quality control mechanism of the folding state of proteins in the secretory pathway that targets unfolded/misfolded polypeptides for proteasomal degradation. The cytosolic p97/valosin-containing protein is an essential ATPase for degradation of ERAD substrates. It has been considered necessary during retro-translocation to extract proteins from the endoplasmic reticulum that are otherwise supposed to accumulate in the endoplasmic reticulum lumen. The activity of the p97-associated deubiquitinylase YOD1 is also required for substrate disposal. We used the in vivo biotinylation retro-translocation assay in mammalian cells under conditions of impaired p97 or YOD1 activity to directly discriminate their requirements and diverse functions in ERAD. Using different ERAD substrates, we found that both proteins participate in two distinct retro-translocation steps. For CD4 and MHC-Iα, which are induced to degradation by the HIV-1 protein Vpu and by the CMV immunoevasins US2 and US11, respectively, p97 and YOD1 have a retro-translocation-triggering role. In contrast, for three other spontaneous ERAD model substrates (NS1, NHK-α1AT, and BST-2/Tetherin), p97 and YOD1 are required in the downstream events of substrate deglycosylation and proteasomal degradation.

CD4 and BST-2/tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules.

CD4 and BST-2/Tetherin are cellular membrane proteins targeted to degradation by the HIV-1 protein Vpu. In both cases proteasomal degradation following recruitment into the ERAD pathway has been described. CD4 is a type I transmembrane glycoprotein, with four extracellular immunoglobulin-like domains containing three intrachain disulfide bridges. BST-2/Tetherin is an atypical type II transmembrane glycoprotein with an N-terminal transmembrane domain and a C-terminal glycophosphatidylinositol anchor, which dimerizes through three interchain bridges. We investigated spontaneous and Vpu-induced retro-translocation of CD4 and BST-2/Tetherin using our novel biotinylation technique in living cells to determine ER-to-cytosol retro-translocation of proteins. We found that CD4 retro-translocates with oxidized intrachain disulfide bridges, and only upon proteasomal inhibition does it accumulate in the cytosol as already reduced and deglycosylated molecules. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric oxidized complex and then becomes deglycosylated and reduced to monomers. These results raise questions on the required features of the putative retro-translocon, suggesting alternative retro-translocation mechanisms for membrane proteins in which complete cysteine reduction and unfolding are not always strictly required before ER to cytosol dislocation.