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.

High fat diet ameliorates mitochondrial cardiomyopathy in CHCHD10 mutant mice.

Mutations in CHCHD10 , a mitochondrial protein with undefined functions, are associated with autosomal dominant mitochondrial diseases. Chchd10 knock-in mice harboring a heterozygous S55L mutation (equivalent to human pathogenic S59L) develop a fatal mitochondrial cardiomyopathy caused by CHCHD10 aggregation and proteotoxic mitochondrial integrated stress response (mtISR). In mutant hearts, mtISR is accompanied by a metabolic rewiring characterized by increased reliance on glycolysis rather than fatty acid oxidation. To counteract this metabolic rewiring, heterozygous S55L mice were subjected to chronic high fat diet (HFD) to decrease insulin sensitivity and glucose uptake and enhance fatty acid utilization in the heart. HFD ameliorated the ventricular dysfunction of mutant hearts and significantly extended the survival of mutant female mice affected by severe pregnancy-induced cardiomyopathy. Gene expression profiles confirmed that HFD increased fatty acid utilization and ameliorated cardiomyopathy markers. Importantly, HFD also decreased accumulation of aggregated CHCHD10 in the S55L heart, suggesting activation of quality control mechanisms. Overall, our findings indicate that metabolic therapy can be effective in mitochondrial cardiomyopathies associated with proteotoxic stress.

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.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

Open radial artery harvesting better preserves endothelial function compared to the endoscopic approach.

OBJECTIVES: Both the open and endovascular techniques are commonly used for harvesting the radial artery (ORAH and ERAH, respectively), and yet, very little is known about the effects of these 2 techniques on endothelial integrity and function of the radial artery (RA). The aim of this study was to assess the endothelial integrity and function of RA harvested using the 2 approaches. METHODS: Two independent surgical teams working in the same institution routinely use the RA for coronary artery bypass grafting exclusively employing either ORAH or ERAH. Thirty-nine consecutive patients were enrolled in this comparative study. Endothelial function after ORAH or ERAH was assessed by using the wire myograph system. The integrity of the RA endothelium was evaluated by immunohistochemical staining for erythroblast transformation specific-related gene. RESULTS: The vasodilation in response to acetylcholine was significantly higher in RA harvested with ORAH (P ≤ 0.001 versus ERAH). Endothelial integrity was not different between the 2 groups. CONCLUSIONS: ORAH is associated with a significantly higher endothelium-dependent vasodilation. Further investigation on the potential implications of these findings in terms of graft spasm and patency as well as clinical outcomes are needed.

Pressure overload leads to coronary plaque formation, progression, and myocardial events in ApoE-/- mice.

Hypercholesterolemia and hypertension are two major risk factors for coronary artery diseases, which remain the major cause of mortality in the industrialized world. Current animal models of atherosclerosis do not recapitulate coronary plaque disruption, thrombosis, and myocardial infarction occurring in humans. Recently, we demonstrated that exposure of the heart to high pressure, by transverse aortic constriction (TAC), induced coronary lesions in ApoE-/- mice on chow diet. The aim of this study was to characterize the magnitude and location of coronary lesions in ApoE-/- mice after TAC and to assess the susceptibility of coronary plaque to disruption, leading to myocardial events. Here, we describe a reliable pathological condition in mice characterized by the development of coronary lesions and its progression, leading to myocardial infarction; this model better recapitulates human disease. Following TAC surgery, about 90% of ApoE-/- mice developed coronary lesions, especially in the left anterior descending artery, with 59% of the mice manifesting a different magnitude of LAD stenosis. Myocardial events, identified in 74% of the mice, were mainly due to coronary plaque thrombosis and occlusion. That TAC-induced development and progression of coronary lesions in ApoE-/- mice, leading to myocardial events, represents a potentially novel and important tool to investigate the development of coronary lesions and its sequelae in a setting that better resemble human conditions.

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.

ADAM10 controls the differentiation of the coronary arterial endothelium.

The coronary vasculature is crucial for normal heart function, yet much remains to be learned about its development, especially the maturation of coronary arterial endothelium. Here, we show that endothelial inactivation of ADAM10, a key regulator of Notch signaling, leads to defects in coronary arterial differentiation, as evidenced by dysregulated genes related to Notch signaling and arterial identity. Moreover, transcriptome analysis indicated reduced EGFR signaling in A10ΔEC coronary endothelium. Further analysis revealed that A10ΔEC mice have enlarged dysfunctional hearts with abnormal myocardial compaction, and increased expression of venous and immature endothelium markers. These findings provide the first evidence for a potential role for endothelial ADAM10 in cardioprotective homeostatic EGFR signaling and implicate ADAM10/Notch signaling in coronary arterial cell specification, which is vital for normal heart development and function. The ADAM10/Notch signaling pathway thus emerges as a potential therapeutic target for improving the regenerative capacity and maturation of the coronary vasculature.

Targeting skeletal endothelium to ameliorate bone loss.

Recent studies have identified a specialized subset of CD31hiendomucinhi (CD31hiEMCNhi) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3-/- mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

An engineered S1P chaperone attenuates hypertension and ischemic injury.

Endothelial dysfunction, a hallmark of vascular disease, is restored by plasma high-density lipoprotein (HDL). However, a generalized increase in HDL abundance is not beneficial, suggesting that specific HDL species mediate protective effects. Apolipoprotein M-containing HDL (ApoM+HDL), which carries the bioactive lipid sphingosine 1-phosphate (S1P), promotes endothelial function by activating G protein-coupled S1P receptors. Moreover, HDL-bound S1P is limiting in several inflammatory, metabolic, and vascular diseases. We report the development of a soluble carrier for S1P, ApoM-Fc, which activated S1P receptors in a sustained manner and promoted endothelial function. In contrast, ApoM-Fc did not modulate circulating lymphocyte numbers, suggesting that it specifically activated endothelial S1P receptors. ApoM-Fc administration reduced blood pressure in hypertensive mice, attenuated myocardial damage after ischemia/reperfusion injury, and reduced brain infarct volume in the middle cerebral artery occlusion model of stroke. Our proof-of-concept study suggests that selective and sustained targeting of endothelial S1P receptors by ApoM-Fc could be a viable therapeutic strategy in vascular diseases.

S1PR1 (Sphingosine-1-Phosphate Receptor 1) Signaling Regulates Blood Flow and Pressure.

Nitric oxide is one of the major endothelial-derived vasoactive factors that regulate blood pressure (BP), and the bioactive lipid mediator S1P (sphingosine-1-phosphate) is a potent activator of endothelial nitric oxide synthase through G protein-coupled receptors. Endothelial-derived S1P and the autocrine/paracrine activation of S1PR (S1P receptors) play an important role in preserving vascular functions and BP homeostasis. Furthermore, FTY720 (fingolimod), binding to 4 out of 5 S1PRs recently approved by the Food and Drug Administration to treat autoimmune conditions, induces a modest and transient decrease in heart rate in both animals and humans, suggesting that drugs targeting sphingolipid signaling affect cardiovascular functions in vivo. However, the role of specific S1P receptors in BP homeostasis remains unknown. The aim of this study is to determine the role of the key vascular S1P receptors, namely, S1PR1 and S1PR3, in BP regulation in physiological and hypertensive conditions. The specific loss of endothelial S1PR1 decreases basal and stimulated endothelial-derived nitric oxide and resets BP to a higher-than-normal value. Interestingly, we identified a novel and important role for S1PR1 signaling in flow-mediated mechanotransduction. FTY720, acting as functional antagonist of S1PR1, markedly decreases endothelial S1PR1, increases BP in control mice, and exacerbates hypertension in angiotensin II mouse model, underlining the antihypertensive functions of S1PR1 signaling. Our study identifies S1P-S1PR1-nitric oxide signaling as a new regulatory pathway in vivo of vascular relaxation to flow and BP homeostasis, providing a novel therapeutic target for the treatment of hypertension.

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.

S1P Signaling and De Novo Biosynthesis in Blood Pressure Homeostasis.

Initially discovered as abundant components of eukaryotic cell membranes, sphingolipids are now recognized as important bioactive signaling molecules that modulate a variety of cellular functions, including those relevant to cancer and immunologic, inflammatory, and cardiovascular disorders. In this review, we discuss recent advances in our understanding of the role of sphingosine-1-phosphate (S1P) receptors in the regulation of vascular function, and focus on how de novo biosynthesized sphingolipids play a role in blood pressure homeostasis. The therapeutic potential of new drugs that target S1P signaling is also discussed.