RESUMEN
One hundred and fifty years ago, Johann Thudichum described sphingolipids as unusual "Sphinx-like" lipids from the brain. Today, we know that thousands of sphingolipid molecules mediate many essential functions in embryonic development and normal physiology. In addition, sphingolipid metabolism and signalling pathways are dysregulated in a wide range of pathologies, and therapeutic agents that target sphingolipids are now used to treat several human diseases. However, our understanding of sphingolipid regulation at cellular and organismal levels and their functions in developmental, physiological and pathological settings is rudimentary. In this Review, we discuss recent advances in sphingolipid pathways in different organelles, how secreted sphingolipid mediators modulate physiology and disease, progress in sphingolipid-targeted therapeutic and diagnostic research, and the trans-cellular sphingolipid metabolic networks between microbiota and mammals. Advances in sphingolipid biology have led to a deeper understanding of mammalian physiology and may lead to progress in the management of many diseases.
RESUMEN
Respiratory viral infections are frequent causes of acute respiratory distress syndrome (ARDS), a disabling condition with a mortality of up to 46%. The pulmonary endothelium plays an important role in the development of ARDS as well as the pathogenesis of pulmonary fibrosis; however, the therapeutic potential to modulate endothelium-dependent signaling to prevent deleterious consequences has not been well explored. Here, we used a clinically relevant influenza A virus infection model, endothelial cell-specific transgenic gain-of-function and loss-of-function mice as well as pharmacologic approaches and in vitro modeling, to define the mechanism by which S1PR1 expression is dampened during influenza virus infection and determine whether therapeutic augmentation of S1PR1 has the potential to reduce long-term postviral fibrotic complications. We found that the influenza virus-induced inflammatory milieu promoted internalization of S1PR1, which was pharmacologically inhibited with paroxetine, an inhibitor of GRK2. Moreover, genetic overexpression or administration of paroxetine days after influenza virus infection was sufficient to reduce postviral pulmonary fibrosis. Taken together, our data suggest that endothelial S1PR1 signaling provides critical protection against long-term fibrotic complications after pulmonary viral infection. These findings support the development of antifibrotic strategies that augment S1PR1 expression in virus-induced ARDS to improve long-term patient outcomes.
Asunto(s)
Infecciones por Orthomyxoviridae , Fibrosis Pulmonar , Síndrome de Dificultad Respiratoria , Animales , Humanos , Ratones , Endotelio/metabolismo , Paroxetina , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
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Artritis Reumatoide/inmunología , FN-kappa B/metabolismo , Proteína Metiltransferasas/metabolismo , Factor de Transcripción ReIA/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Ensamble y Desensamble de Cromatina/genética , Metilación de ADN , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Inflamación , Lisina/metabolismo , FN-kappa B/genética , FN-kappa B/inmunología , Unión Proteica/genética , Proteína Metiltransferasas/genética , Proteína Metiltransferasas/inmunología , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunologíaRESUMEN
BACKGROUND: The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)-an unconventional Rho family GTPase GEF in vascular function. METHODS: We generated mice deficient in DOCK4' used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells' used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation. RESULTS: Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)-induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation. CONCLUSIONS: Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1-mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.
Asunto(s)
Células Endoteliales , Proteínas de Unión al GTP rho , Uniones Adherentes/metabolismo , Animales , Permeabilidad Capilar/fisiología , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Pulmón/metabolismo , Ratones , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.
Asunto(s)
Permeabilidad Capilar , Células Endoteliales/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Bleomicina , Coagulación Sanguínea , Eliminación de Gen , Fibrosis Pulmonar Idiopática/sangre , Pulmón/irrigación sanguínea , Pulmón/patología , Lisofosfolípidos/sangre , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , RNA-Seq , Análisis de la Célula Individual , Esfingosina/análogos & derivados , Esfingosina/sangreRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule regulating organogenesis, angiogenesis, cell proliferation, and apoptosis. S1P is generated by sphingosine kinases (SPHK1 and SPHK2) through the phosphorylation of ceramide-derived sphingosine. Phenotypes caused by manipulating S1P metabolic enzymes and receptors suggested several possible functions for S1P in embryonic stem cells (ESCs), yet the mechanisms by which S1P and related sphingolipids act in ESCs are controversial. We designed a rigorous test to evaluate the requirement of S1P in murine ESCs by knocking out both Sphk1 and Sphk2 to create cells incapable of generating S1P. To accomplish this, we created lines mutant for Sphk2 and conditionally mutant (floxed) for Sphk1, allowing evaluation of ESCs that transition to double-null state. The Sphk1/2-null ESCs lack S1P and accumulate the precursor sphingosine. The double-mutant cells fail to grow due to a marked cell cycle arrest at G2/M. Mutant cells activate expression of telomere elongation factor genes Zscan4, Tcstv1, and Tcstv3 and display longer telomeric repeats. Adding exogenous S1P to the medium had no impact, but the cell cycle arrest is partially alleviated by the expression of a ceramide synthase 2, which converts excess sphingosine into ceramide. The results indicate that sphingosine kinase activity is essential in mouse ESCs for limiting the accumulation of sphingosine that otherwise drives cell cycle arrest.
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Puntos de Control del Ciclo Celular/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos adversos , Animales , Proliferación Celular , Femenino , Humanos , Ratones , Ratones NoqueadosRESUMEN
Objective- Arteriovenous fistulae (AVF) are the most common access created for hemodialysis; however, many AVF fail to mature and require repeated intervention, suggesting a need to improve AVF maturation. Eph-B4 (ephrin type-B receptor 4) is the embryonic venous determinant that is functional in adult veins and can regulate AVF maturation. Cav-1 (caveolin-1) is the major scaffolding protein of caveolae-a distinct microdomain that serves as a mechanosensor at the endothelial cell membrane. We hypothesized that Cav-1 function is critical for Eph-B4-mediated AVF maturation. Approach and Results- In a mouse aortocaval fistula model, both Cav-1 mRNA and protein were increased in the AVF compared with control veins. Cav-1 KO (knockout) mice showed increased fistula wall thickening ( P=0.0005) and outward remodeling ( P<0.0001), with increased eNOS (endothelial NO synthase) activity compared with WT (wild type) mice. Ephrin-B2/Fc inhibited AVF outward remodeling in WT mice but not in Cav-1 KO mice and was maintained in Cav-1 RC (Cav-1 endothelial reconstituted) mice (WT, P=0.0001; Cav-1 KO, P=0.7552; Cav-1 RC, P=0.0002). Cavtratin-a Cav-1 scaffolding domain peptide-decreased AVF wall thickness in WT mice and in Eph-B4 het mice compared with vehicle alone (WT, P=0.0235; Eph-B4 het, P=0.0431); cavtratin also increased AVF patency (day 42) in WT mice ( P=0.0275). Conclusions- Endothelial Cav-1 mediates Eph-B4-mediated AVF maturation. The Eph-B4-Cav-1 axis regulates adaptive remodeling during venous adaptation to the fistula environment. Manipulation of Cav-1 function may be a translational strategy to enhance AVF patency.
Asunto(s)
Derivación Arteriovenosa Quirúrgica , Caveolina 1/fisiología , Receptor EphB4/fisiología , Transducción de Señal/fisiología , Vena Cava Inferior/fisiología , Animales , Aorta Abdominal/cirugía , Caveolas/metabolismo , Caveolina 1/biosíntesis , Caveolina 1/deficiencia , Caveolina 1/genética , Caveolina 1/farmacología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Hemorreología , Humanos , Pulmón/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/fisiología , Fragmentos de Péptidos/farmacología , Remodelación Vascular/fisiología , Vena Cava Inferior/cirugíaRESUMEN
HDL-bound ApoM and albumin are protein chaperones for the circulating bioactive lipid, sphingosine 1-phosphate (S1P); in this role, they support essential extracellular S1P signaling functions in the vascular and immune systems. We previously showed that ApoM- and albumin-bound S1P exhibit differences in receptor activation and biological functions. Whether the physiological functions of S1P require chaperones is not clear. We examined ApoM-deficient, albumin-deficient, and double-KO (DKO) mice for circulatory S1P and its biological functions. In albumin-deficient mice, ApoM was upregulated, thus enabling S1P functions in embryonic development and postnatal adult life. The Apom:Alb DKO mice reproduced, were viable, and exhibited largely normal vascular and immune functions, which suggested sufficient extracellular S1P signaling. However, Apom:Alb DKO mice had reduced levels (â¼25%) of plasma S1P, suggesting that novel S1P chaperones exist to mediate S1P functions. In this study, we report the identification of ApoA4 as a novel S1P binding protein. Recombinant ApoA4 bound to S1P, activated multiple S1P receptors, and promoted vascular endothelial barrier function, all reflective of its function as a S1P chaperone in the absence of ApoM and albumin. We suggest that multiple S1P chaperones evolved to support complex and essential extracellular signaling functions of this lysolipid mediator in a redundant manner.
Asunto(s)
Apolipoproteínas A/metabolismo , Apolipoproteínas M/deficiencia , Lisofosfolípidos/metabolismo , Albúmina Sérica/deficiencia , Esfingosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Apolipoproteínas A/química , Apolipoproteínas M/genética , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Lipid droplets (LD) are dynamic organelles involved in intracellular lipid metabolism in almost all eukaryotic cells, and LD-associated proteins tightly regulate their dynamics. One LD coat protein is caveolin-1 (Cav-1), an essential component for caveola assembly in highly differentiated cells, including adipocytes, smooth muscle cells, and endothelial cells (EC). However, the role of Cav-1 in LD dynamics is unclear. Here we report that EC lacking Cav-1 exhibit impaired LD formation. The decreased LD formation is due to enhanced lipolysis and not caused by reduced triglyceride synthesis or fatty acid uptake. Mechanistically, the absence of Cav-1 increased cAMP/PKA signaling in EC, as indicated by elevated phosphorylation of hormone-sensitive lipase and increased lipolysis. Unexpectedly, we also observed enhanced autocrine production of prostaglandin I2 (PGI2, also called prostacyclin) in Cav-1 KO EC, and this PGI2 increase appeared to stimulate cAMP/PKA pathways, contributing to the enhanced lipolysis in Cav-1 KO cells. Our results reveal an unanticipated role of Cav-1 in regulating lipolysis in non-adipose tissue, indicating that Cav-1 is required for LD metabolism in EC and that it regulates cAMP-dependent lipolysis in part via the autocrine production of PGI2.
Asunto(s)
Caveolina 1/metabolismo , AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Epoprostenol/farmacología , Lipólisis/efectos de los fármacos , Animales , Western Blotting , Caveolina 1/genética , Línea Celular , Células Cultivadas , Metabolismo de los Lípidos/efectos de los fármacos , Espectrometría de Masas , Ratones , Ratones Mutantes , FosforilaciónRESUMEN
RATIONALE: Fatty acids (FA) are transported across the capillary endothelium to parenchymal tissues. However, it is not known how endothelial cells (EC) from large vessels process a postprandial surge of FA. OBJECTIVE: This study was designed to characterize lipid droplet (LD) formation in EC by manipulating pathways leading to the formation and degradation of LD. In addition, several functions of LD-derived FA were assessed. METHODS AND RESULTS: LD were present in EC lining the aorta after the peak in plasma triglycerides initiated by a gavage of olive oil in mice, in vivo. Similarly, in isolated aorta, oleic acid treatment generates LD in EC ex vivo. Cultured EC readily form LD largely via the enzyme DGAT (diacylglycerol O-acyltransferase 1) and degrade LD via ATGL (adipocyte triglyceride lipase) after FA loading. Functionally, LD-derived FA are dynamically regulated and function to protect EC from lipotoxic stress and provide FA for metabolic needs. CONCLUSIONS: Our results delineate endothelial LD dynamics for the first time in vivo and in vitro. Moreover, LD formation protects EC from lipotoxic stress, regulates EC glycolysis, and provides a source of FA for adjacent cells in the vessel wall or tissues.
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Aorta Torácica/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Gotas Lipídicas/metabolismo , Aceite de Oliva/metabolismo , Administración Oral , Animales , Células Cultivadas , Diacilglicerol O-Acetiltransferasa/metabolismo , Glucólisis , Humanos , Hidrólisis , Intubación Gastrointestinal , Lipasa/metabolismo , Lipólisis , Ratones Endogámicos C57BL , Ácido Oléico/metabolismo , Aceite de Oliva/administración & dosificación , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca(2+) levels via SOCE and therefor are less susceptible to Ca(2+) overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca(2+) homeostasis.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Mielina/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Apoptosis , Línea Celular , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Técnicas de Inactivación de Genes , Homeostasis , Ratones , Proteínas de la Mielina/deficiencia , Proteínas de la Mielina/genética , Receptor Nogo 1 , Proteína ORAI1 , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Molécula de Interacción Estromal 1RESUMEN
Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis.
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Dolicoles/biosíntesis , Receptores de Superficie Celular/fisiología , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/deficiencia , Transferasas Alquil y Aril/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Chlorocebus aethiops , Fosfatos de Dolicol/biosíntesis , Fosfatos de Dolicol/deficiencia , Dolicoles/deficiencia , Activación Enzimática/genética , Glicoproteínas/metabolismo , Humanos , Conformación Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/deficiencia , Proteínas de Transporte VesicularRESUMEN
Cerebral venous sinus thrombosis (CVST) is an exceedingly rare complication of epidural anesthesia, with only a handful of known cases after epidural steroid injection (ESI). We report a case of CVST in a 33-year-old male patient that presented with headache after lumbar ESI. His clinical status initially improved on anticoagulation in the intensive care unit. However, he had a sudden worsening of cerebral edema that required an emergent hemicraniectomy. Ultimately, the patient was pronounced dead by neurologic criteria. This case highlights the importance of keeping this rare but potentially fatal diagnosis in the differential even in lower-risk patient populations.
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Trombosis de los Senos Intracraneales , Esteroides , Humanos , Masculino , Adulto , Trombosis de los Senos Intracraneales/tratamiento farmacológico , Trombosis de los Senos Intracraneales/inducido químicamente , Inyecciones Epidurales/efectos adversos , Esteroides/administración & dosificación , Esteroides/uso terapéutico , Resultado Fatal , Anticoagulantes/efectos adversos , Anticoagulantes/administración & dosificación , Anticoagulantes/uso terapéuticoRESUMEN
High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.
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Apolipoproteínas , Lipocalinas , Humanos , Ratones , Animales , Apolipoproteínas/metabolismo , Apolipoproteínas/farmacología , Lipocalinas/metabolismo , Lipocalinas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Apolipoproteínas M , Inflamación , Lipoproteínas HDL/farmacología , Lipoproteínas HDL/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismo , EsfingosinaRESUMEN
In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.
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Proteína ADAM10 , Antígenos CD , Artritis Experimental , Artritis Reumatoide , Cadherinas , Células Endoteliales , Receptores de Esfingosina-1-Fosfato , Animales , Cadherinas/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Ratones , Artritis Experimental/metabolismo , Artritis Experimental/patología , Antígenos CD/metabolismo , Antígenos CD/genética , Células Endoteliales/metabolismo , Humanos , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Artritis Reumatoide/genética , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Transducción de Señal , Ratones Noqueados , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Masculino , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolípidos/metabolismo , Permeabilidad Capilar , FemeninoRESUMEN
Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelial-dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.
Asunto(s)
Aterosclerosis , Lipasa , Ratones , Animales , Triglicéridos/metabolismo , Lipasa/genética , Lipasa/metabolismo , Lipólisis , Metabolismo de los Lípidos , Endotelio Vascular/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P), the circulating HDL-bound lipid mediator that acts via S1P receptors (S1PR), is required for normal vascular development. The role of this signaling axis in vascular retinopathies is unclear. Here, we show in a mouse model of oxygen-induced retinopathy (OIR) that endothelial overexpression of S1pr1 suppresses while endothelial knockout of S1pr1 worsens neovascular tuft formation. Furthermore, neovascular tufts are increased in Apom-/- mice which lack HDL-bound S1P while they are suppressed in ApomTG mice which have more circulating HDL-S1P. These results suggest that circulating HDL-S1P activation of endothelial S1PR1 suppresses neovascular pathology in OIR. Additionally, systemic administration of ApoM-Fc-bound S1P or a small-molecule Gi-biased S1PR1 agonist suppressed neovascular tuft formation. Circulating HDL-S1P activation of endothelial S1PR1 may be a key protective mechanism to guard against neovascular retinopathies that occur not only in premature infants but also in diabetic patients and aging people.
Asunto(s)
Neovascularización Retiniana , Ratones , Animales , Receptores de Esfingosina-1-Fosfato , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/agonistas , Lipoproteínas HDL , Esfingosina , LisofosfolípidosRESUMEN
Serine palmitoyl transferase (SPT), the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL), is needed for embryonic development, physiological homeostasis, and response to stress. The functions of de novo SL synthesis in vascular endothelial cells (EC), which line the entire circulatory system, are not well understood. Here, we show that the de novo SL synthesis in EC not only regulates vascular development but also maintains circulatory and peripheral organ SL levels. Mice with an endothelial-specific gene knockout of SPTLC1 (Sptlc1 ECKO), an essential subunit of the SPT complex, exhibited reduced EC proliferation and tip/stalk cell differentiation, resulting in delayed retinal vascular development. In addition, Sptlc1 ECKO mice had reduced retinal neovascularization in the oxygen-induced retinopathy model. Mechanistic studies suggest that EC SL produced from the de novo pathway are needed for lipid raft formation and efficient VEGF signaling. Post-natal deletion of the EC Sptlc1 also showed rapid reduction of several SL metabolites in plasma, red blood cells, and peripheral organs (lung and liver) but not in the retina, part of the central nervous system (CNS). In the liver, EC de novo SL synthesis was important for acetaminophen-induced rapid ceramide elevation and hepatotoxicity. These results suggest that EC-derived SL metabolites are in constant flux between the vasculature, circulatory elements, and parenchymal cells of non-CNS organs. Taken together, our data point to the central role of the endothelial SL biosynthesis in maintaining vascular development, neovascular proliferation, non-CNS tissue metabolic homeostasis, and hepatocyte response to stress.
Asunto(s)
Serina C-Palmitoiltransferasa , Esfingolípidos , Animales , Ratones , Acetaminofén , Ceramidas , Células Endoteliales/metabolismo , Homeostasis , Oxígeno , Serina , Serina C-Palmitoiltransferasa/genética , Esfingolípidos/metabolismo , Factor A de Crecimiento Endotelial VascularRESUMEN
The neurogenic areas of the brain are highly organized structures in which there is dynamic reciprocal modulation of neural stem cells (NSC) and microvascular endothelial cells (BEC) resulting in control of neural stem cell and vascular proliferation, survival and differentiation throughout the life of the individual. Select molecules such as GSK-3ß, functioning as signaling nodes, and their downstream signaling components including HIF-1α, HIF-2α and ß-catenin participate in regulating and orchestrating the diverse responses involved in this complex process. In this report we demonstrate GSK-3ß's role as a signaling node in two mouse strains (C57BL/6, which have been found to respond to and recover from a hypoxic insult from P3 to P11 poorly and CD-1, which have been found to respond to and recover from a hypoxic insult from P3 to P11 well both in vivo and in vitro) which mimic the wide range of responsiveness to hypoxic insult observed in the very low birth weight premature infant population. Differences in levels of neural stem cell and microvascular endothelial cell GSK-3ß activation, ß-catenin serine phosphorylation, HIF-1α and 2α, BDNF, SDF-1 and VEGF, ß-III-tubulin and cleaved notch-1 expression in C57BL/6 and CD-1 subventricular zone tissues, and cultured NSC and BEC were noted. Specifically, CD1 pups, SVZ tissues and isolated NSC and BEC exhibit less GSK-3ß and ß-catenin serine phoslphorylation and greater HIF-1α and 2α, BDNF, SDF-1 and VEGF, ß-III-tubulin and cleaved notch-1 expression compared to C57BL/6. Correlating with these changes were differences of several neural stem cell and microvascular endothelial cell behaviors including proliferation, apoptosis, migration and differentiation with CD1 NSC exhibiting greater proliferation and migration and decreased apoptosis and differentiation and CD1 BEC exhibiting greater angiogenesis. Further, upon treatment with nanomolar concentrations of a GSK-3ß inhibitor (SB412682), C57 NSC and BEC behaviors could be brought to CD1 levels, consistent with the concept of GSK-3ß functioning as a multifunctional signaling pathway node, modulating several behaviors in these cells. Lastly, the therapeutic potential of targeting GSK-3ß is discussed.
Asunto(s)
Comunicación Celular , Células Endoteliales/citología , Células Endoteliales/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/enzimología , Transducción de Señal , Aminofenoles/farmacología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/citología , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Maleimidas/farmacología , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Receptor Cross-Talk/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Solubilidad/efectos de los fármacos , Especificidad de la Especie , beta Catenina/metabolismoRESUMEN
Acute lung injury (ALI) causes high mortality and lacks any pharmacological intervention. Here, we found that pazopanib ameliorated ALI manifestations and reduced mortality in mouse ALI models and reduced edema in human lung transplantation recipients. Pazopanib inhibits mitogen-activated protein kinase kinase kinase 2 (MAP3K2)- and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208 to increase reactive oxygen species (ROS) formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic mutation of p47phox Ser208 to alanine attenuated ALI manifestations and abrogates anti-ALI effects of pazopanib. This myeloid MAP3K2/MAP3K3-p47phox pathway acted via paracrine H2O2 to enhance pulmonary vasculature integrity and promote lung epithelial cell survival and proliferation, leading to increased pulmonary barrier function and resistance to ALI. Thus, pazopanib has the potential to be effective for treating ALI.