The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes ...vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.
RATIONALE:Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to ...define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood.
OBJECTIVE:To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells.
METHODS AND RESULTS:LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO.
CONCLUSIONS:Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible ...glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
RATIONALE:The mosquito-borne Zika virus (ZIKV) is now recognized as a blood-borne pathogen, raising an important question about how the virus gets into human bloodstream. The imminent threat of the ...ZIKV epidemic to the global blood supply also demands novel therapeutics to stop virus transmission though transfusion.
OBJECTIVE:We intend to characterize ZIKV tropism for human endothelial cells (ECs) and provide potential targets for intervention.
METHODS AND RESULTS:We conducted immunostaining, plaque assay, and quantitative reverse transcription-polymerase chain reaction of ZIKV RNA to evaluate the possible infection of ECs by ZIKV. Both the African and the South American ZIKV strains readily infect human umbilical vein endothelial cells and human ECs derived from aortic and coronary artery, as well as the saphenous vein. Infected ECs released infectious progeny virus. Compared with the African strains, South American ZIKV isolates replicate faster in ECs and are partially cytopathic, suggesting enhanced virulence of these isolates. Flow cytometric analyses showed that the susceptibility of ECs positively correlated with the cell surface levels of tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase. Gain- and loss-of-function studies further revealed that AXL is required for ZIKV entry at a postbinding step. Finally, small-molecule inhibitors of the AXL kinase significantly reduced ZIKA infection of ECs.
CONCLUSIONS:We identified EC as a key cell type for ZIKV infection. These data support the view of hematogenous dissemination of ZIKV and implicate AXL as a new target for antiviral therapy.
Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or ...inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine‐derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine‐deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.
Synopsis
Vessel sprouting during angiogenesis is highly dependent on endothelial cell glutamine metabolism, which serves multiple metabolic functions but is especially interlinked with asparagine synthesis.
Glutamine is required for vessel sprouting in vitro and in vivo.
Glutamine mediates multiple metabolic functions in endothelial cells (ECs).
Asparagine and tricarboxylic acid cycle anaplerosis are both required for glutamine‐restricted ECs.
Glutamine‐dependent asparagine synthesis is indispensable for EC growth.
Vessel sprouting during angiogenesis is highly dependent on endothelial cell glutamine metabolism, which serves multiple metabolic functions but is especially interlinked with asparagine synthesis.
OBJECTIVE—Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal ...anti–human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups.
APPROACH AND RESULTS—In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)–derived αvβ3. By absorption experiments, we subsequently identified anti–HPA-1a antibodies of anti-αvβ3 specificity in the +ICH but not in the −ICH cohort. Only the anti–αvβ3 subtype, but not the anti–β3 subtype, induced EC apoptosis of HPA-1a–positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti–αvβ3 subtype, but not the anti-β3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation.
CONCLUSIONS—We conclude that the composition of the anti–HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti–HPA-1a antibodies of the anti–αvβ3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia.
Increased aerobic glycolysis in endothelial cells of atheroprone areas of blood vessels has been hypothesized to drive increased inflammation and lesion burden but direct links remain to be ...established. Here we show that endothelial cells exposed to disturbed flow in vivo and in vitro exhibit increased levels of protein kinase AMP-activated (PRKA)/AMP-activated protein kinases (AMPKs). Selective deletion of endothelial Prkaa1, coding for protein kinase AMP-activated catalytic subunit alpha1, reduces glycolysis, compromises endothelial cell proliferation, and accelerates the formation of atherosclerotic lesions in hyperlipidemic mice. Rescue of the impaired glycolysis in Prkaa1-deficient endothelial cells through Slc2a1 overexpression enhances endothelial cell viability and integrity of the endothelial cell barrier, and reverses susceptibility to atherosclerosis. In human endothelial cells, PRKAA1 is upregulated by disturbed flow, and silencing PRKAA1 reduces glycolysis and endothelial viability. Collectively, these results suggest that increased glycolysis in the endothelium of atheroprone arteries is a protective mechanism.
Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus ...disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients.
Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.
Although the mechanisms linking cardiopulmonary diseases to ambient fine particles (PM2.5) are still unclear, inflammation and oxidative stress play important roles in PM2.5-induced injury. It is ...well known that inflammation and oxidative stress could be restricted by vitamin E (Ve) or omega-3 fatty acids (Ω-3 FA) consumption. This study investigated the effects of Ve and Ω-3 FA on PM2.5-induced inflammation and oxidative stress in vascular endothelial cells. The underlying mechanisms linking PM2.5 to vascular endothelial injury were also explored. Human umbilical vein endothelial cells (HUVECs) were treated with 50 μg/mL PM2.5 in the presence or absence of different concentrations of Ve and Ω-3 FA. The inflammatory cytokines and oxidative stress markers were determined. The results showed that Ve induced a significant decrease in PM2.5-induced inflammation and oxidative stress. Malondialdehyde (MDA) in supernatant and reactive oxygen species (ROS) in cytoplasm decreased by Ve, while the superoxide dismutase (SOD) activity elevated. The inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) also reduced by Ve. Moreover, Ω-3 FA played the same role on decreasing the inflammation and oxidative stress. IL-6 and TNF-α expressions were significantly lower in combined Ve with Ω-3 FA than treatment with Ve or Ω-3 FA alone. The Ve and Ω-3 FA intervention might abolish the PM2.5-induced oxidative stress and inflammation in vascular endothelial cells. There might be an additive effect of these two nutrients in mediating the PM2.5-induced injury in vascular endothelial cells. The results suggested that inflammation and oxidative stress might be parts of the mechanisms linking PM2.5 to vascular endothelial injury.
Blood and lymphatic vessels pervade almost all body tissues and have numerous essential roles in physiology and disease. The inner lining of these networks is formed by a single layer of endothelial ...cells, which is specialized according to the needs of the tissue that it supplies. Whereas the general mechanisms of blood and lymphatic vessel development are being defined with increasing molecular precision, studies of the processes of endothelial specialization remain mostly descriptive. Recent insights from genetic animal models illuminate how endothelial cells interact with each other and with their tissue environment, providing paradigms for vessel type- and organ-specific endothelial differentiation. Delineating these governing principles will be crucial for understanding how tissues develop and maintain, and how their function becomes abnormal in disease.
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