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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. This study identifies a crucial role for fatty acid oxidation (FAO) in endothelial cells during angiogenesis, and reveals that fatty-acid-derived carbons are used for the de novo synthesis of nucleotides, and hence FAO stimulates vessel sprouting by increasing endothelial cell proliferation. Fatty acids a carbon source for angiogenesis Peter Carmeliet and colleagues identify a crucial role for the oxidation of fatty acids in endothelial cells during angiogenesis. They show that fatty acids provide the carbons for the de novo synthesis of nucleotides, and hence fatty acid oxidation stimulates vessel sprouting by increasing endothelial cell proliferation. Pharmacological blockade of fatty acid oxidation can reduce pathological angiogenesis in a mouse model of retinopathy of prematurity.

Background Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. Methods This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45 dim CD34 + VEGFR-2 + and CD45 dim CD133 − CD34 + CD144 + respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill’s colonies). Results At baseline TG had lower cEPCs, PACs, CFU-Hills’ colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill’s colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill’s colonies with cECs. Conclusions Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill’s colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132

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. The enzyme glutamine synthetase is active in endothelial cell migration during angiogenesis, through autopalmitoylation and the regulation of RHOJ signalling.

Background CD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes. Methods Exosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments. Results Exosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells. Conclusions Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide 1 . Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated 2 , 3 . The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1–Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy. Under stressful conditions, mesenchymal stromal cells transfer mitochondria to endothelial cells through tunnelling nanotubes, and artificially transplanting mitochondria into endothelial cells improves the ability of these cells to engraft and to revascularize ischaemic tissues.

Persistent endothelial injury promotes maladaptive responses by favoring the release of factors leading to perturbation in vascular homeostasis and tissue architecture. Caspase-3 dependent death of microvascular endothelial cells leads to the release of unique apoptotic exosome-like vesicles (ApoExo). Here, we evaluate the impact of ApoExo on endothelial gene expression and function in the context of a pro-apoptotic stimulus. Endothelial cells exposed to ApoExo differentially express genes involved in cell death, inflammation, differentiation, and cell movement. Endothelial cells exposed to ApoExo showed inhibition of apoptosis, improved wound closure along with reduced angiogenic activity and reduced expression of endothelial markers consistent with the first phase of endothelial-to-mesenchymal transition (endoMT). ApoExo interaction with endothelial cells also led to NF-κB activation. NF-κB is known to participate in endothelial dysfunction in numerous diseases. Silencing NF-κB reversed the anti-apoptotic effect and the pro-migratory state and prevented angiostatic properties and CD31 downregulation in endothelial cells exposed to ApoExo. This study identifies vascular injury-derived extracellular vesicles (ApoExo) as novel drivers of NF-κB activation in endothelial cells and demonstrates the pivotal role of this signaling pathway in coordinating ApoExo-induced functional changes in endothelial cells. Hence, targeting ApoExo-mediated NF-κB activation in endothelial cells opens new avenues to prevent endothelial dysfunction.

The blood–retina barrier and blood–brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-β pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in themouse retina during developmentwhen delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-β, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibitingmultiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis. The formation of new blood vessels requires both polarized cell migration and coordinated control of endothelial cell contacts. Here, Cao and colleagues describe at the sub-cellular level the cytoskeletal and cell junction dynamics regulating these processes upon VEGF-induced cell elongation.

PurposeTo compare results of two ophthalmic viscosurgical devices (OVDs)—Viscoat (a dispersive OVD, Alcon) and FR-Pro (a viscous-cohesive OVD, Rayner), in phacoemulsification surgery.MethodsA prospective randomized controlled study. Patients undergoing phacoemulsification were randomly assigned to receive one of the two OVDs. Exclusion criteria were age under 40, preoperative endothelial cell count (ECC) below 1,500 cells/mm2 and an eventful surgery.The primary outcome was change in ECC from baseline to postoperative month one and month three. Secondary outcomes were the difference between ECC at postoperative month one and month three, changes in IOP and occurrence of an IOP spike ≥ 30 mmHg after surgery.ResultsThe study included 84 eyes—43 in the Viscoat group and 41 in the FR-Pro group. Mean cell density loss at month one and month three was 17.0 and 19.2%, respectively, for the Viscoat group and 18.4 and 18.8%, respectively, for the FR-Pro group, with no statistically significant difference between the groups (p = 0.772 and p = 0.671, respectively). The mean ECC difference between the month one and month three visits was 50.5 cells/mm2 and was not statistically significant (p = 0.285). One eye in each group had an IOP spike ≥ 30 mmHg, both normalized by postoperative week one.ConclusionsViscoat and FR-Pro have comparable results following phacoemulsification surgery, suggesting that while FR-Pro is not a dispersive OVD, its endothelial cell protection may be comparable to one, perhaps due to the addition of sorbitol. Furthermore, a one-month follow-up of ECC seems sufficient in such trials.

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial–mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart–irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity. Mechanisms underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin treatment during anticancer therapy remain poorly understood. Here the authors show that treatment with an antibody against the L1 cell adhesion molecule inhibits nuclear L1CAM translocation, thereby controlling vascular DNA damage and preventing cardiotoxicity.