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Autophagy is a highly conserved cellular recycling process which enables eukaryotes to maintain both cellular and overall homeostasis through the catabolic breakdown of intracellular components or the selective degradation of damaged organelles. In recent years, the importance of autophagy in vascular endothelial cells (ECs) has been increasingly recognized, and numerous studies have linked the dysregulation of autophagy to the development of endothelial dysfunction and vascular disease. Here, we provide an overview of the molecular mechanisms underlying autophagy in ECs and our current understanding of the roles of autophagy in vascular biology and review the implications of dysregulated autophagy for vascular disease. Finally, we summarize the current state of the research on compounds to modulate autophagy in ECs and identify challenges for their translation into clinical use.

Arterial-venous malformations (AVMs) are direct connections between arteries and veins without an intervening capillary bed. Either familial inherited or sporadically occurring, localized pericytes (PCs) drop is among the AVMs' hallmarks. Whether impaired PC coverage triggers AVMs or it is a secondary event is unclear. Here we evaluated the role of the master regulator of PC recruitment, Platelet derived growth factor B (PDGFB) in AVM pathogenesis. Using tamoxifen-inducible deletion of Pdgfb in endothelial cells (ECs), we show that disruption of EC Pdgfb-mediated PC recruitment and maintenance leads to capillary enlargement and organotypic AVM-like structures. These vascular lesions contain non-proliferative hyperplastic, hypertrophic and miss-oriented capillary ECs with an altered capillary EC fate identity. Mechanistically, we propose that PDGFB maintains capillary EC size and caliber to limit hemodynamic changes, thus restricting expression of Krüppel like factor 4 and activation of Bone morphogenic protein, Transforming growth factor β and NOTCH signaling in ECs. Furthermore, our study emphasizes that inducing or activating PDGFB signaling may be a viable therapeutic approach for treating vascular malformations., (© 2023. The Author(s).)

Circulating endothelial cells (CEC) are arising as biomarkers for vascular diseases. However, whether they can be utilized as markers of endothelial cell (EC) senescence in vivo remains unknown. Here, we present a protocol to isolate circulating endothelial cells for a characterization of their senescent signature. Further, we characterize different models of EC senescence induction in vitro and show similar patterns of senescence being upregulated in CECs of aged patients as compared to young volunteers. Replication-(ageing), etoposide-(DNA damage) and angiotensin II-(ROS) induced senescence models showed the expected cell morphology and proliferation-reduction effects. Expression of senescence-associated secretory phenotype markers was specifically upregulated in replication-induced EC senescence. All models showed reduced telomere lengths and induction of the INK4a/ARF locus. Additional p14ARF-p21 pathway activation was observed in replication- and etoposide-induced EC senescence. Next, we established a combined magnetic activated- and fluorescence activated cell sorting (MACS-FACS) based protocol for CEC isolation. Interestingly, CECs isolated from aged volunteers showed similar senescence marker patterns as replication- and etoposide-induced senescence models. Here, we provide first proof of senescence in human blood derived circulating endothelial cells. These results hint towards an exciting future of using CECs as mirror cells for in vivo endothelial cell senescence, of particular interest in the context of endothelial dysfunction and cardiovascular diseases., (© 2024. The Author(s).)

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-β signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-β signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-β-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-β-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-β receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-β signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Magnesium deficiency is associated with a greater risk of developing cardiovascular diseases since this cation is fundamental in regulating vascular function. This clinical evidence is sustained by in vitro studies showing that culturing endothelial cells in low concentrations of magnesium promotes the acquisition of a pro-oxidant and pro-inflammatory phenotype. Here, we show that the increase in reactive oxygen species in endothelial cells in low-magnesium-containing medium is due to the upregulation of the pro-oxidant protein thioredoxin interacting protein (TXNIP), with a consequent accumulation of lipid droplets and increase in endothelial permeability through the downregulation and relocalization of junctional proteins. Silencing TXNIP restores the endothelial barrier and lipid content. Because (i) mitochondria serve multiple roles in shaping cell function, health and survival and (ii) mitochondria are the main intracellular stores of magnesium, it is of note that no significant alterations were detected in their morphology and dynamics in our experimental model. We conclude that TXNIP upregulation contributes to low-magnesium-induced endothelial dysfunction in vitro.

A key aspect of cytokine-induced changes as observed in sepsis is the dysregulated activation of endothelial cells (ECs), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. The therapeutic targeting of ECs has been hampered by concerns regarding organ-specific EC heterogeneity and their response to inflammation. Using in vitro and in silico analysis, we present a comprehensive analysis of the proteomic changes in mouse lung, liver and kidney ECs following exposure to a clinically relevant cocktail of proinflammatory cytokines. Mouse lung, liver and kidney ECs were incubated with TNF-α/IL-1β/IFN-γ for 4 or 24 h to model the cytokine-induced changes. Quantitative label-free global proteomics and bioinformatic analysis performed on the ECs provide a molecular framework for the EC response to inflammatory stimuli over time and organ-specific differences. Gene Ontology and PANTHER analysis suggest why some organs are more susceptible to inflammation early on, and show that, as inflammation progresses, some protein expression patterns become more uniform while additional organ-specific proteins are expressed. These findings provide an in-depth understanding of the molecular changes involved in the EC response to inflammation and can support the development of drugs targeting ECs within different organs. Data are available via ProteomeXchange (identifier PXD031804).

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45- cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

Endothelial cell senescence is involved in endothelial dysfunction and vascular diseases. However, the detailed mechanisms of endothelial senescence are not fully understood. Here, we demonstrated that deficiency of developmentally regulated GTP-binding protein 2 ( DRG2 ) induces senescence and dysfunction of endothelial cells. DRG2 knockout (KO) mice displayed reduced cerebral blood flow in the brain and lung blood vessel density. We also determined, by Matrigel plug assay, aorta ring assay, and in vitro tubule formation of primary lung endothelial cells, that deficiency in DRG2 reduced the angiogenic capability of endothelial cells. Endothelial cells from DRG2 KO mice showed a senescence phenotype with decreased cell growth and enhanced levels of p21 and phosphorylated p53, γH2AX, senescence-associated β-galactosidase (SA-β-gal) activity, and senescence-associated secretory phenotype (SASP) cytokines. DRG2 deficiency in endothelial cells upregulated arginase 2 ( Arg2 ) and generation of reactive oxygen species. Induction of SA-β-gal activity was prevented by the antioxidant N-acetyl cysteine in endothelial cells from DRG2 KO mice. In conclusion, our results suggest that DRG2 is a key regulator of endothelial senescence, and its downregulation is probably involved in vascular dysfunction and diseases.

Type 2 diabetes (T2D)-related neurological complication is the risk factor for neurodegenerative disorders. The pathological changes from T2D-caused blood-brain barrier (BBB) dysfunction plays a critical role in developing neurodegeneration. The hyper-activation of the Angiotensin II type 1 receptor (AT1R) in the brain is associated with neurovascular impairment. The AT1R antagonist Valsartan is commonly prescribed to control high blood pressure, heart failure, and diabetic kidney diseases. In this study, we investigated the beneficial effects of Valsartan in db/db diabetic mice and isolated brain endothelial cells. We showed that 2 weeks of Valsartan administration (30 mg/Kg body weight) mitigated the increased permeability of the brain-blood barrier and the reduction of gap junction proteins VE-Cadherin and Claudin 2. In human brain microvascular cells (HBMVECs), we found that Valsartan treatment ameliorated high glucose-induced hyperpermeability by measuring Dextran uptake and transendothelial electrical resistance (TEER). Furthermore, Valsartan treatment recovered high glucose-repressed endothelial VE-Cadherin and Claudin 2 expression. Moreover, Valsartan significantly suppressed the expressions of pro-inflammatory cytokines such as macrophage chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) against high glucose. Mechanistically, Valsartan ameliorated high glucose-repressed endothelial cAMP-responsive element-binding protein (CREB) signaling activation. The blockage of CREB activation by PKA inhibitor H89 abolished the action of Valsartan, suggesting its dependence on CREB signaling. In conclusion, Valsartan shows a neuroprotective effect in diabetic mice by ameliorating BBB dysfunction. These effects of Valsartan require cellular CREB signaling in brain endothelial cells.

Aims: Previous studies have shown the intake of omega-3 polyunsaturated fatty acids is associated with low rates of obesity and ischaemic pathologies. Omega-3 also have anti-inflammatory and plaque-stabilization effects and regulate vasodilation and constriction. However, there are few studies of the role of omega-3 in flow-induced vasodilation involving Ca2+-permeable ion channel TRPV4 in high-fat diet-induced obese (DIO) mouse. Here, we determined whether omega-3 protect against vascular dysfunction induced by a high-fat diet by enhancing TRPV4 activity and subsequently improving flow-mediated vasodilation., Methods and Results: Flow-mediated vasodilation in second-order mesenteric arteries from mice was measured using a pressure myograph. The intracellular Ca2+ concentration in response to flow and GSK1016790A (a TRPV4 agonist) was measured by Fluo-4 fluorescence. Whole-cell current was measured by patch clamp. Cell membrane tether force was measured by atomic force microscopy. Impairment of flow-mediated vasodilation in arteries and Ca2+ influx in endothelial cells from DIO mice was restored by omega-3 treatment. The improved flow-induced vasodilation was inhibited by the TRPV4 antagonist HC067047 and in TRPV4-/- mice. Omega-3 treatment enhanced endothelial TRPV4 activity and altered cell membrane mechanic property, as indicated by enhanced GSK1016790A-induced Ca2+ influx and whole-cell current and altered membrane mean tether force in endothelial cells from DIO mice., Conclusion: Omega-3 improve vascular function by improving flow-induced vasodilation via enhancing TRPV4 activity in the endothelium of obese mice which may be related to improved cell membrane physical property. Activation of TRPV4 in endothelium plays an important role in the protective mechanisms of omega-3 against vascular dysfunction in obesity by improving flow-mediated vasodilation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2/angiopoietin axis. Primary HUVECs treated with plasma from patients with severe COVID-19 upregulated the expression of thromboinflammatory genes, inhibited the expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from patients with COVID-19 demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity, and the highest levels were associated with worse survival. These data highlight the disruption of Tie2/angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.

Purpose of Review: Endothelial cell (EC) front-rear (axial) polarization in response to chemokines and shear stress is fundamental for angiogenesis. This review provides an overview of the in vitro and in vivo methods that are currently available to quantify EC axial polarity., Recent Findings: Innovative methodologies and new animal models have been developed to evaluate EC axial polarity. Micropatterning, wound healing and microfluidic assays allow interrogation of signalling mechanisms in vitro. Mouse and zebrafish transgenic lines, in combination with advances in imaging techniques and computational tools, enable interrogation of physiological functions of EC axial polarity in vascular biology during development and in pathology in vivo., Summary: We present a literature-based review of the methods available to study EC polarity. Further refinement of quantitative methods to analyse EC axial polarity using deep learning-based computational tools will generate new understanding on the aetiology of vascular malformations., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Aims: Endothelial microparticles (EMPs) are extracellular vesicles secreted by endothelial cells. The purpose of this research is to explore that the clinical significance and roles in angiogenesis and endothelial dysfunction of circulating microparticles in Perthes disease., Main Methods: We collected platelet-poor plasma (PPP) from patients and controls, then microparticles (MPs) were extracted. Flow cytometry was performed to calculate the concentrations of CD31+/CD42b-, CD62E+ and CD31+/CD42b+ MPs. ELISA was performed to detect the expression level of biomarkers of endothelial dysfunction and inflammatory factors in plasma. In vitro experiments to evaluate the effect of circulating MPs and EMPs derived from IL-6-stimulated human umbilical vein endothelial cells (HUVECs) on angiogenesis and endothelial dysfunction., Key Findings: Our results revealed that the CD31+/CD42b- EMPs were significantly higher in Perthes disease group than in the control group. The Perthes-MPs being taken up by HUVECs promoted endothelial cell apoptosis, endothelial dysfunction and inhibited angiogenesis in vitro. Moreover, the level of IL-6 in plasma significantly increased in patients with Perthes, which was tightly correlated with the elevated level of circulating CD31+/CD42b- EMPs. IL-6 promoted HUVECs to secrete CD31+/CD42b- MPs, and EMPs derived from high concentration IL-6-stimulated (100 and 1000 pg/mL) HUVECs promoted endothelial cell apoptosis, endothelial dysfunction and inhibited angiogenesis., Significance: In summary, our study suggests that circulating EMPs in the phenotypic spectrum revealed unique phenotypes of endothelial dysfunction, showing close correlation with the secretion of IL-6. These circulating EMPs may give rise to endothelial cell apoptosis, endothelial dysfunction and angiogenesis in Perthes disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Calciprotein particles (CPPs), which increasingly arise in the circulation during the disorders of mineral homeostasis, represent a double-edged sword protecting the human organism from extraskeletal calcification but potentially causing endothelial dysfunction. Existing models, however, failed to demonstrate the detrimental action of CPPs on endothelial cells (ECs) under flow. Here, we applied a flow culture system, where human arterial ECs were co-incubated with CPPs for 4 h, and a normolipidemic and normotensive rat model (10 daily intravenous injections of CPPs) to simulate the scenario occurring in vivo in the absence of confounding cardiovascular risk factors. Pathogenic effects of CPPs were investigated by RT-qPCR and Western blotting profiling of the endothelial lysate. CPPs were internalised within 1 h of circulation, inducing adhesion of peripheral blood mononuclear cells to ECs. Molecular profiling revealed that CPPs stimulated the expression of pro-inflammatory cell adhesion molecules VCAM1 and ICAM1 and upregulated transcription factors of endothelial-to-mesenchymal transition (Snail, Slug and Twist1). Furthermore, exposure to CPPs reduced the production of atheroprotective transcription factors KLF2 and KLF4 and led to YAP1 hypophosphorylation, potentially disturbing the mechanisms responsible for the proper endothelial mechanotransduction. Taken together, our results suggest the ability of CPPs to initiate endothelial dysfunction at physiological flow conditions.

Dihydromyricetin (DHY), a flavonoid component isolated from Ampelopsis grossedentata, exerts versatile pharmacological activities. However, the possible effects of DHY on diabetic vascular endothelial dysfunction have not yet been fully elucidated. In the present study, male C57BL/6 mice, wild type (WT) 129S1/SvImJ mice and sirtuin 3 (SIRT3) knockout (SIRT3 -/- ) mice were injected with streptozotocin (STZ, 60 mg/kg/day) for 5 consecutive days. Two weeks later, DHY were given at the doses of 250 mg/kg by gavage once daily for 12 weeks. Fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) level, endothelium-dependent relaxation of thoracic aorta, reactive oxygen species (ROS) production, SIRT3, and superoxide dismutase 2 (SOD2) protein expressions, as well as mitochondrial Deoxyribonucleic Acid (mtDNA) copy number, in thoracic aorta were detected. Our study found that DHY treatment decreased FBG and HbA1c level, improved endothelium-dependent relaxation of thoracic aorta, inhibited oxidative stress and ROS production, and enhanced SIRT3 and SOD2 protein expression, as well as mtDNA copy number, in thoracic aorta of diabetic mice. However, above protective effects of DHY were unavailable in SIRT3 -/- mice. The study suggested DHY improved endothelial dysfunction in diabetic mice via oxidative stress inhibition in a SIRT3-dependent manner.

Gut microbiota have been emerging as important contributors to the regulation of host homeostasis. Accordingly, several substances converted by gut microbiota can have beneficial or adverse effects on human health. Among them, S-equol, which is produced from the isoflavone daidzein in the human and animal gut by certain microbiota, exerts estrogenic and antioxidant activities. Indoxyl sulfate, which is metabolized in the liver from indole converted from dietary tryptophan by bacterial tryptophanases in the colon, is known as a protein-bound uremic toxin. Trimethylamine N-oxide, which is generated via the oxidization of gut microbiota-derived trimethylamine by hepatic flavin monooxygenases, is known as an accelerator of atherosclerosis. The aforementioned gut-derived substances could be potential regulators of systematic tissue/organ function, including the vascular system. Macro- and microvascular complications of cardiovascular and metabolic diseases, including atherosclerosis, hypertension, and diabetes, occur systemically and represent the principal cause of morbidity and mortality. Vascular endothelial and smooth muscle dysfunction play pivotal roles in the development and progression of vasculopathies. We herein review the link between the aforementioned gut-derived substances and endothelial and vascular smooth muscle cell function. This information will provide a conceptual framework that would allow the development of novel preventive and/or therapeutic approaches against vasculopathies., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd.)

The phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), has long been established as a major contributor to intracellular signaling, primarily by virtue of its role as a substrate for phospholipase C (PLC). Signaling by G q -protein-coupled receptors triggers PLC-mediated hydrolysis of PIP 2 into inositol 1,4,5-trisphosphate and diacylglycerol, which are well known to modulate vascular ion channel activity. Often overlooked, however, is the role PIP 2 itself plays in this regulation. Although numerous reports have demonstrated that PIP 2 is critical for ion channel regulation, how it impacts vascular function has received scant attention. In this review, we focus on PIP 2 as a regulator of ion channels in smooth muscle cells and endothelial cells-the two major classes of vascular cells. We further address the concerted effects of such regulation on vascular function and blood flow control. We close with a consideration of current knowledge regarding disruption of PIP 2 regulation of vascular ion channels in disease., Competing Interests: The authors declare no competing interest.

Chronic unresolved inflammation is the primary determinant of cardiovascular disease. Precise mechanisms that define the genesis of unresolved inflammation in heart failure with preserved ejection fraction (HFpEF) are of interest due to the obesity epidemic. To examine the obesity phenotype and its direct/indirect consequences, multiple approaches were employed using the lipoxin receptor (abbreviated as ALX) dysfunction mouse model. Indirect calorimetry analyses revealed that the deletion of ALX dysregulated energy metabolism driving toward age-related obesity. Heart function data suggest that obesity-prone ALX deficient mice had impaired myocardium strain. Comprehensive measurement of chemokines, extracellular matrix, and arrhythmogenic arrays confirmed the dysregulation of multiple ion channels gene expression with amplified inflammatory chemokines and cytokines response at the age of 4 months compared with WT counterparts. Quantitative analyses of leukocytes demonstrated an increase of proinflammatory Ly6C hi CCR2 + macrophages in the spleen and heart at a steady-state resulting in an inflamed splenocardiac axis. Signs of subtle inflammation were marked with cardiorenal, endothelial defects with decreased CD31 and eNOS and an increased iNOS and COX2 expression. Thus, ALX receptor deficiency serves as an experimental model that defines multiple cellular and molecular mechanisms in HFpEF that could be a target for the development of HFpEF therapy in cardiovascular medicine., (© 2020 Federation of American Societies for Experimental Biology.)

Protein bound uremic toxins, such as p-cresol, cannot be effectively removed by conventional dialysis techniques and are accumulated in plasma, thus contributing to progression of both chronic kidney disease (CKD) and cardiovascular disease (CVD). Pathological effects of uremic toxins include activation of inflammatory response, endothelial dysfunction and release of endothelial microvesicles. To date, the role of p-cresol in endothelial microvesicles formation has not been analyzed. The aim of the present study was evaluate the effects of endothelial microvesicles released by p-cresol (PcEMV) on endothelial dysfunction. An in vitro model of endothelial damage mediated by p-cresol was proposed to evaluate the functional effect of PcEMV on the endothelial repair process carried out by endothelial cells and microRNA (miRNA) that could be involved in this process. We observed that p-cresol induced a greater release of microvesicles in endothelial cells. These microvesicles altered regenerative capacity of endothelial cells, decreasing their capacity for cell migration and their potential to form vascular structures in vitro. Moreover, we observed increased cellular senescence and a deregulation of miRNA-146b-5p and miRNA-223-3p expression in endothelial cells treated with endothelial microvesicles released by p-cresol. In summary our data show that microvesicles generated in endothelial cells treated with p-cresol (PcEMV) interfere with the endothelial repair process by decreasing the migratory capacity, the ability to form new vessels and increasing the senescence of mature endothelial cells. These alterations could be mediated by the upregulation of miRNA-146b-5p and miRNA-223-3p.

Vascular diseases, such as myocardial infarction and cerebral infarction, are most commonly caused by atherosclerosis, one of the leading causes of death worldwide. Risk factors for atherosclerosis include lifestyle and aging. It has been reported that lifespan could be extended in mice by targeting senescent cells, which led to the suppression of aging-related diseases, such as vascular diseases. However, the molecular mechanisms underlying the contribution of aging to vascular diseases are still not well understood. Several types of cells, such as vascular (endothelial cell), vascular-associated (smooth muscle cell and fibroblast) and inflammatory cells, are involved in plaque formation, plaque rupture and thrombus formation, which result in atherosclerosis. Gangliosides, a group of glycosphingolipids, are expressed on the surface of vascular, vascular-associated and inflammatory cells, where they play functional roles. Clarifying the role of gangliosides in atherosclerosis and their relationship with aging is fundamental to develop novel prevention and treatment methods for vascular diseases based on targeting gangliosides. In this review, we highlight the involvement and possible contribution of gangliosides to vascular diseases and further discuss their relationship with aging.

Endothelial-to-mesenchymal transition (EndoMT) is a process of transdifferentiation where endothelial cells gradually adopt the phenotypic characteristics of mesenchymal cells. This phenomenon was first discovered in embryonic heart development. The mechanisms underlying EndoMT are due to the activation of transforming growth factor-β, bone morphogenetic protein, Wingless/Integrated, or Notch signaling pathways. The EndoMT can be modulated by pathological processes, including inflammation, disturbed shear stress, vascular stiffness, and metabolic dysregulation. Recent studies have shown that EndoMT is implicated in the pathogenesis of chronic lung diseases, including pulmonary hypertension and lung fibrosis. Lung pathology of bronchopulmonary dysplasia can be mimicked in rodents exposed to hyperoxia as neonates. Although hyperoxic exposure reduces an endothelial cell marker platelet and endothelial cell adhesion molecule but increases a mesenchymal cell biomarker α-smooth muscle actin in vitro in human pulmonary endothelial cells, there is no direct evidence showing EndoMT in the development of bronchopulmonary dysplasia. Both pulmonary hypertension and lung fibrosis occur in long-term survivors with bronchopulmonary dysplasia. In this review, we discuss the EndoMT and its modulation by pathological processes. We then focus on the role of EndoMT in the pathogenesis of these chronic lung diseases, and discuss therapeutic approaches targeting the EndoMT using its negative regulators., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In 2009, it was postulated that endothelial cells (ECs) would only be able to execute the orders of growth factors if these cells would accordingly adapt their metabolism. Ten years later, it has become clear that ECs, often differently from other cell types, rely on distinct metabolic pathways to survive and form new blood vessels; that manipulation of EC metabolic pathways alone (even without changing angiogenic signaling) suffices to alter vessel sprouting; and that perturbations of these metabolic pathways can underlie excess formation of new blood vessels (angiogenesis) in cancer and ocular diseases. Initial proof of evidence has been provided that targeting (normalizing) these metabolic perturbations in diseased ECs and delivery of metabolites deserve increasing attention as novel therapeutic approaches for inhibiting or stimulating vessel growth in multiple disorders., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein tyrosine (Tyr) nitration, the covalent addition of a nitro group ( • NO 2 ) to Tyr residues, is emerging as a candidate mechanism of endothelial dysfunction. Previous studies have shown that Tyr nitration is primarily induced by nitrosative stress, a process characterized by the production of reactive nitrogen species, especially peroxynitrite anion (ONOO - ), which is considered a secondary product of NO in the presence of superoxide radicals (O 2 •- ). However, the impact of nitrosative stress-induced Tyr nitration on endothelial dysfunction has not been thoroughly elucidated to date. We developed an endothelial dysfunction model, a process called "endothelial-to-mesenchymal transition (EndMT)," and evaluated the production of NO, O 2 production as well as nitration of the catalytic subunit of protein phosphatase (PP)2A. Mass spectrometry analysis showed that Tyr265 was the nitration site in the catalytic subunit of protein phosphatase (PP)2A, and this Tyr nitration increased PP2A activity and disrupted endothelial integrity. To devise an endothelial-targeted anti-PP2Ac nitration strategy, a mimic peptide, tyrosine 265 wild type (Y265WT), conjugated with the cell-penetrating peptide HIV-1 TAT protein (TAT) was synthesized. PP2Ac nitration and PP2A activity were significantly inhibited by pretreatment with TAT-265WT, and the integrity of endothelial cells was maintained. Furthermore, injection of TAT-265WT attenuated renal nitration formation and caused anticapillary rarefaction in a unilateral urethral obstructive nephropathy model. Taken together, these results offer preclinical proof of concept for TAT-265WT as a tractable agent to protect against nitrosative stress-induced endothelial dysfunction in renal microvessels.-Deng,Y., Cai, Y., Liu, L., Lin, X., Lu, P., Guo, Y., Han, M., Xu, G. Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress-induced endothelial dysfunction in renal microvessels.•- , and protein nitration during EndMT. The results showed that TGF-β1 stimulation induced EndMT and elevated endothelial NO and O 2 •- production as well as nitration of the catalytic subunit of protein phosphatase (PP)2A. Mass spectrometry analysis showed that Tyr265 was the nitration site in the catalytic subunit of protein phosphatase (PP)2A, and this Tyr nitration increased PP2A activity and disrupted endothelial integrity. To devise an endothelial-targeted anti-PP2Ac nitration strategy, a mimic peptide, tyrosine 265 wild type (Y265WT), conjugated with the cell-penetrating peptide HIV-1 TAT protein (TAT) was synthesized. PP2Ac nitration and PP2A activity were significantly inhibited by pretreatment with TAT-265WT, and the integrity of endothelial cells was maintained. Furthermore, injection of TAT-265WT attenuated renal nitration formation and caused anticapillary rarefaction in a unilateral urethral obstructive nephropathy model. Taken together, these results offer preclinical proof of concept for TAT-265WT as a tractable agent to protect against nitrosative stress-induced endothelial dysfunction in renal microvessels.-Deng,Y., Cai, Y., Liu, L., Lin, X., Lu, P., Guo, Y., Han, M., Xu, G. Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress-induced endothelial dysfunction in renal microvessels.

The role of endothelial metabolism represents a crucial element governing the formation and the differentiation of blood vessels, termed angiogenesis. Besides glycolysis and fatty acid oxidation, endothelial cells rely on specific amino acids to proliferate, migrate, and survive. In this review we focus on the metabolism of those amino acids and the intermediates that hold an established function within angiogenesis and endothelial pathophysiology. We also discuss recent work which provides a rationale for specific amino acid-restricted diets and its beneficial effects on vascular tissues, including extending the life span and preventing the development of a variety of diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The onset of many disease processes depends on the function of the endothelial cell (EC) glycocalyx (GCX) which acts as a flow-dependent barrier to cellular infiltration and molecular transport across the blood vessel wall., Objective: This review aims to examine these processes with the potential end goal of implementing GCX repair to restore EC barrier function and slow the progression of disease., Methods: Cell and mouse studies were employed to examine the state of EC GCX in healthy versus disruptive flow conditions. Correlations of observations of the GCX with a number of EC functions were sought with an emphasis on studies of trans-endothelial barrier integrity against vessel wall infiltration of cells and molecules from the circulation. To demonstrate the importance of GCX as a regulator of trans-endothelial infiltration, assays were performed using ECs with an intact GCX and compared to assays of ECs with an experimentally degraded GCX. Studies were also conducted of ECs in which a degraded GCX was repaired., Results: In healthy flow conditions, the EC GCX was found to be thick and substantially covered the endothelial surface. GCX expression dropped significantly in complex flow conditions and coincided with a disease-like cellular and molecular accumulation in the endothelium or within the blood vessel wall. Therapeutic repair of the GCX abolished this accumulation., Conclusions: Regenerating the degraded GCX reverses EC barrier dysfunction and may attenuate the progression of vascular disease.

Metabolic disorders such as obesity are accompanied by endothelial cell (EC) dysfunction and decreased vascular density. The current paradigm posits that metabolic alterations associated with obesity secondarily lead to EC dysfunction. However, in view of recent evidence reporting that EC dysfunction per se is able to cause metabolic dysregulation, this paradigm should be revisited and further elaborated. In this article we summarize current views and discuss evidence in favor of a causal role for ECs in systemic metabolic dysregulation. We also integrate and contextualize current research in a pathophysiological framework and discuss potential therapeutic strategies targeting angiogenesis to help to counteract obesity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dysfunctional endothelial cells are an essential contributor to the progression of diverse chronic cardiovascular, renal, and metabolic diseases. It manifests in impairment of nitric oxide-dependent vasorelaxation, vascular permeability, and leukocytes deterrent. While endothelial glycocalyx is known to regulate these functions, glycocalyx has been shown to be impaired in pathologic settings leading to endothelial dysfunction. Are these findings coincidental or are they indicative of a potential cooperation of the glycocalyx and the endothelium in inducing a dysfunctional phenotype? The main thrust of this overview is to advance a hypothesis on the existence of vicious circle relations between impaired endothelial glycocalyx and endothelial cell dysfunction. We briefly introduce physiology and pathology of blood flow-induced components of mechanotransduction in endothelial cells, as this function is dependent on glycocalyx and is critically involved in the development of endothelial dysfunction. Next, we present a series of experimental findings and arguments favoring the view on the impairment of mechanotransduction in dysfunctional endothelia. We advance the concept of feedback reinforcement between perturbed endothelial glycocalyx and progression of endothelial dysfunction and sketch therapeutic approaches to restore them. Among those we introduce our recently designed liposomal nanocarriers of preassembled glycocalyx and present evidence of their ability to expeditiously restore endothelial mechanotransduction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose of Review: Since the first discovery of microRNAs (miRNAs) in 1993, the involvement of miRNAs in different aspects of vascular disease has emerged as an important research field. In this review, we summarize the fundamental roles of miRNAs in controlling endothelial cell functions and their implication with several aspects of vascular dysfunction., Recent Findings: MiRNAs have been found to be critical modulators of endothelial homeostasis. The dysregulation of miRNAs has been linked to endothelial dysfunction and the development and progression of vascular disease which and open new opportunities of using miRNAs as potential therapeutic targets for vascular disease., Summary: Further determination of miRNA regulatory circuits and defining miRNAs-specific target genes remains key to future miRNA-based therapeutic applications toward vascular disease prevention. Many new and unanticipated roles of miRNAs in the control of endothelial functions will assist clinicians and researchers in developing potential therapeutic applications.

MPO (myeloperoxidase) is a peroxidase enzyme secreted by activated leukocytes that plays a pathogenic role in cardiovascular disease, mainly by initiating endothelial dysfunction. The molecular mechanisms of the endothelial damaging action of MPO remain though largely elusive. Calpain is a calcium-dependent protease expressed in the vascular wall. Activation of calpains has been implicated in inflammatory disorders of the vasculature. Using endothelial cells and genetically modified mice, this study identifies the µ-calpain isoform as novel downstream signaling target of MPO in endothelial dysfunction. Mouse lung microvascular endothelial cells were stimulated with 10 nmol/L MPO for 180 minutes. MPO denitrosylated µ-calpain C-terminus domain, and time dependently activated µ-calpain, but not the m-calpain isoform. MPO also reduced Thr 172 AMPK (AMP-activated protein kinase) and Ser 1177 eNOS (endothelial nitric oxide synthase) phosphorylation via upregulation of PP2A (protein phosphatase 2) expression. At the functional level, MPO increased endothelial VCAM-1 (vascular cell adhesion molecule 1) abundance and the adhesion of leukocytes to the mouse aorta. In MPO-treated endothelial cells, pharmacological inhibition of calpain activity attenuated expression of VCAM-1 and PP2A, and restored Thr 172 AMPK and Ser 1177 eNOS phosphorylation. Compared with wild-type mice, µ-calpain deficient mice experienced reduced leukocyte adhesion to the aortic endothelium in response to MPO. Our data first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO. The MPO/calpain/PP2A signaling pathway may provide novel pharmacological targets for the treatment of inflammatory vascular disorders., (© 2018 American Heart Association, Inc.)

Endothelial cells (ECs) are more than inert blood vessel lining material. Instead, they are active players in the formation of new blood vessels (angiogenesis) both in health and (life-threatening) diseases. Recently, a new concept arose by which EC metabolism drives angiogenesis in parallel to well-established angiogenic growth factors (e.g., vascular endothelial growth factor). 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3-driven glycolysis generates energy to sustain competitive behavior of the ECs at the tip of a growing vessel sprout, whereas carnitine palmitoyltransferase 1a-controlled fatty acid oxidation regulates nucleotide synthesis and proliferation of ECs in the stalk of the sprout. To maintain vascular homeostasis, ECs rely on an intricate metabolic wiring characterized by intracellular compartmentalization, use metabolites for epigenetic regulation of EC subtype differentiation, crosstalk through metabolite release with other cell types, and exhibit EC subtype-specific metabolic traits. Importantly, maladaptation of EC metabolism contributes to vascular disorders, through EC dysfunction or excess angiogenesis, and presents new opportunities for anti-angiogenic strategies. Here we provide a comprehensive overview of established as well as newly uncovered aspects of EC metabolism., (Copyright © 2018 the American Physiological Society.)

In the vascular system, the endothelial surface layer (ESL) as the inner surface of blood vessels affects mechanotransduction, vascular permeability, rheology, thrombogenesis, and leukocyte adhesion. It creates barriers between endothelial cells and blood and neighbouring cells. The glycocalyx, composed of glycoconjugates and proteoglycans, is an integral component of the ESL and a key element in inter- and intracellular communication and tissue homeostasis. In pathophysiological conditions (atherosclerosis, infection, ischemia/reperfusion injury, diabetes, trauma and acute lung injury) glycocalyx-degrading factors, i.e. reactive oxygen and nitrogen species, matrix metalloproteinases, heparanase and sialidases, damage the ESL, thereby impairing endothelial functions. This leads to increased capillary permeability, leucocyte-endothelium interactions, thrombosis and vascular inflammation, the latter further driving glycocalyx destruction. The present review highlights current knowledge on the vasculoprotective role of the ESL, with specific emphasis on its remodelling in inflammatory vascular diseases and discusses its potential as a novel therapeutic target to treat vascular pathologies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A seamless tube is a very narrow-bore tube that is composed of a single cell with an intracellular lumen and no adherens or tight junctions along its length. Many capillaries in the vertebrate vascular system are seamless tubes. Seamless tubes also are found in invertebrate organs, including the Drosophila trachea and the Caenorhabditis elegans excretory system. Seamless tube cells can be less than a micron in diameter, and they can adopt very simple "doughnut-like" shapes or very complex, branched shapes comparable to those of neurons. The unusual topology and varied shapes of seamless tubes raise many basic cell biological questions about how cells form and maintain such structures. The prevalence of seamless tubes in the vascular system means that answering such questions has significant relevance to human health. In this review, we describe selected examples of seamless tubes in animals and discuss current models for how seamless tubes develop and are shaped, focusing particularly on insights that have come from recent studies in Drosophila and C. elegans., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Hemolysis triggers the onset of several clinical manifestations of sickle cell anemia (SCA). During hemolysis, heme, which is derived from hemoglobin (Hb), accumulates due to the inability of detoxification systems to scavenge sufficiently. Heme exerts multiple harmful effects, including leukocyte activation and migration, enhanced adhesion molecule expression by endothelial cells and the production of pro-oxidant molecules. Area covered: In this review, we describe the effects of heme on leukocytes and endothelial cells, as well as the features of vascular endothelial cells related to vaso-occlusion in SCA. Expert commentary: Free Hb, heme and iron, potent cytotoxic intravascular molecules released during hemolysis, can exacerbate, modulate and maintain the inflammatory response, a main feature of SCA. Endothelial cells in the vascular environment, as well as leukocytes, can become activated via the molecular signaling effects of heme. Due to the hemolytic nature of SCA, hemolysis represents an interesting therapeutic target for heme-scavenging purposes.

Under physiological conditions, the arterial endothelium exerts a powerful protective influence to maintain vascular homeostasis. However, during the development of vascular disease, these protective activities are lost, and dysfunctional endothelial cells actually promote disease pathogenesis. Numerous investigations have analyzed the characteristics of dysfunctional endothelium with a view to understanding the processes responsible for the dysfunction and to determining their role in vascular pathology. This review adopts an alternate approach: reviewing the mechanisms that contribute to the initial formation of a healthy protective endothelium and on how those mechanisms may be disrupted, precipitating the appearance of dysfunctional endothelial cells and the progression of vascular disease. This approach, which highlights the role of endothelial adherens junctions and vascular endothelial-cadherin in endothelial maturation and endothelial dysfunction, provides new insight into the remarkable biology of this important cell layer and its role in vascular protection and vascular disease.

Since the introduction of various endovascular graft materials such as expanded polytetrafluoroethylene (e-PTFE) and Dacron ® polyester, they have been rapidly applied in endovascular devices for treating a variety of clinical situations. While present endovascular grafts have been successful in treating large blood vessels, there are still significant challenges and limitations for small and tortuous vessels to their use. Recently, our group has demonstrated the potential to use thin-film nitinol (TFN) as a novel material to develop endografts used in the treatment of a wide range of small vascular diseases because TFN is ultralow profile (that is, a few micrometers thick), relatively thromboresistant, and superelastic. While TFN has shown superior thromboresistance, its surface endothelialization is not rapid and sufficient. Therefore, our laboratory has been exploring the feasibility of using thin-film silk as a novel coating for facilitating rapid and confluent endothelial cell growth. The purpose of this study is to fabricate a low-profile composite endograft using thin layers of nitinol and silk, and to evaluate both thrombogenicity as well as endothelial cell and smooth muscle cell responses. This study also evaluates the functionality of the composite endograft using an in vitro blood circulation model. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 575-584, 2017., (© 2015 Wiley Periodicals, Inc.)

RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.

The importance of endothelial cell (EC) metabolism and its regulatory role in the angiogenic behavior of ECs during vessel formation and in the function of different EC subtypes determined by different vascular beds has been recognized only in the last few years. Even more importantly, apart from a role of nitric oxide and reactive oxygen species in EC dysfunction, deregulations of EC metabolism in disease only recently received increasing attention. Although comprehensive metabolic characterization of ECs still needs further investigation, the concept of targeting EC metabolism to treat vascular disease is emerging. In this overview, we summarize EC-specific metabolic pathways, describe the current knowledge on their deregulation in vascular diseases, and give an outlook on how vascular endothelial metabolism can serve as a target to normalize deregulated endothelium., (©2017 Int. Union Physiol. Sci./Am. Physiol. Soc.)

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function., Competing Interests: M.G. is a senior scientist at Angiocrine Bioscience. The remaining other authors declare no competing financial interests.

Although vascular disease is a leading cause of mortality, in vitro tools for controlled, quantitative studies of vascular biological processes in an environment that reflects physiological complexity remain limited. We developed a novel in vitro artery that exhibits a number of unique features distinguishing it from tissue-engineered or organ-on-a-chip constructs, most notably that it allows deployment of endovascular devices including stents, quantitative real-time tracking of cellular responses and detailed measurement of flow velocity and lumenal shear stress using particle image velocimetry. The wall of the stentable in vitro artery consists of an annular collagen hydrogel containing smooth muscle cells (SMCs) and whose lumenal surface is lined with a monolayer of endothelial cells (ECs). The system has in vivo dimensions and physiological flow conditions and allows automated high-resolution live imaging of both SMCs and ECs. To demonstrate proof-of-concept, we imaged and quantified EC wound healing, SMC motility and altered shear stresses on the endothelium after deployment of a coronary stent. The stentable in vitro artery provides a unique platform suited for a broad array of research applications. Wide-scale adoption of this system promises to enhance our understanding of important biological events affecting endovascular device performance and to reduce dependence on animal studies., (© 2016 The Author(s).)

Ageing leads to a progressive deterioration of structure and function of all organs over the time. During this process endothelial cells undergo senescence and manifest significant changes in their properties, resulting in impairment of the vascular functionality and neo-angiogenic capability. This ageing-dependent impairment of endothelial functions is considered a key factor contributing to vascular dysfunctions, which is responsible of several age-related diseases of the vascular system and other organs. Several mechanisms have been described to control ageing-related endothelial cell senescence including microRNAs, mitochondrial dysfunction and micro environmental stressors, such as hypoxia. In this review, we attempt to summarize the recent literature in the field, discussing the major mechanisms involved in endothelial cell senescence. We also underline key molecular aspects of ageing-associated vascular dysfunction in the attempt to highlight potential innovative therapeutic targets to delay the onset of age-related diseases., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Background: The NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism, C242T of the p22(phox) subunit of NADPH oxidase, has been reported to be negatively associated with coronary heart disease and may predict disease prevalence. However, the underlying mechanisms remain unknown., Methods and Results: With the use of computer molecular modeling, we discovered that C242T single-nucleotide polymorphism causes significant structural changes in the extracellular loop of p22(phox) and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22(phox) significantly reduced Nox2 expression but had no significant effect on basal endothelial O2 (.-) production or the expression of Nox1 and Nox4. When cells were stimulated with tumor necrosis factor-α (or high glucose), C242T p22(phox) significantly inhibited tumor necrosis factor-α-induced Nox2 maturation, O2 (.-) production, mitogen-activated protein kinases and nuclear factor κB activation, and inflammation (all P<0.05). These C242T effects were further confirmed using p22(phox) short-hairpin RNA-engineered HeLa cells and Nox2(-/-) coronary microvascular endothelial cells. Clinical significance was investigated by using saphenous vein segments from non-coronary heart disease subjects after phlebotomies. TT (C242T) allele was common (prevalence of ≈22%) and, in comparison with CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2 (.-) generation in response to high-glucose challenge., Conclusions: C242T single-nucleotide polymorphism causes p22(phox) structural changes that inhibit endothelial Nox2 activation and oxidative response to tumor necrosis factor-α or high-glucose stimulation. C242T single-nucleotide polymorphism may represent a natural protective mechanism against inflammatory cardiovascular diseases., (© 2016 American Heart Association, Inc.)

Objective: Increased vascular permeability is a hallmark of sepsis and acute respiratory distress syndrome. Angiopoietin (Ang2) induces vascular leak, and excess Ang2 generation is associated with patient mortality from these diseases. However, mechanisms dampening Ang2 generation during injury remain unclear. Interestingly, microRNA (miR)-150 levels were decreased in septic patients. miR regulate signaling networks by silencing mRNAs containing complementary sequences. Thus, we hypothesized that miR-150 suppresses Ang2 generation and thereby resolves vascular injury., Approach and Results: Wild-type or miR-150(-/-) mice or endothelial cells were exposed to lipopolysaccharide or sepsis, and Ang2 levels, adherens junction reannealing, endothelial barrier function, and mortality were determined. Although Ang2 transiently increased during lipopolysaccharide-induced injury in wild-type endothelial cells and lungs, miR-150 expression was elevated only during recovery from injury. Deletion of miR-150 caused a persistent increase in Ang2 levels and impaired adherens junctions reannealing after injury, resulting thereby in an irreversible increase in vascular permeability. Also, miR-150(-/-) mice died rapidly after sepsis. Rescuing miR-150 expression in endothelial cells prevented Ang2 generation, thereby restoring vascular barrier function in miR-150(-/-) mice. miR-150 terminated Ang2 generation by targeting the transcription factor, early growth response 2. Thus, early growth response 2 or Ang2 depletion in miR-150(-/-) endothelial cells restored junctional reannealing and reinstated barrier function. Importantly, upregulating miR-150 expression by injecting a chemically synthesized miR-150 mimic into wild-type mice vasculature decreased early growth response 2 and Ang2 levels and hence mortality from sepsis., Conclusions: miR-150 is a novel suppressor of Ang2 generation with a key role in resolving vascular injury and reducing mortality resulting from sepsis., (© 2016 American Heart Association, Inc.)

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis.

Poor vascular homeostasis drives many clinical disorders including diabetes, arthritis, atherosclerosis, and peripheral artery disease. Local tissue ischemia resultant of insufficient blood flow is a potent stimulus for recruitment of endothelial progenitor cells (EPCs). This mobilization and homing is a multi-step process involving EPC detachment from their steady state bone marrow niches, entry into circulation, rolling along vessel endothelium, transmigration, and adhesion to denuded extracellular matrix (ECM) where they may participate in neovessel formation. However, these events are often interrupted in pathological conditions partly due to an imbalance in factor presentation at the tissue level. EPC number and function is impaired in patients with vascular diseases and this dysfunction has been proposed as a prominent contributor to disease pathogenesis. Research approaches aimed at providing therapeutic angiogenesis commonly involve the delivery of proangiogenic cells and/or soluble factors. Nevertheless, greater understanding of the mechanisms involved in EPC homing in both healthy and diseased states is critical for improving efficacy of such strategies. This review underscores the matrix-related signals necessary for enhancing EPC recruitment to ischemic tissue and provides an overview of the development of synthetic ECMs that aim to mimic functions of the local native microenvironment for use in therapeutic angiogenesis.

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD., (© 2015 by The American Society of Hematology.)

To provide pharmacological data for future clinical studies, this study investigated the protective effects of diltiazem on vascular endothelial cell (VEC) injury induced by angiotensin-II (AngII), hypoxia, and a combination of both treatments. The concentration of intracellular free calcium and the mitochondrial membrane potential in VEC were assessed as indicators of cell injury. An in vivo hypoxic animal model was used to test the protective effect of diltiazem on vascular endothelial tissues. Our study showed that AngII and hypoxia decreased the mitochondrial membrane potential in VEC, which was significantly inhibited by diltiazem. Diltiazem protected against VEC injury induced by the increased concentration of intracellular free calcium, which was associated with AngII and hypoxia. Diltiazem reduced the apoptosis of rat VEC under a sustained hypoxic condition. In addition, it reduced AngII and endothelin I levels in rat vascular endothelial tissues. Our study confirmed that AngII and hypoxia induced VEC injury by regulating the levels of mitochondrial membrane potential and intracellular free calcium. Diltiazem, a calcium channel blocker, protected VEC from AngII- and hypoxia-induced injury., (© 2015 Wiley Publishing Asia Pty Ltd.)

Higher organisms rely on a closed cardiovascular circulatory system with blood vessels supplying vital nutrients and oxygen to distant tissues. Not surprisingly, vascular pathologies rank among the most life-threatening diseases. At the crux of most of these vascular pathologies are (dysfunctional) endothelial cells (ECs), the cells lining the blood vessel lumen. ECs display the remarkable capability to switch rapidly from a quiescent state to a highly migratory and proliferative state during vessel sprouting. This angiogenic switch has long been considered to be dictated by angiogenic growth factors (eg, vascular endothelial growth factor) and other signals (eg, Notch) alone, but recent findings show that it is also driven by a metabolic switch in ECs. Furthermore, these changes in metabolism may even override signals inducing vessel sprouting. Here, we review how EC metabolism differs between the normal and dysfunctional/diseased vasculature and how it relates to or affects the metabolism of other cell types contributing to the pathology. We focus on the biology of ECs in tumor blood vessel and diabetic ECs in atherosclerosis as examples of the role of endothelial metabolism in key pathological processes. Finally, current as well as unexplored EC metabolism-centric therapeutic avenues are discussed., (© 2015 American Heart Association, Inc.)

Severe symptoms of cerebral and cardiorenal vascular diseases can be triggered when cerebral, coronary, or glomerular arterioles grow inappropriately as a result of abnormal cell proliferation. The risk factor(s) and molecular mechanisms responsible for microvascular lesion formation are largely unknown. Although controversial, both animal and epidemiological studies have shown that estrogen increases the risk of stroke which may be due to microvascular lesions. Since microvascular diseases are characterized by excessive vessel growth, it is plausible that estrogen-induced neovascularization contributes to the growth of microvascular lesions. We present evidence for how ID3 overexpression in endothelial cells contributes to the development of an estrogen-induced neovascular phenotype with an additional focus on Pyk2 kinase. Our data showed that ID3 overexpression increased neovascularization, cell migration, and spheroid growth of human cerebral microvascular endothelial cells, hCMEC/D3. ID3-overexpressing cells showed significant estrogen-induced G2/M phase transition. Estrogen treatment increased both ID3 phosphorylation; total protein that was inhibited by tamoxifen, and Pyk2-mediated estrogen-induced ID3 mRNA expression. These findings suggest that Pyk2 signals ID3 expression and ID3 is necessary for estrogen-induced neovascularization in hCMEC/D3 cells. A better understanding of how microvascular lesions depend on ID3 may open new avenues for prevention and treatment of neurological diseases.

Attenuation of T cell-mediated damage of blood endothelial cells (BECs) in transplanted organs is important to prevent transplant vasculopathy (TV) and chronic rejection. Here, we assessed the importance of minor histocompatibility antigen (mHA) distribution and different coinhibitory molecules for T cell-BEC interaction. A transgenic mHA was directed specifically to BECs using the Tie2 promoter and cellular interactions were assessed in graft-versus-host disease-like and heterotopic heart transplantation settings. We found that cognate CD4(+) T-cell help was critical for the activation of BEC-specific CD8(+) T cells. However, systemic mHA expression on BECs efficiently attenuated adoptively transferred, BEC-specific CD4(+) and CD8(+) T cells and hence prevented tissue damage, whereas restriction of mHA expression to heart BECs precipitated the development of TV. Importantly, the lack of the coinhibitory molecules programmed death-1 (PD-1) and B and T lymphocyte attenuator fostered the initial activation of BEC-specific CD4(+) T cells, but did not affect development of TV. In contrast, TV was significantly augmented in the absence of PD-1 on BEC-specific CD8(+) T cells. Taken together, these results indicate that antigen distribution in the vascular bed determines the impact of coinhibition and, as a consequence, critically impinges on T cell-mediated vascular immunopathology., (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)