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The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to 'see' (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells-specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and, as such, provides a semi-selective barrier between the blood and the interstitial space. Compromise of the lung EC barrier due to inflammatory or toxic events may result in pulmonary edema, which is a cardinal feature of acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). The EC functions are controlled, at least in part, via epigenetic mechanisms mediated by histone deacetylases (HDACs). Zinc-dependent HDACs represent the largest group of HDACs and are activated by Zn2+. Members of this HDAC group are involved in epigenetic regulation primarily by modifying the structure of chromatin upon removal of acetyl groups from histones. In addition, they can deacetylate many non-histone histone proteins, including those located in extranuclear compartments. Recently, the therapeutic potential of inhibiting zinc-dependent HDACs for EC barrier preservation has gained momentum. However, the role of specific HDAC subtypes in EC barrier regulation remains largely unknown. This review aims to provide an update on the role of zinc-dependent HDACs in endothelial dysfunction and its related diseases. We will broadly focus on biological contributions, signaling pathways and transcriptional roles of HDACs in endothelial pathobiology associated mainly with lung diseases, and we will discuss the potential of their inhibitors for lung injury prevention.

Background In endothelial cells (ECs), glycolysis, regulated by PFKFB3 (6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase, isoform‐3), is the major metabolic pathway for ATP generation. In preclinical peripheral artery disease models, VEGF165a (vascular endothelial growth factor165a) and microRNA‐93 both promote angiogenesis. Methods and Results Mice following hind‐limb ischemia (HLI) and ECs with, and without, hypoxia and serum starvation were examined with, and without, microRNA‐93 and VEGF165a. Post‐HLI perfusion recovery was monitored. EC metabolism was studied using seahorse assay, and the expression and activity of major metabolism genes were assessed. Reactive oxygen species levels and EC permeability were evaluated. C57Bl/6J mice generated a robust angiogenic response to HLI, with ECs from ischemic versus nonischemic muscle demonstrating no increase in glycolysis. Balb/CJ mice generated a poor angiogenic response post‐HLI; ischemic versus nonischemic ECs demonstrated significant increase in glycolysis. MicroRNA‐93‐treated Balb/CJ mice post‐HLI showed better perfusion recovery, with ischemic versus nonischemic ECs showing no increase in glycolysis. VEGF165a‐treated Balb/CJ mice post‐HLI showed no improvement in perfusion recovery with ischemic versus nonischemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF165a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR‐93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA‐93 versus VEGF165a upregulated glucose‐6‐phosphate dehydrogenase expression and activity, activating the pentose phosphate pathway. MicroRNA‐93 versus control increased reduced nicotinamide adenine dinucleotide phosphate and virtually eliminated the increase in reactive oxygen species. In ECs under hypoxia and serum starvation, VEGF165a significantly increased and miR‐93 decreased EC permeability. Conclusions In peripheral artery disease, activation of the pentose phosphate pathway to promote angiogenesis may offer potential therapeutic advantages.

Vascular barrier dysfunction is characterized by increased permeability and inflammation of endothelial cells (ECs), which are prominent features of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis, and a major complication of the SARS-CoV-2 infection and COVID-19. Functional impairment of the EC barrier and accompanying inflammation arises due to microbial toxins and from white blood cells of the lung as part of a defensive action against pathogens, ischemia-reperfusion or blood product transfusions, and aspiration syndromes-based injury. A loss of barrier function results in the excessive movement of fluid and macromolecules from the vasculature into the interstitium and alveolae resulting in pulmonary edema and collapse of the architecture and function of the lungs, and eventually culminates in respiratory failure. Therefore, EC barrier integrity, which is heavily dependent on cytoskeletal elements (mainly actin filaments, microtubules (MTs), cell-matrix focal adhesions, and intercellular junctions) to maintain cellular contacts, is a critical requirement for the preservation of lung function. EC cytoskeletal remodeling is regulated, at least in part, by Ser/Thr phosphorylation/dephosphorylation of key cytoskeletal proteins. While a large body of literature describes the role of phosphorylation of cytoskeletal proteins on Ser/Thr residues in the context of EC barrier regulation, the role of Ser/Thr dephosphorylation catalyzed by Ser/Thr protein phosphatases (PPases) in EC barrier regulation is less documented. Ser/Thr PPases have been proposed to act as a counter-regulatory mechanism that preserves the EC barrier and opposes EC contraction. Despite the importance of PPases, our knowledge of the catalytic and regulatory subunits involved, as well as their cellular targets, is limited and under-appreciated. Therefore, the goal of this review is to discuss the role of Ser/Thr PPases in the regulation of lung EC cytoskeleton and permeability with special emphasis on the role of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) as major mammalian Ser/Thr PPases. Importantly, we integrate the role of PPases with the structural dynamics of the cytoskeleton and signaling cascades that regulate endothelial cell permeability and inflammation.

ABSTRACT The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro. Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to 'see' (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells—specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell. Author summary: Receptors and their ligands are at the heart of cell-to-cell signaling, but can only interact if they are in the same place at the same time. This is controlled in part by expression–only if a cell expresses a receptor can the cell perceive and respond to that ligand. But it is also controlled in part by where in the cell that receptor is. If the receptor is on the cell surface, it may be able to bind extracellular ligands. If inside the cell, e.g. on endosomal vesicles, it may not. Similarly, the intracellular portions of receptors at the cell surface and inside the cell are exposed to different local environments and can initiate different signaling pathways. Therefore, understanding receptor localization and trafficking (how the receptors move within the cell) is an important starting point for understanding receptor function. Here, we use mechanistic computational modeling and quantitative experimental data to define the localization and trafficking of the VEGF receptors, which are key drivers of blood vessel growth and remodeling. We show that these receptors each has different trafficking and patterns of localization, which has significant implications for the differential responses of these receptors to extracellular ligands. [ABSTRACT FROM AUTHOR]

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and, as such, provides a semi-selective barrier between the blood and the interstitial space. Compromise of the lung EC barrier due to inflammatory or toxic events may result in pulmonary edema, which is a cardinal feature of acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). The EC functions are controlled, at least in part, via epigenetic mechanisms mediated by histone deacetylases (HDACs). Zinc-dependent HDACs represent the largest group of HDACs and are activated by Zn2+. Members of this HDAC group are involved in epigenetic regulation primarily by modifying the structure of chromatin upon removal of acetyl groups from histones. In addition, they can deacetylate many non-histone histone proteins, including those located in extranuclear compartments. Recently, the therapeutic potential of inhibiting zinc-dependent HDACs for EC barrier preservation has gained momentum. However, the role of specific HDAC subtypes in EC barrier regulation remains largely unknown. This review aims to provide an update on the role of zinc-dependent HDACs in endothelial dysfunction and its related diseases. We will broadly focus on biological contributions, signaling pathways and transcriptional roles of HDACs in endothelial pathobiology associated mainly with lung diseases, and we will discuss the potential of their inhibitors for lung injury prevention. [ABSTRACT FROM AUTHOR]

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

Vascular barrier dysfunction is characterized by increased permeability and inflammation of endothelial cells (ECs), which are prominent features of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis, and a major complication of the SARS-CoV-2 infection and COVID-19. Functional impairment of the EC barrier and accompanying inflammation arises due to microbial toxins and from white blood cells of the lung as part of a defensive action against pathogens, ischemia-reperfusion or blood product transfusions, and aspiration syndromes-based injury. A loss of barrier function results in the excessive movement of fluid and macromolecules from the vasculature into the interstitium and alveolae resulting in pulmonary edema and collapse of the architecture and function of the lungs, and eventually culminates in respiratory failure. Therefore, EC barrier integrity, which is heavily dependent on cytoskeletal elements (mainly actin filaments, microtubules (MTs), cell-matrix focal adhesions, and intercellular junctions) to maintain cellular contacts, is a critical requirement for the preservation of lung function. EC cytoskeletal remodeling is regulated, at least in part, by Ser/Thr phosphorylation/dephosphorylation of key cytoskeletal proteins. While a large body of literature describes the role of phosphorylation of cytoskeletal proteins on Ser/Thr residues in the context of EC barrier regulation, the role of Ser/Thr dephosphorylation catalyzed by Ser/Thr protein phosphatases (PPases) in EC barrier regulation is less documented. Ser/Thr PPases have been proposed to act as a counter-regulatory mechanism that preserves the EC barrier and opposes EC contraction. Despite the importance of PPases, our knowledge of the catalytic and regulatory subunits involved, as well as their cellular targets, is limited and under-appreciated. Therefore, the goal of this review is to discuss the role of Ser/Thr PPases in the regulation of lung EC cytoskeleton and permeability with special emphasis on the role of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) as major mammalian Ser/Thr PPases. Importantly, we integrate the role of PPases with the structural dynamics of the cytoskeleton and signaling cascades that regulate endothelial cell permeability and inflammation. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a multisystemic respiratory disease that is associated with progressive airway and pulmonary vascular remodeling due to the increased proliferation of bronchial smooth muscles cells (BSMCs) and pulmonary arterial smooth muscle cells (PASMCs) and the overproduction of extracellular matrix (e.g., collagen). Cigarette smoke (CS) and several mediators, such as PDGF (platelet-derived growth factor) and IL-6, play critical roles in COPD pathogenesis. HDAC6 has been shown to be implicated in vascular remodeling. However, the role of airway HDAC6 signaling in pulmonary vascular remodeling in COPD and the underlying mechanisms remain undetermined. Here, we show that HDAC6 expression is upregulated in the lungs of patients with COPD and a COPD animal model. We also found that CS extract (CSE), PDGF, and IL-6 increase the protein levels and activation of HDAC6 in BSMCs and PASMCs. Furthermore, CSE and these stimulants induced deacetylation and phosphorylation of ERK1/2 and increased collagen synthesis and BSMC and PASMC proliferation, which were outcomes that were prevented by HDAC6 inhibition. Inhibition of ERK1/2 also diminished the CSE-, PDGF-, and IL-6--caused elevation in collagen levels and cell proliferation. Pharmacologic HDAC6 inhibition with tubastatin A prevented the CS-stimulated increases in the thickness of the bronchial and pulmonary arterial wall, airway resistance, emphysema, and right ventricular systolic pressure and right ventricular hypertrophy in a rat model of COPD. These data demonstrate that the upregulated HDAC6 governs the collagen synthesis and BSMC and PASMC proliferation that lead to airway and vascular remodeling in COPD. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is an acute inflammatory process arises from a wide range of lung insults. A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier but the mechanisms involved are incompletely understood. The therapeutic potential of histone deacetylase (HDAC) inhibitors for the treatment of cardiovascular and inflammatory diseases is increasingly apparent, but the mechanisms by which HDACs regulate pulmonary vascular barrier function remain to be resolved. We found that specific Class IIa HDACs inhibitor, TMP269, significantly attenuated the lipopolysaccharide (LPS)‐induced human lung microvascular endothelial cells (HLMVEC) barrier compromise in vitro and improved vascular barrier integrity and lung function in murine model of ALI in vivo. TMP269 decreased LPS‐induced myosin light chain phosphorylation suggesting the role for Class IIa HDACs in LPS‐induced cytoskeleton reorganization. TMP269 did not affect microtubule structure and tubulin acetylation in contrast to the HDAC6‐specific inhibitor, Tubastatin A suggesting that Class IIa HDACs and HDAC6 (Class IIb) regulate endothelial cytoskeleton and permeability via different mechanisms. Furthermore, LPS increased the expression of ArgBP2 which has recently been attributed to HDAC‐mediated activation of Rho. Depletion of ArgBP2 abolished the ability of LPS to disrupt barrier function in HLMVEC and both TMP269 and Tubastatin A decreased the level of ArgBP2 expression after LPS stimulation suggesting that both Class IIa and IIb HDACs regulate endothelial permeability via ArgBP2‐dependent mechanism. Collectively, our data strongly suggest that Class IIa HDACs are involved in LPS‐induced ALI in vitro and in vivo via specific mechanism which involved contractile responses, but not microtubule reorganization. [ABSTRACT FROM AUTHOR]

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Giphosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH. [ABSTRACT FROM AUTHOR]

Rationale: Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH.Objectives: To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH.Methods: PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH.Measurements and Main Results: Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs.Conclusions: Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2. [ABSTRACT FROM AUTHOR]

We have previously shown that Gs‐coupled adenosine receptors (A2a) are primarily involved in adenosine‐induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine‐induced Rac1 activation and EC barrier enhancement is mediated by Gs‐dependent stimulation of cAMP‐dependent Epac1‐mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively‐active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine‐induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine‐induced increase in TER. We next examined the role of cAMP‐activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine‐induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine‐induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine‐induced increases in TER and Tiam1 depletion had no effect on adenosine‐induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine‐induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP‐dependent Epac1/Vav2/Rap1‐mediated signaling. [ABSTRACT FROM AUTHOR]

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a "calm" or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a "activated" state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells. [ABSTRACT FROM AUTHOR]

Background: Endothelial cells (ECs) use glycolysis to produce energy. In preclinical models of peripheral arterial disease, further activation of EC glycolysis was ineffective or deleterious in promoting hypoxia-dependent angiogenesis, whereas pentose phosphate pathway activation was effective. Hexosamine biosynthesis pathway, pentose phosphate pathway, and glycolysis are closely linked. Glucosamine directly activates hexosamine biosynthesis pathway., Methods: Hind-limb ischemia in endothelial nitric oxide synthase knockout (eNOS -/- ) and BALB/c mice was used. Glucosamine (600 μg/g per day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging and angiogenesis was studied by CD31 immunostaining. In vitro, human umbilical vein ECs and mouse microvascular ECs with glucosamine, L-glucose, or vascular endothelial growth factor (VEGF 165 a) were tested under hypoxia and serum starvation. Cell Counting Kit-8, tube formation, intracellular reactive oxygen species, electric cell-substrate impedance sensing, and fluorescein isothiocyanate dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time quantitative polymerase chain reaction, and Western blot. Human muscle biopsies from patients with peripheral arterial disease were assessed for EC O-GlcNAcylation before and after supervised exercise versus standard medical care., Results: On day 3 after hind-limb ischemia, glucosamine-treated versus control eNOS -/- mice had less necrosis (n=4 or 5 per group). Beginning on day 7 after hind-limb ischemia, glucosamine-treated versus control BALB/c mice had higher blood flow, which persisted to day 21, when ischemic muscles showed greater CD31 staining per muscle fiber (n=8 per group). In vitro, glucosamine versus L-glucose ECs showed improved survival (n=6 per group) and tube formation (n=6 per group). RNA sequencing of glucosamine versus L-glucose ECs showed increased amino acid metabolism (n=3 per group). That resulted in increased oxidative phosphorylation (n=8-12 per group) and serine biosynthesis pathway without an increase in glycolysis or pentose phosphate pathway genes (n=6 per group). This was associated with better barrier function (n=6-8 per group) and less reactive oxygen species (n=7 or 8 per group) compared with activating glycolysis by VEGF 165 a. These effects were mediated by activating transcription factor 4, a driver of exercise-induced angiogenesis. In muscle biopsies from humans with peripheral arterial disease, EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise versus standard medical care (n=6 per group)., Conclusion: In cells, mice, and humans, activation of hexosamine biosynthesis pathway by glucosamine in peripheral arterial disease induces an "exercise-like" angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder.

The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro . Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.

Chronic obstructive pulmonary disease (COPD) is a multisystemic respiratory disease that is associated with progressive airway and pulmonary vascular remodeling due to the increased proliferation of bronchial smooth muscles cells (BSMCs) and pulmonary arterial smooth muscle cells (PASMCs) and the overproduction of extracellular matrix (e.g., collagen). Cigarette smoke (CS) and several mediators, such as PDGF (platelet-derived growth factor) and IL-6, play critical roles in COPD pathogenesis. HDAC6 has been shown to be implicated in vascular remodeling. However, the role of airway HDAC6 signaling in pulmonary vascular remodeling in COPD and the underlying mechanisms remain undetermined. Here, we show that HDAC6 expression is upregulated in the lungs of patients with COPD and a COPD animal model. We also found that CS extract (CSE), PDGF, and IL-6 increase the protein levels and activation of HDAC6 in BSMCs and PASMCs. Furthermore, CSE and these stimulants induced deacetylation and phosphorylation of ERK1/2 and increased collagen synthesis and BSMC and PASMC proliferation, which were outcomes that were prevented by HDAC6 inhibition. Inhibition of ERK1/2 also diminished the CSE-, PDGF-, and IL-6-caused elevation in collagen levels and cell proliferation. Pharmacologic HDAC6 inhibition with tubastatin A prevented the CS-stimulated increases in the thickness of the bronchial and pulmonary arterial wall, airway resistance, emphysema, and right ventricular systolic pressure and right ventricular hypertrophy in a rat model of COPD. These data demonstrate that the upregulated HDAC6 governs the collagen synthesis and BSMC and PASMC proliferation that lead to airway and vascular remodeling in COPD.

Acute lung injury (ALI) is a severe progressive disorder that arises from a wide range of causes such as toxins or inflammation, resulting in significant morbidity and mortality. There are no effective therapeutic options apart from mechanical ventilation strategies. While the mechanisms that govern the clinically relevant process of increased EC permeability and remodeling associated with ALI are under intense investigation, our knowledge of the processes that determine barrier enhancement or preservation are far from completion. Recently, epigenetic mechanisms have emerged as a major regulator of enduring changes in cell behavior and the therapeutic potential of inhibiting histone deacetylases (HDACs) for the treatment of cardiovascular and inflammatory diseases has gained remarkable attention. Although HDACs have been shown to play an important role in regulating EC barrier function, the involved HDAC subtypes and mechanisms remain undefined. Further investigation of the HDAC signaling may provide therapeutic approaches for the prevention and treatment of ALI.