Local Complement Contributes to Pathogenic Activation of Lung Endothelial Cells in SARS-CoV-2 Infection.

Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.

Isolation of vasa vasorum endothelial cells from pulmonary artery adventitia: Implementation to vascular biology research.

Isolated endothelial cells are valuable in vitro model for vascular research. At present, investigation of disease-relevant changes in vascular endothelium at the molecular level requires established endothelial cell cultures, preserving vascular bed-specific phenotypic characteristics. Vasa vasorum (VV) form a microvascular network around large blood vessels, in both the pulmonary and systemic circulations, that are critically important for maintaining the integrity and oxygen supply of the vascular wall. However, despite the pathophysiological significance of the VV, methods for the isolation and culture of vasa vasorum endothelial cells (VVEC) have not yet been reported. In our prior studies, we demonstrated the presence of hypoxia-induced angiogenic expansion of the VV in the pulmonary artery (PA) of neonatal calves; an observation which has been followed by a series of in vitro studies on isolated PA VVEC. Here we present a detailed protocol for reproducible isolation, purification, and culture of PA VVEC. We show these cells to express generic endothelial markers, (vWF, eNOS, VEGFR2, Tie1, and CD31), as well as progenitor markers (CD34 and CD133), bind lectin Lycopersicon Esculentum, and incorporate acetylated low-density lipoproteins labeled with acetylated LDL (DiI-Ac-LDL). qPCR analysis additionally revealed the expression of CD105, VCAM-1, ICAM-1, MCAM, and NCAM. Ultrastructural electron microscopy and immunofluorescence staining demonstrated that VVEC are morphologically characterized by a developed actin and microtubular cytoskeleton, mitochondrial network, abundant intracellular vacuolar/secretory system, and cell-surface filopodia. VVEC exhibit exponential growth in culture and can be mitogenically activated by multiple growth factors. Thus, our protocol provides the opportunity for VVEC isolation from the PA, and potentially from other large vessels, enabling advances in VV research.

The Short-Chain Fatty Acid Butyrate Attenuates Pulmonary Vascular Remodeling and Inflammation in Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive cardiovascular disorder in which local vascular inflammation leads to increased pulmonary vascular remodeling and ultimately to right heart failure. The HDAC inhibitor butyrate, a product of microbial fermentation, is protective in inflammatory intestinal diseases, but little is known regarding its effect on extraintestinal diseases, such as PH. In this study, we tested the hypothesis that butyrate is protective in a Sprague-Dawley (SD) rat model of hypoxic PH. Treatment with butyrate (220 mg/kg intake) prevented hypoxia-induced right ventricular hypertrophy (RVH), hypoxia-induced increases in right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, and permeability. A reversal effect of butyrate (2200 mg/kg intake) was observed on elevated RVH. Butyrate treatment also increased the acetylation of histone H3, 25-34 kDa, and 34-50 kDa proteins in the total lung lysates of butyrate-treated animals. In addition, butyrate decreased hypoxia-induced accumulation of alveolar (mostly CD68+) and interstitial (CD68+ and CD163+) lung macrophages. Analysis of cytokine profiles in lung tissue lysates showed a hypoxia-induced upregulation of TIMP-1, CINC-1, and Fractalkine and downregulation of soluble ICAM (sICAM). The expression of Fractalkine and VEGFα, but not CINC-1, TIMP-1, and sICAM was downregulated by butyrate. In rat microvascular endothelial cells (RMVEC), butyrate (1 mM, 2 and 24 h) exhibited a protective effect against TNFα- and LPS-induced barrier disruption. Butyrate (1 mM, 24 h) also upregulated tight junctional proteins (occludin, cingulin, claudin-1) and increased the acetylation of histone H3 but not α-tubulin. These findings provide evidence of the protective effect of butyrate on hypoxic PH and suggest its potential use as a complementary treatment for PH and other cardiovascular diseases.

Extracellular adenosine enhances pulmonary artery vasa vasorum endothelial cell barrier function via Gi/ELMO1/Rac1/PKA-dependent signaling mechanisms.

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 Gi-phosphoinositide 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.

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases.

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.

Sustained Activation of Rho GTPases Promotes a Synthetic Pulmonary Artery Smooth Muscle Cell Phenotype in Neprilysin Null Mice.

OBJECTIVE: Pulmonary artery smooth muscle cells (PASMCs) from neprilysin (NEP) null mice exhibit a synthetic phenotype and increased activation of Rho GTPases compared with their wild-type counterparts. Although Rho GTPases are known to promote a contractile SMC phenotype, we hypothesize that their sustained activity decreases SM-protein expression in these cells. APPROACH AND RESULTS: PASMCs isolated from wild-type and NEP-/- mice were used to assess levels of SM-proteins (SM-actin, SM-myosin, SM22, and calponin) by Western blotting, and were lower in NEP-/- PASMCs compared with wild-type. Rac and Rho (ras homology family member) levels and activity were higher in NEP-/- PASMCs, and ShRNA to Rac and Rho restored SM-protein, and attenuated the enhanced migration and proliferation of NEP-/- PASMCs. SM-gene repressors, p-Elk-1, and Klf4 (Kruppel lung factor 4), were higher in NEP-/- PASMCs and decreased by shRNA to Rac and Rho. Costimulation of wild-type PASMCs with PDGF (platelet-derived growth factor) and the NEP substrate, ET-1 (endothelin-1), increased Rac and Rho activity, and decreased SM-protein levels mimicking the NEP knock-out phenotype. Activation of Rac and Rho and downstream effectors was observed in lung tissue from NEP-/- mice and humans with chronic obstructive pulmonary disease. CONCLUSIONS: Sustained Rho activation in NEP-/- PASMCs is associated with a decrease in SM-protein levels and increased migration and proliferation. Inactivation of RhoGDI (Rho guanine dissociation inhibitor) and RhoGAP (Rho GTPase activating protein) by phosphorylation may contribute to prolonged activation of Rho in NEP-/- PASMCs. Rho GTPases may thus have a role in integration of signals between vasopeptides and growth factor receptors and could influence pathways that suppress SM-proteins to promote a synthetic phenotype.

Endothelial cells cope with hypoxia-induced depletion of ATP via activation of cellular purine turnover and phosphotransfer networks.

Intravascular ATP and adenosine have emerged as important regulators of endothelial barrier function, vascular remodeling and neovascularization at various pathological states, including hypoxia, inflammation and oxidative stress. By using human umbilical vein endothelial cells (HUVEC) and bovine vasa vasorum endothelial cells (VVEC) as representatives of macro- and microvessel phenotypes, this study was undertaken to evaluate cellular mechanisms contributing to physiological adaptation of vascular endothelium to hypoxia, with a particular emphasis on ectoenzymatic purine-converting activities and their link to intracellular ATP homeostasis and signaling pathways. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), ecto-5'-nucleotidase/CD73 and ecto-adenylate kinase activities were determined by thin-layer chromatography (TLC) with 3H-labelled nucleotide substrates. Exposure of HUVEC and VVEC to 1% O2 for 4-24 h triggered rather moderate activation of ATP breakdown into adenosine via the CD39-CD73 axis. Additional TLC analysis of salvage pathways revealed the enhanced ability of hypoxic HUVEC to convert cell-incorporated [3H]adenosine into [3H]ADP/ATP. Furthermore, following a period of hypoxia, HUVEC underwent concurrent changes in intracellular signaling manifested in the depletion of putative ATP stores and targeted up-regulation of phospho-p53, p70S6K/mTOR and other tyrosine kinases. The revealed complex implication of both extrinsic and intrinsic mechanisms into a tuned hypoxia-induced control of purine homeostasis and signaling may open up further research for the development of pharmacological treatments to improve endothelial cell function under disease conditions associated with a loss of cellular ATP during oxygen deprivation.

Glycolysis and oxidative phosphorylation are essential for purinergic receptor-mediated angiogenic responses in vasa vasorum endothelial cells.

Angiogenesis is an energy-demanding process; however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose) and oxidative phosphorylation (OXPHOS) (oligomycin, rotenone, and FCCP), we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP, and 2-methylthioadenosine diphosphate trisodium salt (MeSADP), but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase B3, hexokinase (HK), and GLUT-1, but not lactate dehydrogenase. Moreover, extracellular ATP and MeSADP, and to a lesser extent adenosine, increased basal and maximal oxygen consumption rates in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5 mM glucose compared with 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PDH)-E1α and increased succinate dehydrogenase (SDH), cytochrome oxidase IV, and ß-subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial Ca2+ concentration, implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of phosphatidylinositol 3-kinase and Akt pathways in lactate production, PDH-E1α phosphorylation, and the expression of HK, SDH, and GLUT-1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways is essential for VV angiogenesis and may contribute to pathologic vascular remodeling in pulmonary hypertension.

The adventitia: essential regulator of vascular wall structure and function.

The vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. It is the most complex compartment of the vessel wall and is composed of a variety of cells, including fibroblasts, immunomodulatory cells (dendritic cells and macrophages), progenitor cells, vasa vasorum endothelial cells and pericytes, and adrenergic nerves. In response to vascular stress or injury, resident adventitial cells are often the first to be activated and reprogrammed to influence the tone and structure of the vessel wall; to initiate and perpetuate chronic vascular inflammation; and to stimulate expansion of the vasa vasorum, which can act as a conduit for continued inflammatory and progenitor cell delivery to the vessel wall. This review presents the current evidence demonstrating that the adventitia acts as a key regulator of vascular wall function and structure from the outside in.

High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization.

Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

Histone deacetylase inhibitors synergize with sildenafil to suppress purine metabolism and proliferation in pulmonary hypertension.

RATIONALE: Sildenafil, a well-known vasodilator known to interfere with purinergic signaling through effects on cGMP, is a mainstay in the treatment of pulmonary hypertension (PH). However, little is known regarding its effects on the metabolic reprogramming of vascular cells, which is a hallmark of PH. Purine metabolism, especially intracellular de novo purine biosynthesis is essential for vascular cell proliferation. Since adventitial fibroblasts are critical contributors to proliferative vascular remodeling in PH, in this study we aimed to investigate if sildenafil, beyond its well-known vasodilator role in smooth muscle cells, impacts intracellular purine metabolism and proliferation of fibroblasts derived from human PH patients. METHODS: Integrated omics approaches (plasma and cell metabolomics) and pharmacological inhibitor approaches were employed in plasma samples and cultured pulmonary artery fibroblasts from PH patients. MEASUREMENTS AND MAIN RESULTS: Plasma metabolome analysis of 27 PH patients before and after treatment with sildenafil, demonstrated a partial, but specific effect of sildenafil on purine metabolites, especially adenosine, adenine, and xanthine. However, circulating markers of cell stress, including lactate, succinate, and hypoxanthine were only decreased in a small subset of sildenafil-treated patients. To better understand potential effects of sildenafil on pathological changes in purine metabolism (especially purine synthesis) in PH, we performed studies on pulmonary fibroblasts from PAH patients (PH-Fibs) and corresponding controls (CO-Fibs), since these cells have previously been shown to demonstrate stable and marked PH associated phenotypic and metabolic changes. We found that PH-Fibs exhibited significantly increased purine synthesis. Treatment of PH-Fibs with sildenafil was insufficient to normalize cellular metabolic phenotype and only modestly attenuated the proliferation. However, we observed that treatments which have been shown to normalize glycolysis and mitochondrial abnormalities including a PKM2 activator (TEPP-46), and the histone deacetylase inhibitors (HDACi), SAHA and Apicidin, had significant inhibitory effects on purine synthesis. Importantly, combined treatment with HDACi and sildenafil exhibited synergistic inhibitory effects on proliferation and metabolic reprogramming in PH-Fibs. CONCLUSIONS: While sildenafil alone partially rescues metabolic alterations associated with PH, treatment with HDACi, in combination with sildenafil, represent a promising and potentially more effective strategy for targeting vasoconstriction, metabolic derangement and pathological vascular remodeling in PH.

Macrophage Xanthine Oxidoreductase Links LPS Induced Lung Inflammatory Injury to NLRP3 Inflammasome Expression and Mitochondrial Respiration.

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) remain poorly treated inflammatory lung disorders. Both reactive oxygen species (ROS) and macrophages are involved in the pathogenesis of ALI/ARDS. Xanthine oxidoreductase (XOR) is an ROS generator that plays a central role in the inflammation that contributes to ALI. To elucidate the role of macrophage-specific XOR in endotoxin induced ALI, we developed a conditional myeloid specific XOR knockout in mice. Myeloid specific ablation of XOR in LPS insufflated mice markedly attenuated lung injury demonstrating the essential role of XOR in this response. Macrophages from myeloid specific XOR knockout exhibited loss of inflammatory activation and increased expression of anti-inflammatory genes/proteins. Transcriptional profiling of whole lung tissue of LPS insufflated XOR fl/fl//LysM-Cre mice demonstrated an important role for XOR in expression and activation of the NLRP3 inflammasome and acquisition of a glycolytic phenotype by inflammatory macrophages. These results identify XOR as an unexpected link between macrophage redox status, mitochondrial respiration and inflammatory activation.

Interplay of macrophages and T cells in the lung vasculature.

In severe pulmonary arterial hypertension (PAH), vascular lesions are composed of phenotypically altered vascular and inflammatory cells that form clusters or tumorlets. Because macrophages are found in increased numbers in intravascular and perivascular space in human PAH, here we address the question whether macrophages play a role in pulmonary vascular remodeling and whether accumulation of macrophages in the lung vasculature could be compromised by the immune system. We used the mouse macrophage cell line RAW 264.7 because these cells are resistant to apoptosis, have high proliferative capacity, and resemble cells in the plexiform lesions that tend to pile up instead of maintaining a monolayer. Cells were characterized by immunocytochemistry with cell surface markers (Lycopersicon Esculentum Lectin, CD117, CD133, FVIII, CD31, VEGFR-2, and S100). Activated, but not quiescent, T cells were able to suppress RAW 264.7 cell proliferative and migration activity in vitro. The carboxyfluorescein diacetate-labeled RAW 264.7 cells were injected into the naïve Sprague Dawley (SD) rat and athymic nude rat. Twelve days later, cells were found in the lung vasculature of athymic nude rats that lack functional T cells, contributing to vascular remodeling. No labeled RAW 264.7 cells were detected in the lungs of immune-competent SD rats. Our data demonstrate that T cells can inhibit in vitro migration and in vivo accumulation of macrophage-like cells.

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension.

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)ß(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Chronic hypoxia impairs extracellular nucleotide metabolism and barrier function in pulmonary artery vasa vasorum endothelial cells.

Vascular remodeling plays a pivotal role in a variety of pathophysiological conditions where hypoxia and inflammation are prominent features. Intravascular ATP, ADP and adenosine are known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signalling to endothelial cell growth and angiogenesis remains poorly understood. By using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitia of control and chronically hypoxic neonatal calves, these studies were aimed to evaluate the effect of hypoxia on biochemical and functional properties of microvascular endothelial network at the sites of angiogenesis. In comparison with normoxic controls, VVEC from hypoxic animals are characterized by (1) drastically impaired nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5'-nucleotidase/CD73 activities with respective increases in basal extracellular ATP and ADP levels (2) higher proliferative responses to low micromolar concentrations of ATP and ADP; and (3) enhanced permeability and disordered adenosinergic control of vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran). Together, these results suggest that unique pattern of purine-mediated angiogenic activation and enhanced leakiness of VVEC from chronically hypoxic vessels may be defined by disordered endothelial nucleotide homeostasis at sites of active neovascularization.

Metabolite G-Protein Coupled Receptors in Cardio-Metabolic Diseases.

G protein-coupled receptors (GPCRs) have originally been described as a family of receptors activated by hormones, neurotransmitters, and other mediators. However, in recent years GPCRs have shown to bind endogenous metabolites, which serve functions other than as signaling mediators. These receptors respond to fatty acids, mono- and disaccharides, amino acids, or various intermediates and products of metabolism, including ketone bodies, lactate, succinate, or bile acids. Given that many of these metabolic processes are dysregulated under pathological conditions, including diabetes, dyslipidemia, and obesity, receptors of endogenous metabolites have also been recognized as potential drug targets to prevent and/or treat metabolic and cardiovascular diseases. This review describes G protein-coupled receptors activated by endogenous metabolites and summarizes their physiological, pathophysiological, and potential pharmacological roles.

Mechanisms of SARS-CoV-2-induced lung vascular disease: potential role of complement.

The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory distress syndrome, with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to acute respiratory distress syndrome secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation, and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be "conditional innate immune cells" centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillaritis-like tissue damage, clotting, and hyperinflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas.

c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells.

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells.

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1-3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.