The role of globins in cardiovascular physiology.

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

Experimental TET2 Clonal Hematopoiesis Predisposes to Renal Hypertension Through an Inflammasome-Mediated Mechanism.

BACKGROUND: Hypertension incidence increases with age and represents one of the most prevalent risk factors for cardiovascular disease. Clonal events in the hematopoietic system resulting from somatic mutations in driver genes are prevalent in elderly individuals who lack overt hematologic disorders. This condition is referred to as age-related clonal hematopoiesis (CH), and it is a newly recognized risk factor for cardiovascular disease. It is not known whether CH and hypertension in the elderly are causally related and, if so, what are the mechanistic features. METHODS: A murine model of adoptive bone marrow transplantation was employed to examine the interplay between Tet2 (ten-eleven translocation methylcytosine dioxygenase 2) clonal hematopoiesis and hypertension. RESULTS: In this model, a subpressor dose of Ang II (angiotensin II) resulted in elevated systolic and diastolic blood pressure as early as 1 day after challenge. These conditions led to the expansion of Tet2-deficient proinflammatory monocytes and bone marrow progenitor populations. Tet2 deficiency promoted renal CCL5 (C-C motif ligand 5) chemokine expression and macrophage infiltration into the kidney. Consistent with macrophage involvement, Tet2 deficiency in myeloid cells promoted hypertension when mice were treated with a subpressor dose of Ang II. The hematopoietic Tet2-/- condition led to sodium retention, renal inflammasome activation, and elevated levels of IL (interleukin)-1ß and IL-18. Analysis of the sodium transporters indicated NCC (sodium-chloride symporter) and NKCC2 (Na+-K+-Cl- cotransporter 2) activation at residues Thr53 and Ser105, respectively. Administration of the NLRP3 (NLR family pyrin domain containing 3) inflammasome inhibitor MCC950 reversed the hypertensive state, sodium retention, and renal transporter activation. CONCLUSIONS: Tet2-mediated CH sensitizes mice to a hypertensive stimulus. Mechanistically, the expansion of hematopoietic Tet2-deficient cells promotes hypertension due to elevated renal immune cell infiltration and activation of the NLRP3 inflammasome, with consequences on sodium retention. These data indicate that carriers of TET2 CH could be at elevated risk for the development of hypertension and that immune modulators could be useful in treating hypertension in this patient population.

Heme metabolism in nonerythroid cells.

Heme is an iron-containing prosthetic group necessary for the function of several proteins termed "hemoproteins." Erythrocytes contain most of the body's heme in the form of hemoglobin and contain high concentrations of free heme. In nonerythroid cells, where cytosolic heme concentrations are 2 to 3 orders of magnitude lower, heme plays an essential and often overlooked role in a variety of cellular processes. Indeed, hemoproteins are found in almost every subcellular compartment and are integral in cellular operations such as oxidative phosphorylation, amino acid metabolism, xenobiotic metabolism, and transcriptional regulation. Growing evidence reveals the participation of heme in dynamic processes such as circadian rhythms, NO signaling, and the modulation of enzyme activity. This dynamic view of heme biology uncovers exciting possibilities as to how hemoproteins may participate in a range of physiologic systems. Here, we discuss how heme is regulated at the level of its synthesis, availability, redox state, transport, and degradation and highlight the implications for cellular function and whole organism physiology.

Obesogenic diet disrupts tissue-specific mitochondrial gene signatures in the artery and capillary endothelium.

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.

Generation and characterization of a conditional eNOS knock out mouse model for cell-specific reactivation of eNOS in gain-of-function studies.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in the vessel wall regulates blood pressure and cardiovascular hemodynamics. In this study, we generated conditional eNOS knock out (KO) mice characterized by a duplicated/inverted exon 2 flanked with two pairs of loxP regions (eNOSinv/inv); a Cre-recombinase activity induces cell-specific reactivation of eNOS, as a result of a flipping of the inverted exon 2 (eNOSfl). This work aimed to test the efficiency of the Cre-mediated cell-specific recombination and the resulting eNOS expression/function. As proof of concept, we crossed eNOSinv/inv mice with DeleterCrepos (DelCrepos) mice, expressing Cre recombinase in all cells. We generated heterozygous eNOSfl/inv or homozygous eNOSfl/fl mice, and eNOSinv/inv littermate mice. We found that both eNOSfl/fl and eNOSfl/inv mice express eNOS and the overall expression level depends on the number of mutated alleles, while eNOSinv/inv mice did not show any eNOS expression. Vascular endothelial function was restored in eNOSfl/fl and eNOSfl/inv mice, as determined by ACh-dependent vasodilation of aortic rings. Cre-dependent reactivation of eNOS in eNOSfl/fl and eNOSfl/inv mice rescued eNOSinv/inv (phenotypically global eNOS KO) mice from hypertension. These findings demonstrate that eNOS expression is restored in eNOSfl/fl mice at comparable physiological levels of WT mice, and its functional activity is independent on the number of the reactivated alleles. Therefore, eNOSinv/inv mice are a useful model for studying the effects of conditional reactivation of eNOS and gene dosage effects in specific cells for gain-of-function studies.

The hemodynamic response to nitrite is acute and dependent upon tissue perfusion.

In the vasculature, nitric oxide (NO) is produced in the endothelium by endothelial nitric oxide synthase (eNOS) and is critical for the regulation of blood flow and blood pressure. Blood flow may also be regulated by the formation of nitrite-derived NO catalyzed by hemoproteins under hypoxic conditions. We sought to investigate whether nitrite administration may affect tissue perfusion and systemic hemodynamics in WT and eNOS knockout mice. We found that global eNOS KO mice show decreased tissue perfusion compared to WT mice by using laser speckle contrast imaging. To study both the acute and long-term effects of sodium nitrite (0, 0.1, 1, and 10 mg/kg) on peripheral blood flow and systemic blood pressure, a bolus of nitrite was delivered intraperitoneally every 24 h over 4 consecutive days. We found that nitrite administration resulted in a dose-dependent and acute increase in peripheral blood flow in eNOS KO mice but had no effects in WT mice. The nitrite induced changes in tissue perfusion were transient, as determined by intraindividual comparisons of tissue perfusion 24-h after injection. Accordingly, 10 mg/kg sodium nitrite acutely decreased blood pressure in eNOS KO mice but not in WT mice as determined by invasive Millar catheterization. Interestingly, we found the vasodilatory effects of nitrite to be inversely correlated to baseline tissue perfusion. These results demonstrate the nitrite acutely recovers hypoperfusion and hypertension in global eNOS KO mice and suggest the vasodilatory actions of nitrite are dependent upon tissue hypoperfusion.

Caveolar peroxynitrite formation impairs endothelial TRPV4 channels and elevates pulmonary arterial pressure in pulmonary hypertension.

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH.

Collectrin (Tmem27) deficiency in proximal tubules causes hypertension in mice and a TMEM27 variant associates with blood pressure in males in a Latino cohort.

Collectrin (Tmem27), an angiotensin-converting enzyme 2 homologue, is a chaperone of amino acid transporters in the kidney and endothelium. Global collectrin knockout (KO) mice have hypertension, endothelial dysfunction, exaggerated salt sensitivity, and diminished renal blood flow. This phenotype is associated with altered nitric oxide and superoxide balance and increased proximal tubule (PT) Na+/H+ exchanger isoform 3 (NHE3) expression. Collectrin is located on the X chromosome where genome-wide association population studies have largely been excluded. In the present study, we generated PT-specific collectrin KO (PT KO) mice to determine the precise contribution of PT collectrin in blood pressure homeostasis. We also examined the association of human TMEM27 single-nucleotide polymorphisms with blood pressure traits in 11,926 Hispanic Community Health Study/Study of Latinos (HCHS/SOL) Hispanic/Latino participants. PT KO mice exhibited hypertension, and this was associated with increased baseline NHE3 expression and diminished lithium excretion. However, PT KO mice did not display exaggerated salt sensitivity or a reduction in renal blood flow compared with control mice. Furthermore, PT KO mice exhibited enhanced endothelium-mediated dilation, suggesting a compensatory response to systemic hypertension induced by deficiency of collectrin in the PT. In HCHS/SOL participants, we observed sex-specific single-nucleotide polymorphism associations with diastolic blood pressure. In conclusion, loss of collectrin in the PT is sufficient to induce hypertension, at least in part, through activation of NHE3. Importantly, our model supports the notion that altered renal blood flow may be a determining factor for salt sensitivity. Further studies are needed to investigate the role of the TMEM27 locus on blood pressure and salt sensitivity in humans.NEW & NOTEWORTHY The findings of our study are significant in several ways: 1) loss of an amino acid chaperone in the proximal tubule is sufficient to cause hypertension, 2) the results in global and proximal tubule-specific collectrin knockout mice support the notion that vascular dysfunction is required for salt sensitivity or that impaired renal tubule function causes hypertension but is not sufficient to cause salt sensitivity, and 3) our study is the first to implicate a role of collectrin in human hypertension.

Bedding as a variable affecting fasting blood glucose and vascular physiology in mice.

Rodent husbandry requires careful consideration of environmental factors that may impact colony performance and subsequent physiological studies. Of note, recent reports have suggested corncob bedding may affect a broad range of organ systems. As corncob bedding may contain digestible hemicelluloses, trace sugars, and fiber, we hypothesized that corncob bedding impacts overnight fasting blood glucose and murine vascular function. Here, we compared mice housed on corncob bedding, which were then fasted overnight on either corncob or ALPHA-dri bedding, a virgin paper pulp cellulose alternative. Male and female mice were used from two noninduced, endothelial-specific conditional knockout strains [Cadherin 5-cre/ERT2, floxed hemoglobin-α1 (Hba1fl/fl) or Cadherin 5-cre/ERT2, floxed cytochrome-B5 reductase 3 (CyB5R3fl/fl)] on a C57BL/6J genetic background. After fasting overnight, initial fasting blood glucose was measured, and mice were anesthetized with isoflurane for measurement of blood perfusion via laser speckle contrast analysis using a PeriMed PeriCam PSI NR system. After a 15-min equilibration, the mice were injected intraperitoneally with the α1-adrenergic receptor agonist, phenylephrine (5 mg/kg), or saline, and monitored for changes in blood perfusion. After a 15-min response period, blood glucose was remeasured postprocedure. In both strains, mice fasted on corncob bedding had higher blood glucose than the pulp cellulose group. In the CyB5R3fl/fl strain, mice housed on corncob bedding displayed a significant reduction in phenylephrine-mediated change in perfusion. In the Hba1fl/fl strain, phenylephrine-induced change in perfusion was not different in the corncob group. This work suggests that corncob bedding, in part due to its ingestion by mice, could impact vascular measurements and fasting blood glucose. To promote scientific rigor and improve reproducibility, bedding type should be routinely included in published methods.NEW & NOTEWORTHY This study demonstrates real-time measurement of changes in perfusion to pharmacological treatment using laser speckle contrast analysis. Furthermore, this investigation revealed that fasting mice overnight on corncob bedding has differential effects on vascular function and that there was increased fasting blood glucose in mice fasted on corncob bedding compared with paper pulp cellulose bedding. This highlights the impact that bedding type can have on outcomes in vascular and metabolic research and reinforces the need for thorough and robust reporting of animal husbandry practices.

Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Smooth muscle cell FTO regulates contractile function.

The fat mass and obesity gene (FTO) is a N6-methyladenosine RNA demethylase that was initially linked by Genome-wide association studies to increased rates of obesity. Subsequent studies have revealed multiple mass-independent effects of the gene, including cardiac myocyte contractility. We created a mouse with a conditional and inducible smooth muscle cell deletion of Fto (Myh11 Cre+ Ftofl/fl) and did not observe any changes in mouse body mass or mitochondrial metabolism. However, the mice had significantly decreased blood pressure (hypotensive), despite increased heart rate and sodium, and significantly increased plasma renin. Remarkably, the third-order mesenteric arteries from these mice had almost no myogenic tone or capacity to constrict to smooth muscle depolarization or phenylephrine. Microarray analysis from Fto-/--isolated smooth muscle cells demonstrated a significant decrease in serum response factor (Srf) and the downstream effectors Acta2, Myocd, and Tagln; this was confirmed in cultured human coronary arteries with FTO siRNA. We conclude Fto is an important component to the contractility of smooth muscle cells.NEW & NOTEWORTHY We show a key role for the fat mass obesity (FTO) gene in regulating smooth muscle contractility, possibly by methylation of serum response factor (Srf).

Macrophage metabolic adaptation to heme detoxification involves CO-dependent activation of the pentose phosphate pathway.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase-dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.

Loss of Endothelial FTO Antagonizes Obesity-Induced Metabolic and Vascular Dysfunction.

RATIONALE: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. OBJECTIVES: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. METHODS AND RESULTS: We generated endothelial Fto-deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto-deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D2 levels. Blockade of prostaglandin D2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto-deficient mice on high-fat diet; conversely, direct addition of prostaglandin D2 rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D2 application. CONCLUSIONS: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.

Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium.

The proinflammatory cytokine IL-1ß is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1ß expression and release are tightly controlled by changes in intracellular Ca2+ ([Ca2+]i), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells following treatment with the proinflammatory cytokine TNF-α. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-κB-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1ß. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1ß expression was not altered after direct ATP stimulation in human umbilical vein endothelial cells. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca2+ diffusion into the cell to regulate IL-1ß. High-throughput flow cytometric analysis demonstrated that TNF-α treatments lead to elevated [Ca2+]i, corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-α-associated increases in [Ca2+]i, blocked phosphorylation of the NF-κB-p65 protein, and reduced IL-1ß transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca2+]i regulation via the Panx1 channel induces a feed-forward effect on NF-κB to regulate IL-1ß synthesis and release in endothelium during inflammation.

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

BACKGROUND: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. METHODS: We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. RESULTS: We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. CONCLUSIONS: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Guidelines for the measurement of vascular function and structure in isolated arteries and veins.

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.

Polarized Proteins in Endothelium and Their Contribution to Function.

Protein localization in endothelial cells is tightly regulated to create distinct signaling domains within their tight spatial restrictions including luminal membranes, abluminal membranes, and interendothelial junctions, as well as caveolae and calcium signaling domains. Protein localization in endothelial cells is also determined in part by the vascular bed, with differences between arteries and veins and between large and small arteries. Specific protein polarity and localization is essential for endothelial cells in responding to various extracellular stimuli. In this review, we examine protein localization in the endothelium of resistance arteries, with occasional references to other vessels for contrast, and how that polarization contributes to endothelial function and ultimately whole organism physiology. We highlight the protein localization on the luminal surface, discussing important physiological receptors and the glycocalyx. The protein polarization to the abluminal membrane is especially unique in small resistance arteries with the presence of the myoendothelial junction, a signaling microdomain that regulates vasodilation, feedback to smooth muscle cells, and ultimately total peripheral resistance. We also discuss the interendothelial junction, where tight junctions, adherens junctions, and gap junctions all convene and regulate endothelial function. Finally, we address planar cell polarity, or axial polarity, and how this is regulated by mechanosensory signals like blood flow.

Heterocellular Contact Can Dictate Arterial Function.

RATIONALE: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. OBJECTIVE: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. METHODS AND RESULTS: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. CONCLUSIONS: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.