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In the vascular wall, endothelial nitric oxide synthase (eNOS) produces NO to regulate peripheral vascular resistance, tissue perfusion, and blood pressure. In resistance arteries, eNOS couples with α-globin and, through chemical reactions, modulates NO diffusion needed for vascular smooth muscle relaxation. While α-globin protein alone is known to be unstable, the mechanisms that enable α-globin protein expression remain elusive. Here, Lechauve et al. report that arterial endothelium expresses α hemoglobin-stabilizing protein, which acts as a critical chaperone protein for α-globin expression and vascular function. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) was first described as a bioactive molecule through its ability to stimulate soluble guanylate cyclase, but the revelation that NO was the endothelium derived relaxation factor drove the field to its modern state. The wealth of research conducted over the past 30 years has provided us with a picture of how diverse NO signaling can be within the vascular wall, going beyond simple vasodilation to include such roles as signaling through protein S-nitrosation. This expanded view of NO's actions requires highly regulated and compartmentalized production. Importantly, resistance arteries house multiple proteins involved in the production and transduction of NO allowing for efficient movement of the molecule to regulate vascular tone and reactivity. In this review, we focus on the many mechanisms regulating NO production and signaling action in the vascular wall, with a focus on the control of endothelial nitric oxide synthase (eNOS), the enzyme responsible for synthesizing most of the NO within these confines. We also explore how cross talk between the endothelium and smooth muscle in the microcirculation can modulate NO signaling, illustrating that this one small molecule has the capability to produce a plethora of responses. [ABSTRACT FROM AUTHOR]

A vast amount of investigation has centered on how the endothelium and smooth muscle communicate. From this evidence, myoendothelial junctions have emerged as critical anatomical structures to regulate heterocellular cross talk. Indeed, there is now evidence that the myoendothelial junction serves as a signaling microdomain to organize proteins used to facilitate vascular heterocellular communication. This review highlights the evolving role of myoendothelial junctions in the context of vascular cell-cell communication. [ABSTRACT FROM AUTHOR]

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) ? (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb ? and is abrogated by its genetic depletion. Mechanistically, endothelial Hb ? haem iron in the Fe3+ state permits NO signalling, and this signalling is shut off when Hb ? is reduced to the Fe2+ state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb ? oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells. [ABSTRACT FROM AUTHOR]

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a genetic red blood cell disorder characterized by increased reactive oxygen species (ROS) and a concordant reduction in antioxidant capacity in the endothelium. Superoxide dismutase 2 (SOD2) is a mitochondrial-localized enzyme that catalyzes the dismutation of superoxide to hydrogen peroxide. Decreased peripheral blood expression of SOD2 is correlated with increased hemolysis and cardiomyopathy in SCD. Here, we report for the first time that endothelial cells exhibit reduced SOD2 protein expression in the pulmonary endothelium of SCD patients. To investigate the impact of decreased SOD2 expression in the endothelium, SOD2 was knocked down in human pulmonary microvascular endothelial cells (hPMVECs). We found that SOD2 deficiency in hPMVECs results in endothelial cell dysfunction, including reduced cellular adhesion, diminished migration, integrin protein dysregulation, and disruption of permeability. Furthermore, we uncover that SOD2 mediates changes in endothelial cell function via processing of fibronectin through its inability to facilitate dimerization. These results demonstrate that endothelial cells are deficient in SOD2 expression in SCD patients and suggest a novel pathway for SOD2 in regulating fibronectin processing. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a fatal cardiopulmonary disease characterized by increased vascular cell proliferation with apoptosis resistance and occlusive remodeling of the small pulmonary arteries. The Notch family of proteins subserves proximal signaling of an evolutionarily conserved pathway that effects cell proliferation, fate determination, and development. In endothelial cells (ECs), Notch receptor 2 (Notch2) was shown to promote endothelial apoptosis. However, a pro- or antiproliferative role for Notch2 in pulmonary endothelial proliferation and ensuing PAH is unknown. We postulated that suppressed Notch2 signaling drives pulmonary endothelial proliferation in the context of PAH. We observed that levels of Notch2 are ablated in lungs from PAH subjects compared with non-PAH controls. Notch2 expression was attenuated in human pulmonary artery endothelial cells (hPAECs) exposed to vasoactive stimuli including hypoxia, TGF-b, ET-1, and IGF-1. Notch2-deficient hPAECs activated Akt, Erk1/2, and antiapoptotic protein Bcl-2 and reduced levels of p21cip and Bax associated with increased EC proliferation and reduced apoptosis. In addition, Notch2 suppression elicited a paradoxical activation of Notch1 and canonical Notch target gene Hes1, Hey1, and Hey2 transcription. Furthermore, reduction in Rb and increased E2F1 binding to the Notch1 promoter appear to explain the Notch1 upregulation. Yet, when Notch1 was decreased in Notch2-suppressed cells, the wound injury response was augmented. In aggregate, our results demonstrate that loss of Notch2 in hPAECs derepresses Notch1 and elicits EC hallmarks of PAH. Augmented EC proliferation upon Notch1 knockdown points to a context-dependent role for Notch1 and 2 in endothelial cell homeostasis. [ABSTRACT FROM AUTHOR]

The nitric oxide/soluble guanylyl cyclase (NO-sGC) signaling pathway regulates the cardiovascular, neuronal, and gastrointestinal systems. Impaired sGC signaling can result in disease and system-wide organ failure. This review seeks to examine the redox control of sGC through heme and cysteine regulation while discussing therapeutic drugs that target various conditions. Heme regulation involves mechanisms of insertion of the heme moiety into the sGC protein, the molecules and proteins that control switching between the oxidized (Fe 3+ ) and reduced states (Fe 2+ ), and the activity of heme degradation. Modifications to cysteine residues by S-nitrosation on the α1 and β1 subunits of sGC have been shown to be important in sGC signaling. Moreover, redox balance and localization of sGC is thought to control downstream effects. In response to altered sGC activity due to changes in the redox state, many therapeutic drugs have been developed to target decreased NO-sGC signaling. The importance and relevance of sGC continues to grow as sGC dysregulation leads to numerous disease conditions. [ABSTRACT FROM AUTHOR]

Hemoglobin subunit alpha (HBA) expression in endothelial cells (ECs) has recently been shown to control vascular tone and function. We sought to elucidate the transcriptional regulation of HBA expression in the EC. Gain of KLF2 or KLF4 function studies led to significant induction of HBA in ECs. An opposite effect was observed in ECs isolated from animals with endothelial-specific ablation of Klf2, Klf4 or both. Promoter reporter assays demonstrated that KLF2/ KLF4 transactivated the hemoglobin alpha promoter, an effect that was abrogated following mutation of all four putative KLF-binding sites. Fine promoter mutational studies localized three out of four KLF-binding sites (sites 2, 3, and 4) as critical for the transactivation of the HBA promoter by KLF2/KLF4. Chromatin immunoprecipitation studies showed that KLF4 bound to the HBA promoter in ECs. Thus, KLF2 and KLF4 serve as important regulators that promote HBA expression in the endothelium. [ABSTRACT FROM AUTHOR]

In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP 3 ) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall. [ABSTRACT FROM AUTHOR]

Background--Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. Methods and Results--Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates l-arginine uptake and plasma membrane levels of CAT1 and y+LAT1 amino acid transporters in endothelial cells. Treatment with l-arginine modestly lowers blood pressure of collectrin knockout mice. Conclusions--Collectrin is a consequential link between the transport of l-arginine and endothelial nitric oxide synthase uncoupling in hypertension. [ABSTRACT FROM AUTHOR]

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention. Here we identify cell surface receptor CD47 as a novel age-sensitive driver of vascular and metabolic dysfunction. With the natural aging process, CD47 and its ligand thrombospondin-1 were increased, concurrent with a reduction of self-renewal transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in arteries from aged wild-type mice and older human subjects compared to younger controls. These perturbations were prevented in arteries from aged CD47-null mice. Arterial endothelial cells isolated from aged wild-type mice displayed cellular exhaustion with decreased proliferation, migration and tube formation compared to cells from aged CD47-null mice. CD47 suppressed ex vivo sprouting, in vivo angiogenesis and skeletal muscle blood flow in aged wild-type mice. Treatment of arteries from older humans with a CD47 blocking antibody mitigated the age-related deterioration in angiogenesis. Finally, aged CD47-null mice were resistant to age- and diet-associated weight gain, glucose intolerance and insulin desensitization. These results indicate that the CD47-mediated signaling maladapts during aging to broadly impair endothelial self-renewal, angiogenesis, perfusion and glucose homeostasis. Our findings provide a strong rationale for therapeutically targeting CD47 to minimize these dysfunctions during aging. [ABSTRACT FROM AUTHOR]