Endothelial alpha globin is a nitrite reductase.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle.

Pannexin 1 channels in renin-expressing cells influence renin secretion and blood pressure homeostasis.

Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear.

Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium.

The proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1β expression and release are tightly controlled by changes in intracellular Ca ([Ca]), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified.

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.

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.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis.

Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX).

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation.

Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation.

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by a plethora of signalling processes influencing cell-to-cell interactions between the vascular endothelial cells (ECs) in post-capillary venules and circulating leukocytes. Recently, ATP-sensitive P2Y purinergic receptors have emerged as downstream regulators of EC activation in vascular inflammation. However, the mechanism(s) regulating cellular ATP release in this response remains elusive.

A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells.

Both purinergic signaling through nucleotides such as ATP (adenosine 5'-triphosphate) and noradrenergic signaling through molecules such as norepinephrine regulate vascular tone and blood pressure. Pannexin1 (Panx1), which forms large-pore, ATP-releasing channels, is present in vascular smooth muscle cells in peripheral blood vessels and participates in noradrenergic responses. Using pharmacological approaches and mice conditionally lacking Panx1 in smooth muscle cells, we found that Panx1 contributed to vasoconstriction mediated by the α1 adrenoreceptor (α1AR), whereas vasoconstriction in response to serotonin or endothelin-1 was independent of Panx1.

Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction.

Objective: Hemoglobin α (Hb α) and endothelial nitric oxide synthase (eNOS) form a macromolecular complex at myoendothelial junctions; the functional role of this interaction remains undefined. To test if coupling of eNOS and Hb α regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb α to disrupt this interaction.

Approach And Results: In silico modeling of Hb α and eNOS identified a conserved sequence of interaction.

Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

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) α (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.

Expression of pannexin isoforms in the systemic murine arterial network.

Aims: Pannexins (Panx) form ATP release channels and it has been proposed that they play an important role in the regulation of vascular tone. However, distribution of Panx across the arterial vasculature is not documented.

Methods: We tested antibodies against Panx1, Panx2 and Panx3 on human embryonic kidney cells (which do not endogenously express Panx proteins) transfected with plasmids encoding each Panx isoform and Panx1(-/-) mice.

MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation.

Rationale: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis.

Objective: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.

A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.

Objective: Plasminogen activator inhibitor-1 (PAI-1) has previously been shown to be key to the formation of myoendothelial junctions (MEJs) in normal and pathological states (eg, obesity). We therefore sought to identify the mechanism whereby PAI-1 could be selectively accumulated at the MEJ.

Methods And Results: We identified PAI-1 protein enrichment at the MEJ in obese mice and in response to tumor necrosis factor (TNF-α) with a vascular cell coculture.

Pannexin1 regulates α1-adrenergic receptor- mediated vasoconstriction.

Rationale: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell communication is not clear in the resistance vasculature. Pannexins (Panx) are purine-releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of vascular smooth muscle cell constriction.

Objective: We investigated the role of pannexins in phenylephrine- and KCl-mediated constriction of resistance arteries.

A new method for in vivo visualization of vessel remodeling using a near-infrared dye.

Objectives: Vascular obstructive events can be partially compensated for by remodeling processes that increase vessel diameter and collateral tortuosity. However, methods for visualizing remodeling events in vivo and with temporal comparisons from the same animal remain elusive.

Methods: Using a novel infrared conjugated polyethylene glycol dye, we investigated the possibility of intravital vascular imaging of the mouse ear before and after ligation of the primary feeder artery.

Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.

Objective: To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle.

Methods And Results: Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall.

Enhanced connexin 43 expression delays intra-mitotic duration and cell cycle traverse independently of gap junction channel function.

Connexins (Cxs) and gap junction (GJ)-mediated communication have been linked with the regulation of cell cycle traverse. However, it is not clear whether Cx expression or GJ channel function are the key mediators in this process or at what stage this regulation may occur. We therefore tested the hypothesis that enhanced Cx expression could alter the rate of cell cycle traverse independently of GJ channel function.

Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.

Rationale: Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined.

Objective: Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation.

Methods And Results: MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells.

Site-specific connexin phosphorylation is associated with reduced heterocellular communication between smooth muscle and endothelium.

Background/aims: Myoendothelial junctions (MEJs) represent a specialized signaling domain between vascular smooth muscle cells (VSMC) and endothelial cells (EC). The functional consequences of phosphorylation state of the connexins (Cx) at the MEJ have not been explored.

Methods/results: Application of adenosine 3',5'-cyclic monophosphate sodium (pCPT) to mouse cremasteric arterioles reduces the detection of connexin 43 (Cx43) phosphorylated at its carboxyl terminal serine 368 site (S368) at the MEJ in vivo.

Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.

Although much physiology in resistance vessels has been attributed to the cytoplasmic connection between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), little is known of the protein expression between the two cell types. In an attempt to identify the proteins between ECs and VSMCs, mouse cremaster arterioles were stained with phalloidin-Alexa 594 and viewed on a confocal microscope that resolved "actin bridges" within the internal elastic lamina between ECs and VSMCs. To determine the incidence of protein, the pixel intensity from the antibodies on actin bridges were compared with the pixel intensity from antibodies within ECs or VSMCs.