Joshua T. Butcher

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. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by TNF-α. This process involves activation of type-1 TNF receptors, recruitment of Src family kinases (SFK) and SFK-dependent phosphorylation of Panx1. Using an inducible, EC-specific Panx1 knockout mouse line, we report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation, placing Panx1 channels at the centre of cytokine crosstalk with purinergic signalling in the endothelium.

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

The ATP-releasing channel Panx1 is specifically involved in blood pressure regulation by adrenergic signaling. Regulating blood pressure with ATP Blood pressure is dynamically regulated to enable rapid responses to changes in position and physical or emotional stress, such as exercise or anger and fear. Many signals that activate G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) control vascular tone, including norepinephrine (also known as noradrenaline) released by the sympathetic nervous system, which increases blood pressure. Billaud et al. report that the α1 adrenoreceptor (α1AR)—but not the endothelin-1 or serotonin receptor, which are also Gαq-coupled GPCRs and stimulate vasoconstriction—is specifically coupled to activation of the ATP (adenosine 5′-triphosphate)–releasing channel pannexin1 (Panx1). Mice lacking Panx1 in smooth muscle cells were hypotensive, specifically during their active period of the day. Isolated arteries from these mice did not release ATP and contracted less in response to adrenoreceptor stimulation. Thus, ATP release through Panx1 channels specifically contributes to blood pressure regulation by the sympathetic nervous system. 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. Analysis of the Panx1-deficient mice showed that Panx1 contributed to blood pressure regulation especially during the night cycle when sympathetic nervous activity is highest. Using mimetic peptides and site-directed mutagenesis, we identified a specific amino acid sequence in the Panx1 intracellular loop that is essential for activation by α1AR signaling. Collectively, these data describe a specific link between noradrenergic and purinergic signaling in blood pressure homeostasis.

Spatial heterogeneity in skeletal muscle microvascular blood flow distribution is increased in the metabolic syndrome

Previous studies have demonstrated that the metabolic syndrome is associated with impaired skeletal muscle arteriolar function, although integrating observations into a conceptual framework for impaired perfusion in peripheral vascular disease (PVD) has been limited. This study builds on previous work to evaluate in situ arteriolar hemodynamics in cremaster muscle of obese Zucker rats (OZR) to integrate existing knowledge into a greater understanding of impaired skeletal muscle perfusion. In OZR cremaster muscle, perfusion distribution at microvascular bifurcations (γ) was consistently more heterogeneous than in controls. However, while consistent, the underlying mechanistic contributors were spatially divergent as altered adrenergic constriction was the major contributor to altered γ at proximal microvascular bifurcations, with a steady decay with distance, while endothelial dysfunction was a stronger contributor in distal bifurcations with no discernible role proximally. Using measured values of γ, we found that simulations predict that successive alterations to γ in OZR caused more heterogeneous perfusion distribution in distal arterioles than in controls, an effect that could only be rectified by combined adrenoreceptor blockade and improvements to endothelial dysfunction. Intravascular (125)I-labeled albumin tracer washout from in situ gastrocnemius muscle of OZR provided independent support for these observations, indicating increased perfusion heterogeneity that was corrected only by combined adrenoreceptor blockade and improved endothelial function. These results suggest that a defining element of PVD in the metabolic syndrome may be an altered γ at microvascular bifurcations, that its contributors are heterogeneous and spatially distinct, and that interventions to rectify this negative outcome must take a new conceptual framework into account.

Hemoglobin α/eNOS Coupling at Myoendothelial Junctions Is Required for Nitric Oxide Scavenging During Vasoconstriction

Objective— Hemoglobin &agr; (Hb &agr;) 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 &agr; regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb &agr; to disrupt this interaction. Approach and Results— In silico modeling of Hb &agr; and eNOS identified a conserved sequence of interaction. By mutating portions of Hb &agr;, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb &agr; sequence (Hb &agr; X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb &agr; and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb &agr; X significantly decreased interaction between eNOS and Hb &agr;. Fluorescein isothiocyanate labeling of Hb &agr; X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb &agr; X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb &agr; X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb &agr; X, which caused a significant decrease in blood pressure; injection of Hb &agr; X into eNOS-/- mice had no effect. Conclusions— These results identify a novel sequence on Hb &agr; that is important for Hb &agr;/eNOS complex formation and is critical for nitric oxide signaling at myoendothelial junctions.

Divergence between arterial perfusion and fatigue resistance in skeletal muscle in the metabolic syndrome

The metabolic syndrome is associated with elevated peripheral vascular disease risk, characterized by mismatched blood flow delivery/distribution and local metabolism. The obese Zucker rat (OZR) model of the metabolic syndrome exhibits myriad vascular impairments, although their integrated impact on functional hyperaemia remains unclear. In this study, arterial pressor responses and skeletal muscle perfusion were assessed in lean Zucker rats (LZRs) and OZRs during adrenergic stimulation (phenylephrine), challenge with thromboxane (U46619) and endothelium‐dependent dilatation (methacholine). The OZRs were hypertensive compared with the LZRs, but this was abolished by adrenoreceptor blockade (phentolamine); pressor responses to U46619 were similar between strains and were abolished by blockade with the prostaglandin H2/thromboxane A2 receptor antagonist, SQ‐29548. Depressor reactivity to methacholine was impaired in OZRs, but was improved by antioxidant treatment (TEMPOL). Across levels of metabolic demand, blood flow to in situ gastrocnemius muscle was restrained by adrenergic constriction in OZRs, although this diminished with increased demand. Oxygen extraction, reduced in OZRs compared with LZRs across levels of metabolic demand, was improved by TEMPOL or SQ‐29548; treatment with phentolamine did not impact extraction, and neither TEMPOL nor SQ‐29548 improved muscle blood flow in OZRs. While oxygen uptake and muscle performance were consistently reduced in OZRs versus LZRs, treatment with all three agents improved outcomes, while treatment with individual agents was less effective. These results suggest that contributions of vascular dysfunction to perfusion, oxygen uptake and muscle performance are spatially distinct, with adrenergic constriction impacting proximal resistance and endothelial dysfunction impacting distal microvessel–tissue exchange. Further, these data suggest that increasing skeletal muscle blood flow in OZRs is not sufficient to improve performance, unless distal perfusion inhomogeneities are rectified.

Endothelial nitric oxide synthase in the microcirculation

Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)—a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

Blunted temporal activity of microvascular perfusion heterogeneity in metabolic syndrome: a new attractor for peripheral vascular disease?

A key clinical outcome for peripheral vascular disease (PVD) in patients is a progressive decay in skeletal muscle performance and its ability to resist fatigue with elevated metabolic demand. We have demonstrated that PVD in obese Zucker rats (OZR) is partially due to increased perfusion distribution heterogeneity at successive microvascular bifurcations within skeletal muscle. As this increased heterogeneity (γ) is longitudinally present in the network, its cumulative impact is a more heterogeneous distribution of perfusion between terminal arterioles than normal, causing greater regional tissue ischemia. To minimize this negative outcome, a likely compensatory mechanism against an increased γ should be an increased temporal switching at arteriolar bifurcations to minimize downstream perfusion deficits. Using in situ cremaster muscle, we determined that temporal activity (the cumulative sum of absolute differences between successive values of γ, taken every 20 s) was lower in OZR than in control animals, and this difference was present in both proximal (1A-2A) and distal (3A-4A) arteriolar bifurcations. Although adrenoreceptor blockade (phentolamine) improved temporal activity in 1A-2A arteriolar bifurcations in OZR, this was without impact in the distal microcirculation, where only interventions against oxidant stress (Tempol) and thromboxane A(2) activity (SQ-29548) were effective. Analysis of the attractor for γ indicated that it was not only elevated in OZR but also exhibited severe reductions in range, suggesting that the ability of the microcirculation to respond to any challenge is highly restricted and may represent the major contributor to the manifestation of poor muscle performance at this age in OZR.

Protective effect of sex on chronic stress- and depressive behavior-induced vascular dysfunction in BALB/cJ mice

The presence of chronic, unresolvable stresses leads to negative health outcomes, including development of clinical depression/depressive disorders, with outcome severity being correlated with depressive symptom severity. One of the major outcomes associated with chronic stress and depression is the development of cardiovascular disease (CVD) and an elevated CVD risk profile. However, in epidemiological research, sex disparities are evident, with premenopausal women suffering from depressive symptoms more acutely than men, but also demonstrating a relative protection from the onset of CVD. Given this, we investigated the differential effect of sex on conduit artery and resistance arteriolar function in male and female mice following 8 wk of an unpredictable chronic mild stress (UCMS) protocol. In males, plasma cortisol and depressive symptom severity (e.g., coat status, anhedonia, delayed grooming) were elevated by UCMS. Endothelium-dependent dilation to methacholine/acetylcholine was impaired in conduit arteries and skeletal muscle arterioles, suggesting a severe loss of nitric oxide bioavailability and increased production of thromboxane A2 vs. prostaglandin I2 associated with elevated reactive oxygen species (ROS) and an increased level of systemic inflammation. Endothelium-independent dilation was intact. In females, depressive symptoms and plasma cortisol increases were more severe than in males, although alterations to vascular reactivity were blunted, including the effects of elevated ROS and inflammation on dilator responses. These results suggest that compared with males, female rats are more susceptible to chronic stress in terms of the severity of depressive behaviors, but that the subsequent development of vasculopathy is blunted owing to an improved ability to tolerate elevated ROS and systemic inflammatory stress.

Microvascular perfusion heterogeneity contributes to peripheral vascular disease in metabolic syndrome

A major challenge facing public health is the increased incidence and prevalence of the metabolic syndrome, a clinical condition characterized by excess adiposity, impaired glycaemic control, dyslipidaemia and moderate hypertension. The greatest concern for this syndrome is the profound increase in risk for development of peripheral vascular disease (PVD) in afflicted persons. However, ongoing studies suggest that reductions in bulk blood flow to skeletal muscle may not be the primary contributor to the premature muscle fatigue that is a hallmark of PVD. Compelling evidence has been provided suggesting that an increasingly spatially heterogeneous and temporally stable distribution of blood flow at successive arteriolar bifurcations in metabolic syndrome creates an environment where a large number of the pre‐capillary arterioles have low perfusion, low haematocrit, and are increasingly confined to this state, with limited ability to adapt perfusion in response to a challenged environment. Single pharmacological interventions are unable to significantly restore function owing to a divergence in their spatial effectiveness, although combined therapeutic approaches to correct adrenergic dysfunction, elevated oxidant stress and increased thromboxane A2 improve perfusion‐based outcomes. Integrated, multi‐target therapeutic interventions designed to restore healthy network function and flexibility may provide for superior outcomes in subjects with metabolic syndrome‐associated PVD.

Distinct temporal phases of microvascular rarefaction in skeletal muscle of obese Zucker rats.

Evolution of metabolic syndrome is associated with a progressive reduction in skeletal muscle microvessel density, known as rarefaction. Although contributing to impairments to mass transport and exchange, the temporal development of rarefaction and the contributing mechanisms that lead to microvessel loss are both unclear and critical areas for investigation. Although previous work suggests that rarefaction severity in obese Zucker rats (OZR) is predicted by the chronic loss of vascular nitric oxide (NO) bioavailability, we have determined that this hides a biphasic development of rarefaction, with both early and late components. Although the total extent of rarefaction was well predicted by the loss in NO bioavailability, the early pulse of rarefaction developed before a loss of NO bioavailability and was associated with altered venular function (increased leukocyte adhesion/rolling), and early elevation in oxidant stress, TNF-α levels, and the vascular production of thromboxane A2 (TxA2). Chronic inhibition of TNF-α blunted the severity of rarefaction and also reduced vascular oxidant stress and TxA2 production. Chronic blockade of the actions of TxA2 also blunted rarefaction, but did not impact oxidant stress or inflammation, suggesting that TxA2 is a downstream outcome of elevated reactive oxygen species and inflammation. If chronic blockade of TxA2 is terminated, microvascular rarefaction in OZR skeletal muscle resumes, but at a reduced rate despite low NO bioavailability. These results suggest that therapeutic interventions against inflammation and TxA2 under conditions where metabolic syndrome severity is moderate or mild may prevent the development of a condition of accelerated microvessel loss with metabolic syndrome.