Background: Panx1 (pannexin 1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in smooth muscle cells (SMCs) influences α-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure. Methods: To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Electrical field stimulation on isolated arteries and blood pressure were assessed. Results: Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, overexpression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Connexin 43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone. Conclusions: These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.

Coronary microvascular disease (CMD) caused by obesity and diabetes is major contributor to heart failure with preserved ejection fraction; however, the mechanisms underlying CMD are not well understood. Using cardiac magnetic resonance applied to mice fed a high-fat, high-sucrose diet as a model of CMD, we elucidated the role of inducible nitric oxide synthase (iNOS) and 1400W, an iNOS antagonist, in CMD. Global iNOS deletion prevented CMD along with the associated oxidative stress and diastolic and subclinical systolic dysfunction. The 1400W treatment reversed established CMD and oxidative stress and preserved systolic/diastolic function in mice fed a high-fat, high-sucrose diet. Thus, iNOS may represent a therapeutic target for CMD.

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. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin’s inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36–39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36–39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36–39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.

In Brief Interleukine-1 beta (IL-1β) has been identified as a critical factor that contributes to the inflammatory response in cardiovascular disease (e.g., atherosclerosis). Pannexin 1 (Panx1) channel activity in endothelial cells regulates localized inflammatory cell recruitment. In response to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular calcium to control the production and release of cytokines including IL-1β. GRAPHICAL ABSTRACT Abstract 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 2+ . In addition, purinergic signaling through ATP release has also been reported to promote IL-1β production. Despite this, the mechanisms that control IL-1β synthesis and expression have not been identified. The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, especially during inflammation. However, resolution of purinergic signaling occurs quickly due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that TNF signaling induced NFκβ-associated Panx1 transcription. Genetic inhibition of Panx1 reduced the expression and secretion of IL-1β. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL1-β expression was not altered after direct ATP stimulation, following degradation of ATP by apyrase, or after pharmacological blockade of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, control IL-1β production. Because Panx1 forms a large pore channel, we hypothesized it may act to passively diffuse Ca 2+ into the cell upon opening to regulate IL-1β. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated intracellular Ca 2+ . Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in intracellular Ca 2+ , and IL-1β transcription. Furthermore, we found that the Ca 2+ -sensitive NFκβ-p65 protein failed to localize to the nucleus after genetic or pharmacological block of Panx1. Taken together, our study provides the first evidence that intracellular Ca 2+ regulation via the Panx1 channel induces a feed-forward effect on NFκβ to regulate IL-1β synthesis and release in endothelium during inflammation.

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.