The proinflammatory cytokine IL-1b is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1b expression and release are tightly controlled by changes in intracellular Ca 2+ ([Ca 2+ ] 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-a. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-kB-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1b. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1b 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 Ca 2+ diffusion into the cell to regulate IL-1b. High-throughput flow cytometric analysis demonstrated that TNF-a treatments lead to elevated [Ca 2+ ] i , corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-a-associated increases in [Ca 2+ ] i , blocked phosphorylation of the NF-kB-p65 protein, and reduced IL-1b transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca 2+ ] i regulation via the Panx1 channel induces a feedforward effect on NF-kB to regulate IL-1b 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 D 2 levels. Blockade of prostaglandin D 2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto -deficient mice on high-fat diet; conversely, direct addition of prostaglandin D 2 rescued myogenic tone in high-fat diet–fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D 2 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.
25inflammation, endothelial cell, smooth muscle cell 4 to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 5 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular 6 calcium to control the production and release of cytokines including IL-1β. 7 8 GRAPHICAL ABSTRACT 9 10 Graphical Abstract: The inflammatory process in endothelial cells is controlled by Ca 2+ entry through Pannexin 1 membrane channels. 3 Abstract 1The proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease 2 that can be targeted to reduce major cardiovascular events. IL-1β expression and release 3 are tightly controlled by changes in intracellular Ca 2+ . In addition, purinergic signaling 4 through ATP release has also been reported to promote IL-1β production. Despite this, 5 the mechanisms that control IL-1β synthesis and expression have not been identified. 6The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, 7 especially during inflammation. However, resolution of purinergic signaling occurs quickly 8 due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human 9 endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis 10 alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase 11 in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome 12 sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that 13 TNF signaling induced NFκβ-associated Panx1 transcription. Genetic inhibition of Panx1 14 reduced the expression and secretion of IL-1β. We initially hypothesized that increased 15Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. 16However, our data demonstrate that IL1-β expression was not altered after direct ATP 17 stimulation, following degradation of ATP by apyrase, or after pharmacological blockade 18 of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, 19 control IL-1β production. Because Panx1 forms a large pore channel, we hypothesized it 20 may act to passively diffuse Ca 2+ into the cell upon opening to regulate IL-1β. High-21 throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated 22 intracellular Ca 2+ . Genetic or pharmacological inhibition of Panx1 reduced TNF-23 associated increases in intracellular Ca 2+ , and IL-1β transcription. Furthermore, we found 24 that the Ca 2+ -sensitive NFκβ-p65 protein failed to localize to the nucleus after genetic or 25 pharmacological block of Panx1. Taken together, our study provides the first evidence 26 that intracellular Ca 2+ regulation via the Panx1 channel induces a feed-forward effect on 27NFκβ to regulate IL-1β synthesis and release in endothelium during inflammation. 28 4 (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates
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.
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