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Tumour necrosis factor (TNF) is a ubiquitously expressed cytokine with functions beyond the immune system. In several diseases, the induction of TNF expression in resistance artery smooth muscle cells enhances microvascular myogenic vasoconstriction and perturbs blood flow. This pathological role prompted our hypothesis that constitutively expressed TNF regulates myogenic signalling and systemic haemodynamics under non-pathological settings. Here we show that acutely deleting the TNF gene in smooth muscle cells or pharmacologically scavenging TNF with etanercept (ETN) reduces blood pressure and resistance artery myogenic responsiveness; the latter effect is conserved across five species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF) that connect to a canonical myogenic signalling pathway. Our data positions mTNF ‘reverse signalling’ as an integral element of a microvascular mechanosensor; pathologic or therapeutic perturbations of TNF signalling, therefore, necessarily affect microvascular tone and systemic haemodynamics. TNF is typically viewed as an inflammatory mediator. Here the authors identify a non-inflammatory mechanism conserved across species whereby the constitutively expressed smooth muscle cell TNF mediates myogenic signal transduction in skeletal muscle resistance arteries and regulates mean arterial blood pressure.

The development of cardiac hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response that may eventually lead to ventricular dilation and heart failure. Regulator of G protein signaling 5 (Rgs5) is a negative regulator of G protein-mediated signaling by inactivating Gα(q) and Gα(i), which mediate actions of most known vasoconstrictors. Previous studies have demonstrated that Rgs5 expresses among various cell types within mature heart and showed high levels of Rgs5 mRNA in monkey and human heart tissue by Northern blot analysis. However, the critical role of Rgs5 on cardiac remodeling remains unclear. To specifically determine the role of Rgs5 in pathological cardiac remodeling, we used transgenic mice with cardiac-specific overexpression of human Rgs5 gene and Rgs5⁻ / ⁻ mice. Our results demonstrated that the transgenic mice were resistant to cardiac hypertrophy and fibrosis through inhibition of MEK-ERK1/2 signaling, whereas the Rgs5⁻ / ⁻ mice displayed the opposite phenotype in response to pressure overload. These studies indicate that Rgs5 protein is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure.

In asthma and chronic obstructive pulmonary disease, activation of Gq-protein—coupled receptors causes bronchoconstriction. In each case, the management of moderate-to-severe disease uses inhaled corticosteroid (glucocorticoid)/long-acting β2-adrenoceptor agonist (LABA) combination therapies, which are more efficacious than either monotherapy alone. In primary human airway smooth muscle cells, glucocorticoid/LABA combinations synergistically induce the expression of regulator of G-protein signaling 2 (RGS2), a GTPase-activating protein that attenuates Gq signaling. Functionally, RGS2 reduced intracellular free calcium flux elicited by histamine, methacholine, leukotrienes, and other spasmogens. Furthermore, protection against spasmogen-increased intracellular free calcium, following treatment for 6 h with LABA plus corticosteroid, was dependent on RGS2. Finally, Rgs2-deficient mice revealed enhanced bronchoconstriction to spasmogens and an absence of LABA-induced bronchoprotection. These data identify RGS2 gene expression as a genomic mechanism of bronchoprotection that is induced by glucocorticoids plus LABAs in human airway smooth muscle and provide a rational explanation for the clinical efficacy of inhaled corticosteroid (glucocorticoid)/LABA combinations in obstructive airways diseases.

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-α (PKGI-α), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-α attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-α binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G q , terminating PAR-1 signaling. Disruption of the RGS-2–PKGI-α interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2 −/− mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-α binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.

Right ventricular (RV) pressure and volume loading induce RV fibrosis in association with RV dysfunction, morbidity, and mortality in repaired tetralogy of Fallot. Transforming‐growth factor‐β1 (TGFβ1) and platelet‐derived growth factor (PDGF) activate common downstream signaling pathways via TGFβ canonical and non‐canonical signaling to promote increased fibroblast activation, proliferation, and fibrosis in other organs. However, the role of PDGF and TGFβ canonical and non‐canonical signaling in RV fibrosis is incompletely characterized. Here, we investigate whether dual inhibition of TGFβ and PDGF, using Tranilast (TRN), improves RV remodeling in response to pulmonary artery banding (PAB) or pulmonary regurgitation (PR). TRN reduced TGFβ canonical signaling in PAB rats associated with improved RV fibrosis, hypertrophy, and RV function. In response to PR, TRN reduced PDGFRβ expression and normalized ERK1/2 activity, which were associated with reduced RV hypertrophy and improved diastolic relaxation. We identify that PDGF drives RV fibroblast proliferation and activation via SMAD2/3, JNK, and β‐catenin signaling. Our studies suggest that TGFβ and PDGF are interconnected drivers of RV fibrosis and hence synergistic targets to improve RV remodeling in RV pressure and volume loading.

Gradients of the fast transient outward K+ current (Ito,f) contribute to heterogeneity of ventricular repolarization in a number of species. Cardiac Ito,f levels and gradients change notably with heart disease. Human cardiac Ito,f appears to be encoded by the Kv4.3 pore-forming α-subunit plus the auxiliary KChIP2 β-subunit while mouse cardiac Ito,f requires Kv4.2 and Kv4.3 α-subunits plus KChIP2. Regional differences in cardiac Ito,f are associated with expression differences in Kv4.2 and KChIP2. Although Ito,f was reported to be absent in mouse ventricular cardiomyocytes lacking the Kv4.2 gene (Kv4.2-/-) when short depolarizing voltage pulses were used to activate voltage-gated K+ currents, in the present study, we showed that the use of long depolarization steps revealed a heteropodatoxin-sensitive Ito,f (at ~40% of the wild-type levels). Immunohistological studies further demonstrated membrane expression of Kv4.3 in Kv4.2-/- cardiomyocytes. Transmural Ito,f gradients across the left ventricular wall were reduced by ~3.5-fold in Kv4.2-/- heart, compared to wild-type. The Ito,f gradient in Kv4.2-/- hearts was associated with gradients in KChIP2 mRNA expression while in wild-type there was also a gradient in Kv4.2 expression. In conclusion, we found that Kv4.3-based Ito,f exists in the absence of Kv4.2, although with a reduced transmural gradient. Kv4.2-/- mice may be a useful animal model for studying Kv4.3-based Ito,f as observed in humans.

We have shown that both insulin and resveratrol (RSV) decrease neointimal hyperplasia in chow-fed rodents via mechanisms that are in part overlapping and involve the activation of endothelial nitric oxide synthase (eNOS). However, this vasculoprotective effect of insulin is abolished in high-fat-fed insulin-resistant rats. Since RSV, in addition to increasing insulin sensitivity, can activate eNOS via pathways that are independent of insulin signaling, such as the activation of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), we speculated that unlike insulin, the vasculoprotective effect of RSV would be retained in high-fat-fed rats. We found that high-fat feeding decreased insulin sensitivity and increased neointimal area and that RSV improved insulin sensitivity (p < 0.05) and decreased neointimal area in high-fat-fed rats (p < 0.05). We investigated the role of SIRT1 in the effect of RSV using two genetic mouse models. We found that RSV decreased neointimal area in high-fat-fed wild-type mice (p < 0.05), an effect that was retained in mice with catalytically inactive SIRT1 (p < 0.05) and in heterozygous SIRT1-null mice. In contrast, the effect of RSV was abolished in AMKPα2-null mice. Thus, RSV decreased neointimal hyperplasia after arterial injury in both high-fat-fed rats and mice, an effect likely not mediated by SIRT1 but by AMPKα2.

Supravalvular aortic stenosis is an autosomal-dominant disease of elastin (Eln) insufficiency caused by loss-of-function mutations or gene deletion. Recently, we have modeled this disease in mice (Eln+/-) and found that Eln haploinsufficiency results in unexpected changes in cardiovascular hemodynamics and arterial wall structure. Eln+/- animals were found to be stably hypertensive from birth, with a mean arterial pressure 25-30 mmHg higher than their wild-type counterparts. The animals have only moderate cardiac hypertrophy and live a normal life span with no overt signs of degenerative vascular disease. Examination of arterial mechanical properties showed that the inner diameters of Eln+/- arteries were generally smaller than wild-type arteries at any given intravascular pressure. Because the Eln+/- mouse is hypertensive, however, the effective arterial working diameter is comparable to that of the normotensive wild-type animal. Physiological studies indicate a role for the renin-angiotensin system in maintaining the hypertensive state. The association of hypertension with elastin haploinsufficiency in humans and mice strongly suggests that elastin and other proteins of the elastic fiber should be considered as causal genes for essential hypertension.

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)–/– mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS–/– mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS–/– mice, despite comparable reductions in tissue PO2. Paradoxically, nNOS–/– mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

A number of diverse G-protein signaling pathways have been shown to regulate insulin secretion from pancreatic β-cells. Accordingly, regulator of G-protein signaling (RGS) proteins have also been implicated in coordinating this process. One such protein, RGS4, is reported to show both positive and negative effects on insulin secretion from β-cells depending on the physiologic context under which it was studied. We here use an RGS4-deficient mouse model to characterize previously unknown G-protein signaling pathways that are regulated by RGS4 during glucose-stimulated insulin secretion from the pancreatic islets. Our data show that loss of RGS4 results in a marked deficiency in glucose-stimulated insulin secretion during both phase I and phase II of insulin release in intact mice and isolated islets. These deficiencies are associated with lower cAMP/PKA activity and a loss of normal calcium surge (phase I) and oscillatory (phase II) kinetics behavior in the RGS4-deficient β-cells, suggesting RGS4 may be important for regulation of both Gαi and Gαq signaling control during glucose-stimulated insulin secretion. Together, these studies add to the known list of G-protein coupled signaling events that are controlled by RGS4 during glucose-stimulated insulin secretion and highlight the importance of maintaining normal levels of RGS4 function in healthy pancreatic tissues.