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Idiopathic pulmonary arterial hypertension (IPAH) is usually without an identified genetic cause, despite clinical and molecular similarity to bone morphogenetic protein receptor type 2 mutation-associated heritable pulmonary arterial hypertension (PAH). There is phenotypic heterogeneity in IPAH, with a minority of patients showing long-term improvement with calcium channel-blocker therapy. We sought to identify gene variants (GVs) underlying IPAH and determine whether GVs differ in vasodilator-responsive IPAH (VR-PAH) versus vasodilator-nonresponsive IPAH (VN-PAH). We performed whole-exome sequencing (WES) on 36 patients with IPAH: 17 with VR-PAH and 19 with VN-PAH. Wnt pathway differences were explored in human lung fibroblasts. We identified 1,369 genes with 1,580 variants unique to IPAH. We used a gene ontology approach to analyze variants and identified overrepresentation of several pathways, including cytoskeletal function and ion binding. By mapping WES data to prior genome-wide association study data, Wnt pathway genes were highlighted. Using the connectivity map to define genetic differences between VR-PAH and VN-PAH, we found enrichment in vascular smooth muscle cell contraction pathways and greater genetic variation in VR-PAH versus VN-PAH. Using human lung fibroblasts, we found increased stimulated Wnt activity in IPAH versus controls. A pathway-based analysis of WES data in IPAH demonstrated multiple rare GVs that converge on key biological pathways, such as cytoskeletal function and Wnt signaling pathway. Vascular smooth muscle contraction-related genes were enriched in VR-PAH, suggesting a potentially different genetic predisposition for VR-PAH. This pathway-based approach may be applied to next-generation sequencing data in other diseases to uncover the contribution of unexpected or multiple GVs to a phenotype.

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α 2C -adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates. Blood flow in the skin of mammals changes in response to cold, but the mechanisms driving this response are unclear. Aubdool et al . show that the non-selective cation channel, TRPA1, is a vascular cold sensor and required for the vascular protective response to local cold exposure.

Inhibition of Notch signaling in macrophages is known to reduce inflammation, however, its role in regulating vascular hyporeactivity in sepsis is unknown. Thus we aimed to evaluate the effect of sepsis on vascular Notch signaling. Polymicrobial sepsis was induced by caecal ligation and puncture (CLP) in mice. mRNA expressions of Notch receptors (Notch1,3) and ligands (Jag1, Dll4), and downstream effector genes (Hey1, MLCK, MYPT1) were assessed by RT-qPCR. Protein level of activated Notch (NICD) was assessed by Western blot and immuno-histochemistry. Isometric tension in isolated aortic rings was measured by wire myography.CLP down-regulated aortic expression of Notch3, Jag1 and Dll4 as compared to control mice. Additionally, the protein level of NICD was found to be lesser in aortic tissue sections from CLP mice. Expression of Hey1 and MLCK were attenuated whereas MYPT1 expression was increased in septic mouse aorta. DAPT pretreatment did not improve CLP-induced vascular hyporeactivity to NA, CaCl 2 and high K + (80 mM), rather significantly attenuated the aortic response to these vasoconstrictors in control mice. Treatment with 1400 W reversed attenuated Notch3 (but not Jag1 and MLCK) expression in septic mouse aorta. In conclusion, sepsis significantly attenuated the Notch (especially Notch3) signaling in mouse aorta along with reduction in contractile gene expression and vasoconstriction response. Further, iNOS/NO pathway was involved in sepsis-induced down-regulation of Notch3 receptor. Thus systemic inhibition of Notch signaling during sepsis may have serious impact on sepsis-induced vascular hyporeactivity.

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2cre/+SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.

A high amount of salt in the diet increases blood pressure (BP) and leads to salt-sensitive hypertension in individuals with impaired renal sodium excretion. Small guanosine triphosphatase (GTP)ase Rho and Rac, activated by salt intake, play important roles in the pathogenesis of salt-sensitive hypertension as key switches of intracellular signaling. Focusing on Rho, high salt intake in the central nervous system increases sodium concentrations of cerebrospinal fluid in salt-sensitive subjects via Rho/Rho kinase and renin-angiotensin system activation and causes increased brain salt sensitivity and sympathetic nerve outflow in BP control centers. In vascular smooth muscle cells, Rho-guanine nucleotide exchange factors and Rho determine sensitivity to vasoconstrictors such as angiotensin II (Ang II), and facilitate vasoconstriction via G-protein and Wnt pathways, leading to increased vascular resistance, including in the renal arteries, in salt-sensitive subjects with high salt intake. In the vascular endothelium, Rho/Rho kinase inhibits nitric oxide (NO) production and function, and high salt amounts further augment Rho activity via asymmetric dimethylarginine, an endogenous inhibitor of NO synthetase, causing aberrant relaxation and increased vascular tone. Rho-associated mechanisms are deeply involved in the development of salt-sensitive hypertension, and their further elucidation can help in developing effective protection and new therapies.

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

N-methyl- d -aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A–D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.

Preeclampsia is a major health problem in human pregnancy, severely complicating 5-8% of all pregnancies. The emerging molecular mechanism is that conditions like hypoxic stress trigger the release of placental messengers into the maternal circulation, which causes preeclampsia. Our objective was to develop an in vitro model, which can be used to further elucidate the molecular mechanisms of preeclampsia and which might be used to find a remedy. Human non-complicated term placentas were collected. Placental explants were subjected to severe hypoxia and the conditioned media were added to chorionic arteries that were mounted into a myograph. Contractile responses of the conditioned media were determined, as well as effects on thromboxane-A2 (U46619) induced contractility. To identify the vasoactive compounds present in the conditioned media, specific receptor antagonists were evaluated. Factors released by placental explants generated under severe hypoxia induced an increased vasoconstriction and vascular contractility to thromboxane-A2. It was found that agonists for the angiotensin-I and endothelin-1 receptor released by placental tissue under severe hypoxia provoke vasoconstriction. The dietary antioxidant quercetin could partially prevent the acute and sustained vascular effects in a concentration-dependent manner. Both the acute vasoconstriction, as well as the increased contractility to U46619 are in line with the clinical vascular complications observed in preeclampsia. Data obtained with quercetin supports that our model opens avenues for e.g. nutritional interventions aimed at treating or preventing preeclampsia.

The objective of this study was to systematically investigate cardiac and peripheral vasomotor autonomic functions in late-onset transthyretin Val30Met familial amyloid polyneuropathy (FAP ATTR Val30Met) patients from non-endemic areas. The coefficient of variation of R–R intervals (CVR-R), responses to the Valsalva manoeuvre, head-up tilt test with impedance cardiography, noradrenaline infusion test, and (123)I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy were assessed in eight patients. Although only four patients manifested orthostatic hypotension during the head-up tilt test, CVR-R, responses to the Valsalva manoeuvre, and myocardial MIBG uptake indicated a higher prevalence of cardiac sympathetic and parasympathetic dysfunction. Total peripheral resistance at 60° tilt did not increase from baseline values in five of six examined patients. An infusion of low-dose noradrenaline induced an increase in systolic blood pressure in all patients. The extent of the change in systolic blood pressure negatively correlated to that in total peripheral resistance ( p  < 0.05). Patients with poor vasoconstrictor responses to orthostatic stress tended to exhibit severe reduction of unmyelinated fibres in sural nerve biopsy specimens. In conclusion, both cardiac and peripheral vasomotor autonomic dysfunctions were prevalent in late-onset FAP ATTR Val30Met patients from non-endemic areas, even in those without orthostatic intolerance. However, vasoconstriction by alpha-adrenoceptor agonists was preserved even after denervation, carrying important implications for the management of orthostatic hypotension in FAP.

The importance of excess salt intake in the pathogenesis of hypertension is widely recognized. Blood pressure is controlled primarily by salt and water balance because of the infinite gain property of the kidney to rapidly eliminate excess fluid and salt. Up to fifty percent of patients with essential hypertension are salt-sensitive, as manifested by a rise in blood pressure with salt loading. We conducted a two-stage genetic analysis in hypertensive patients very accurately phenotyped for their salt-sensitivity. All newly discovered never treated before, essential hypertensives underwent an acute salt load to monitor the simultaneous changes in blood pressure and renal sodium excretion. The first stage consisted in an association analysis of genotyping data derived from genome-wide array on 329 subjects. Principal Component Analysis demonstrated that this population was homogenous. Among the strongest results, we detected a cluster of SNPs located in the first introns of PRKG1 gene (rs7897633, p = 2.34E-05) associated with variation in diastolic blood pressure after acute salt load. We further focused on two genetic loci, SLC24A3 and SLC8A1 (plasma membrane sodium/calcium exchange proteins, NCKX3 and NCX1, respectively) with a functional relationship with the previous gene and associated to variations in systolic blood pressure (the imputed rs3790261, p = 4.55E-06; and rs434082, p = 4.7E-03). In stage 2, we characterized 159 more patients for the SNPs in PRKG1, SLC24A3 and SLC8A1. Combined analysis showed an epistatic interaction of SNPs in SLC24A3 and SLC8A1 on the pressure-natriuresis (p interaction = 1.55E-04, p model = 3.35E-05), supporting their pathophysiological link in cellular calcium homeostasis. In conclusions, these findings point to a clear association between body sodium-blood pressure relations and molecules modulating the contractile state of vascular cells through an increase in cytoplasmic calcium concentration.