Stimulation of the calcium-sensing receptor induces relaxations through CGRP and NK1 receptor-mediated pathways in male rat mesenteric arteries.

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.

NONO2P, a novel nitric oxide donor, causes vasorelaxation through NO/sGC/PKG pathway, K+ channels opening and SERCA activation.

BACKGROUND & AIMS: The treatment of cardiovascular diseases (CVD) could greatly benefit from using nitric oxide (NO) donors. This study aimed to investigate the mechanisms of action of NONO2P that contribute to the observed responses in the mesenteric artery. The hypothesis was that NONO2P would have similar pharmacological actions to sodium nitroprusside (SNP) and NO. METHODS: Male Wistar rats were euthanized to isolate the superior mesenteric artery for isometric tension recordings. NO levels were measured using the DAF-FM/DA dye, and cyclic guanosine monophosphate (cGMP) levels were determined using a cGMP-ELISA Kit. RESULTS: NONO2P presented a similar maximum efficacy to SNP. The free radical of NO (NO•) scavengers (PTIO; 100 µM and hydroxocobalamin; 30 µM) and nitroxyl anion (NO-) scavenger (L-cysteine; 3 mM) decreased relaxations promoted by NONO2P. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor (ODQ; 10 µM) nearly abolished the vasorelaxation. The cGMP-dependent protein kinase (PKG) inhibition (KT5823; 1 µM) attenuated the NONO2P relaxant effect. The vasorelaxant response was significantly attenuated by blocking inward rectifying K+ channels (Kir), voltage-operated K+ channels (KV), and large conductance Ca2+-activated K+ channels (BKCa). NONO2P-induced relaxation was attenuated by cyclopiazonic acid (10 µM), indicating that sarcoplasmic reticulum Ca2+-ATPase (SERCA) activation is involved in this relaxation. Moreover, NONO2P increased NO levels in endothelial cells and cGMP production. CONCLUSIONS: NONO2P induces vasorelaxation with the same magnitude as SNP, releasing NO• and NO-. Its vasorelaxant effect involves sGC, PKG, K+ channels opening, and SERCA activation, suggesting its potential as a therapeutic option for CVD.

Fat-Diets in Perinatal Stages Altered Nr3c2-Mediated Ca2+ Currents in Mesenteric Arteries of Offspring Rats.

SCOPE: Perinatal high-fat diets (PHF) can influence fetal/neonate development, resulting in cardiovascular pathogenesis, but precise mechanisms remain unclear. This study tests aldosterone receptor-mediated Ca2+ influx and the underlying mechanisms influenced by PHF. METHODS AND RESULTS: Maternal S.D. rats receive PHF during pregnancy and lactation periods. Their male offspring are fed normal diets after weaning for four months. Mesenteric arteries (MA) are for electrophysiological testing, Ca2+ imaging, target gene expression, and promotor methylation. PHF increases aldosterone receptor gene Nr3c2-mediated Ca2+ currents in the smooth muscle cells (SMCs) of the MA via L-type Ca2+ channels (LTCC) in the offspring. The increased expression of aldosterone-receptors and LTCC are responsible for an activated Nr3c2-LTCC pathway in the vasculature, eventually predisposes an increase of Ca2+ influx in the myocytes of resistance arteries. The inhibitor of aldosterone-receptors suppresses the increased Ca2+ currents in the SMCs. Nr3c2 and LTCC are upregulated through the transcriptional mechanism in methylation, which can be reversed in the functional changes by methylation inhibitor 5AZA. CONCLUSION: The results firstly demonstrate that aldosterone-receptor activation can stimulate Ca2+ currents via LTCC in vascular myocytes, which can be altered by perinatal foods via epigenetic changes of DNA methylation in the promoters of Nr3c2 and LTCC.

Vascular Smooth Muscle TRPV4 (Transient Receptor Potential Vanilloid Family Member 4) Channels Regulate Vasoconstriction and Blood Pressure in Obesity.

BACKGROUND: Vascular endothelium and smooth muscle work together to keep the balance of vasomotor tone and jointly maintain vascular homeostasis. Ca2+-permeable ion channel TRPV4 (transient receptor potential vanilloid family member 4) in endothelial cells regulates endothelium-dependent vasodilation and contraction in various states. However, how vascular smooth muscle cell TRPV4 (TRPV4SMC) contributes to vascular function and blood pressure regulation in physiological and pathologically obese condition has not been fully studied. METHODS: We generated smooth muscle TRPV4-deficient mice and developed diet-induced obese mice model and analyzed the role of TRPV4SMC in intracellular Ca2+ ([Ca2+]i) regulation and vasoconstriction. Vasomotor changes of mouse mesenteric artery were measured by wire, and pressure myography. [Ca2+]i were measured by fluo-4 staining. Blood pressure was recorded by telemetric device. RESULTS: Vascular TRPV4SMC played different roles in regulating vasomotor tone than endothelial TRPV4 due to their different features of [Ca2+]i regulation. Loss of TRPV4SMC attenuated U46619- and phenylephrine-induced contraction, suggesting its involvement in regulating vascular contractility. Mesenteric arteries from obese mice showed SMC hyperplasia, suggesting an increased level of TRPV4SMC. Loss of TRPV4SMC did not influence the development of obesity but protected mice from obesity-induced vasoconstriction and hypertension. In arteries deficient in SMC TRPV4, SMCs F-actin polymerization and RhoA dephosphorylation were attenuated under contractile stimuli. Moreover, SMC-dependent vasoconstriction was inhibited in human resistance arteries with TRPV4 inhibitor application. CONCLUSIONS: Our data identify TRPV4SMC as a regulator of vascular contraction in both physiological states and pathologically obese mice. TRPV4SMC contributes to the ontogeny of vasoconstriction and hypertension induced by TRPV4SMC over-expression in obese mice mesenteric artery.

Food Restriction Augmented Alpha1-Adrenergic Mediated Contraction in Mesenteric Arteries.

Food restriction (FR) enhances sensitivity to cardiopulmonary reflexes and α1-adrenoreceptors in females in the presence of hypotension. However, the effect of FR on cardiopulmonary and vascular function in males is not well-understood. This study examines the effects of FR on cardiopulmonary, isolated arterial function, and potential underlying mechanisms. Male Sprague-Dawley (SD) rats were randomly divided into 3 groups and monitored for 5 weeks: (1) control (n = 30), (2) 20% food reduction (FR20, n = 30), and (3) 40% food reduction (FR40, n = 30). Non-invasive blood pressure was measured twice a week. Pulmonary arterial pressure (PAP) was measured using isolated/perfused lungs. The isolated vascular reactivity was assessed using double-wire myographs. FR rats exhibited a lower mean arterial pressure and heart rate; however, only the FR40 group exhibited statistically significant differences. We observed that FR enhanced sensitivity (EC50) to vasoconstriction induced by the α1-adrenoreceptor phenylephrine (PhE) but not to serotonin, U46619, or high K+ in the mesenteric arteries. PhE-mediated vasoconstriction in the mesenteric arteries was eliminated in the presence of the eNOS inhibitor (L-NAME). In addition, incubation with NOX2/4 inhibitors (apocynin, GKT137831, and VAS2870) and the reactive oxygen species (ROS) scavenger inhibitor (Tiron) eliminated the differences in PhE-mediated vasoconstriction, but the cyclooxygenase inhibitor (indomethacin) in the mesenteric arteries did not. Augmentation of α1-adrenergic-mediated contraction via the inhibition of the eNOS-NO pathway increased the activation of ROS through NOX2/4 in response to FR. Reduced eNOS-NO signaling may be a pathophysiological counterbalance to prevent hypovolemic shock in response to FR.

The Use of Wire Myography to Investigate Vascular Tone and Function.

Wire myography enables the investigation of vascular tone and function of small vessels. The vessel of interest is harvested from the experimental model of choice, and then mounted as ring preparations onto a four-channel wire myograph. This technique enables ex vivo measurements of isometric response of vessels to different pharmacological agents. Here we describe in detail how to dissect, mount, and normalize vessels for the wire myography technique. We will also provide examples of how to construct concentration-response curves to a contractile and vasodilatory pharmacological agent.

Early Inactivation of Membrane Estrogen Receptor Alpha (ERα) Recapitulates the Endothelial Dysfunction of Aged Mouse Resistance Arteries.

Flow-mediated dilation (FMD) of resistance arteries is essential for tissue perfusion but it decreases with ageing. As estrogen receptor alpha (Erα encoded by Esr1), and more precisely membrane ERα, plays an important role in FMD in young mice in a ligand-independent fashion, we evaluated its influence on this arteriolar function in ageing. We first confirmed that in young (6-month-old) mice, FMD of mesenteric resistance arteries was reduced in Esr1-/- (lacking ERα) and C451A-ERα (lacking membrane ERα). In old (24-month-old) mice, FMD was reduced in WT mice compared to young mice, whereas it was not further decreased in Esr1-/- and C451A-ERα mice. Markers of oxidative stress were similarly increased in old WT and C451A-ERα mice. Reduction in oxidative stress with superoxide dismutase plus catalase or Mito-tempo, which reduces mitochondrial superoxide restored FMD to a normal control level in young C451A-ERα mice as well as in old WT mice and old C451A-ERα mice. Estradiol-mediated dilation was absent in old WT mice. We conclude that oxidative stress is a key event in the decline of FMD, and that an early defect in membrane ERα recapitulates phenotypically and functionally ageing of these resistance arteries. The loss of this function could take part in vascular ageing.

The effect of TNF-α inhibitor treatment on microRNAs and endothelial function in collagen induced arthritis.

Chronic inflammation causes dysregulated expression of microRNAs. Aberrant microRNA expression is associated with endothelial dysfunction. In this study we determined whether TNF-α inhibition impacted the expression of miRNA-146a-5p and miRNA-155-5p, and whether changes in the expression of these miRNAs were related to inflammation-induced changes in endothelial function in collagen-induced arthritis (CIA). Sixty-four Sprague-Dawley rats were divided into control (n = 24), CIA (n = 24) and CIA+etanercept (n = 16) groups. CIA and CIA+etanercept groups were immunized with bovine type-II collagen, emulsified in incomplete Freund's adjuvant. Upon signs of arthritis, the CIA+etanercept group received 10mg/kg of etanercept intraperitoneally, every three days. After six weeks of treatment, mesenteric artery vascular reactivity was assessed using wire-myography. Serum concentrations of TNF-α, C-reactive protein, interleukin-6, vascular adhesion molecule-1 (VCAM-1) and pentraxin-3 (PTX-3) were measured by ELISA. Relative expression of circulating miRNA-146a-5p and miRNA-155-5p were determined using RT-qPCR. Compared to controls, circulating miRNA-155-5p, VCAM-1 and PTX-3 concentrations were increased, and vessel relaxation was impaired in the CIA (all p<0.05), but not in the CIA+etanercept (all p<0.05) groups. The CIA group had greater miRNA-146a-5p expression compared to the CIA+etanercept group (p = 0.005). Independent of blood pressure, miRNA-146a-5p expression was associated with increased PTX-3 concentrations (p = 0.03), while miRNA-155-5p expression was associated with impaired vessel relaxation (p = 0.01). In conclusion, blocking circulating TNF-α impacted systemic inflammation-induced increased expression of miRNA-146a-5p and miRNA-155-5p, which were associated with endothelial inflammation and impaired endothelial dependent vasorelaxation, respectively.

Microcirculatory and glycocalyx properties are lowered by high-salt diet but augmented by Western diet in genetically heterogeneous mice.

Many individuals in industrialized societies consume a high-salt, Western diet(WD); however, the effects of this diet on microcirculatory properties and glycocalyx barrier function are unknown. Young genetically heterogeneous male and female mice underwent 12 wk of normal chow (NC) diet, NC diet with 4% salt (NC4%), Western diet (WD), or WD with 4% salt (WD4%). Microcirculatory properties and glycocalyx barrier function were evaluated in the mesenteric microcirculation, using an intravital microscope equipped with an automated capture and analysis system. Total microvascular density summed across 4- to 25-µm microvessel segment diameters was lower in NC4% than in NC and WD (P < 0.05). Perfused boundary region (PBR), a marker of glycocalyx barrier function, averaged across 4- to 25-µm microvessel segment diameters was similar between NC and NC4%, as well as between WD and WD4% (P > 0.05). PBR was lower in WD and WD4% than in NC and NC4% (P < 0.05), indicating augmented glycocalyx barrier function in WD and WD4%. There were strong, inverse relationships between PBR and adiposity and blood glucose (r = -0.44 to -0.61, P < 0.05). In summary, NC4% induces deleterious effects on microvascular density, whereas WD augments glycocalyx barrier function. Interestingly, the combination of high-salt, Western diet in WD4% resulted in lower total microvascular density like NC4% and augmented glycocalyx barrier function like WD. These data suggest distinct microcirculatory adaptations to high-salt and Western diets that coincide when these diets are combined in young genetically heterogeneous male and female mice.NEW & NOTEWORTHY Many individuals in industrialized societies consume a combination of high-salt and Western diet; however, the effects of this diet on microcirculatory and glycocalyx properties are unknown. This study reveals that a high-salt diet lowers microcirculatory and glycocalyx properties, whereas a Western diet augments glycocalyx barrier function and thickness. Taken together, these data indicate that there are distinct microcirculatory adaptations to high-salt and Western diets that coincide when high-salt and Western diets are combined.

Identification of Piezo1 channels in perivascular adipose tissue (PVAT) and their potential role in vascular function.

The vasculature constantly experiences distension/pressure exerted by blood flow and responds to maintain homeostasis. We hypothesized that activation of the stretch sensitive, non-selective cation channel Piezo1 would directly increase vascular contraction in a way that might be modified by perivascular adipose tissue (PVAT). The presence and function of Piezo1 was investigated by RT-PCR, immunohistochemistry, and isolated tissue bath contractility. Superior and mesenteric resistance arteries, aortae, and their PVATs from male Sprague Dawley rats were used. Piezo1 mRNA was detected in aortic vessels, aortic PVAT, mesenteric vessels, and mesenteric PVAT. Both adipocytes and stromal vascular fraction of mesenteric PVAT expressed Piezo1 mRNA. In PVAT, expression of Piezo1 mRNA was greater in magnitude than that of Piezo2, transient receptor potential cation channel, subfamily V, member 4 (TRPV4), anoctamin 1, calcium activated chloride channel (TMEM16), and Pannexin1 (Panx1). Piezo1 protein was present in endothelium and PVAT of rat aortic and in PVAT of mesenteric artery. The Piezo1 agonists Yoda1 and Jedi2 (1 nM - 10 µM) did not stimulate aortic contraction [max < 10% phenylephrine (PE) 10 µM contraction] or relaxation in tissues + or -PVAT. Depolarizing the aorta by modestly elevated extracellular K+ did not unmask aortic contraction to Yoda1 (max <10% PE 10 µM contraction). Finally, the Piezo1 antagonist Dooku1 did not modify PE-induced aorta contraction + or -PVAT. Surprisingly, Dooku1 directly caused aortic contraction in the absence (Dooku1 =26 ± 11; Vehicle = 11 ± 11%PE contraction) but not in the presence of PVAT (Dooku1 = 2 ± 1; Vehicle = 8 ± 5% PE contraction). Thus, Piezo1 is present and functional in the isolated rat aorta but does not serve direct vascular contraction with or without PVAT. We reaffirmed the isolated mouse aorta relaxation to Yoda1, indicating a species difference in Piezo1 activity between mouse and rat.

Cystamine reduces vascular stiffness in Western diet-fed female mice.

Consumption of diets high in fat, sugar, and salt (Western diet, WD) is associated with accelerated arterial stiffening, a major independent risk factor for cardiovascular disease (CVD). Women with obesity are more prone to develop arterial stiffening leading to more frequent and severe CVD compared with men. As tissue transglutaminase (TG2) has been implicated in vascular stiffening, our goal herein was to determine the efficacy of cystamine, a nonspecific TG2 inhibitor, at reducing vascular stiffness in female mice chronically fed a WD. Three experimental groups of female mice were created. One was fed regular chow diet (CD) for 43 wk starting at 4 wk of age. The second was fed a WD for the same 43 wk, whereas a third cohort was fed WD, but also received cystamine (216 mg/kg/day) in the drinking water during the last 8 wk on the diet (WD + C). All vascular stiffness parameters assessed, including aortic pulse wave velocity and the incremental modulus of elasticity of isolated femoral and mesenteric arteries, were significantly increased in WD- versus CD-fed mice, and reduced in WD + C versus WD-fed mice. These changes coincided with respectively augmented and diminished vascular wall collagen and F-actin content, with no associated effect in blood pressure. In cultured human vascular smooth muscle cells, cystamine reduced TG2 activity, F-actin:G-actin ratio, collagen compaction capacity, and cellular stiffness. We conclude that cystamine treatment represents an effective approach to reduce vascular stiffness in female mice in the setting of WD consumption, likely because of its TG2 inhibitory capacity.NEW & NOTEWORTHY This study evaluates the novel role of transglutaminase 2 (TG2) inhibition to directly treat vascular stiffness. Our data demonstrate that cystamine, a nonspecific TG2 inhibitor, improves vascular stiffness induced by a diet rich in fat, fructose, and salt. This research suggests that TG2 inhibition might bear therapeutic potential to reduce the disproportionate burden of cardiovascular disease in females in conditions of chronic overnutrition.

C1q/tumor necrosis factor-related protein-3 improves microvascular endothelial function in diabetes through the AMPK/eNOS/NO· signaling pathway.

The repair of vascular endothelial cell dysfunction is an encouraging approach for the treatment of vascular complications associated with diabetes. It has been demonstrated that members of C1q/tumor necrosis factor-related protein (CTRP) family may improve endothelial function. Nevertheless, the protective properties of CTRPs in diabetic microvascular complications continue to be mostly unknown. Here, we demonstrate that the C1q-like globular domain of CTRP3, CTRP5, and CTRP9 (gCTRP3, 5, 9) exerted a vasorelaxant effect on the microvasculature, of which gCTRP3 was the most powerful one. In a murine model of type 2 diabetes mellitus, serum gCTRP3 level and endothelial function decreased markedly compared with controls. Two weeks of gCTRP3 treatment (0.5 µg/g/d) enhanced endothelium-dependent relaxation in microvessels, increased nitric oxide (NO·) production, and reduced retinal vascular leakage. In addition, Western blotting in human retinal microvascular endothelial cells indicated that gCTRP3 triggered AMP-activated protein kinase-α (AMPKα), hence increasing the endothelial NO synthase (eNOS) level and NO· production. In addition, incubation with gCTRP3 in vitro ameliorated the endothelial dysfunction induced by high glucose in the branch of the mesenteric artery. Blockade of either eNOS or AMPKα completely abolished the effects of gCTRP3 described above. Taken together, we demonstrate for the first time that gCTRP3 improves impaired vasodilatation of microvasculature in diabetes by ameliorating endothelial cell function through the AMPK/eNOS/NO· signaling pathway. This finding may suggest an effective intervention against diabetes-associated microvascular complications.

Dual Role of Thrombospondin-1 in Flow-Induced Remodeling.

(1) Background: Chronic increases in blood flow, as in cardiovascular diseases, induce outward arterial remodeling. Thrombospondin-1 (TSP-1) is known to interact with matrix proteins and immune cell-surface receptors, but its contribution to flow-mediated remodeling in the microcirculation remains unknown. (2) Methods: Mesenteric arteries were ligated in vivo to generate high- (HF) and normal-flow (NF) arteries in wild-type (WT) and TSP-1-deleted mice (TSP-1-/-). After 7 days, arteries were isolated and studied ex vivo. (3) Results: Chronic increases in blood flow induced outward remodeling in WT mice (increasing diameter from 221 ± 10 to 280 ± 10 µm with 75 mmHg intraluminal pressure) without significant effect in TSP-1-/- (296 ± 18 to 303 ± 14 µm), neutropenic or adoptive bone marrow transfer mice. Four days after ligature, pro inflammatory gene expression levels (CD68, Cox2, Gp91phox, p47phox and p22phox) increased in WT HF arteries but not in TSP-1-/- mice. Perivascular neutrophil accumulation at day 4 was significantly lower in TSP-1-/- than in WT mice. (4) Conclusions: TSP-1 origin is important; indeed, circulating TSP-1 participates in vasodilation, whereas both circulating and tissue TSP-1 are involved in arterial wall thickness and diameter expansion.

Age Impairs Soluble Guanylyl Cyclase Function in Mouse Mesenteric Arteries.

Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10-6 mol L-1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10-6 mol L-1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.

Aerobic training-mediated DNA hypermethylation of Agtr1a and Mas1 genes ameliorate mesenteric arterial function in spontaneously hypertensive rats.

BACKGROUND: The imbalance of vasoconstrictor and vasodilator axes of the renin-angiotensin system (RAS) is observed in hypertension. Exercise regulates RAS level and improves vascular function. This study focused on the contribution of RAS axes in vascular function of mesenteric arteries and exercise-induced DNA methylation of the Agtr1a (AT1aR) and Mas1 (MasR) genes in hypertension. METHODS: Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats were randomized into exercise or sedentary group. Levels of plasma RAS components, vascular tone, and DNA methylation markers were measured. RESULTS: Blood pressure of SHR was markedly reduced after 12 weeks of aerobic exercise. RAS peptides in plasma were all increased with an imbalanced upregulation of Ang II and Ang-(1-7) in SHR, exercise revised the level of RAS and increased Ang-(1-7)/Ang II. The vasoconstriction response induced by Ang II was mainly via type 1 receptors (AT1R), while this contraction was inhibited by Mas receptor (MasR). mRNA and protein of AT1R and MasR were both upregulated in SHR, whereas exercise significantly suppressed this imbalanced increase and increased MasR/AT1R ratio. Exercise hypermethylated Agtr1a and Mas1 genes, associating with increased DNMT1 and DNMT3b and SAM/SAH. CONCLUSIONS: Aerobic exercise ameliorates vascular function via hypermethylation of the Agtr1a and Mas1 genes and restores the vasoconstrictor and vasodilator axes balance.

A primer for measuring cGMP signaling and cGMP-mediated vascular relaxation.

Soluble guanylyl cyclase (sGC, also called GC1) is the main receptor for nitric oxide (NO) that catalyzes the production of the second messenger molecule, 3'5' cyclic guanosine monophosphate (cGMP) leading to vasorelaxation, and inhibition of leukocyte recruitment and platelet aggregation. Enhancing cGMP levels, through sGC agonism or inhibition of cGMP breakdown via phosphodiesterase inhibition, has yielded FDA approval for several cGMP modifier therapies for treatment of cardiovascular and pulmonary diseases. While basic research continues to improve our understanding of cGMP signaling and as new therapies evolve to elevate cGMP levels, we provide a short methodological primer for measuring cGMP and cGMP-mediated vascular relaxation for investigators.

Empagliflozin Relaxes Resistance Mesenteric Arteries by Stimulating Multiple Smooth Muscle Cell Voltage-Gated K+ (KV) Channels.

The antidiabetic drug empagliflozin is reported to produce a range of cardiovascular effects, including a reduction in systemic blood pressure. However, whether empagliflozin directly modulates the contractility of resistance-size mesenteric arteries remains unclear. Here, we sought to investigate if empagliflozin could relax resistance-size rat mesenteric arteries and the associated underlying molecular mechanisms. We found that acute empagliflozin application produces a concentration-dependent vasodilation in myogenic, depolarized and phenylephrine (PE)-preconstricted mesenteric arteries. Selective inhibition of smooth muscle cell voltage-gated K+ channels KV1.5 and KV7 abolished empagliflozin-induced vasodilation. In contrast, pharmacological inhibition of large-conductance Ca2+-activated K+ (BKCa) channels and ATP-sensitive (KATP) channels did not abolish vasodilation. Inhibition of the vasodilatory signaling axis involving endothelial nitric oxide (NO), smooth muscle cell soluble guanylyl cyclase (sGC) and protein kinase G (PKG) did not abolish empagliflozin-evoked vasodilation. Inhibition of the endothelium-derived vasodilatory molecule prostacyclin (PGI2) had no effect on the vasodilation. Consistently, empagliflozin-evoked vasodilation remained unaltered by endothelium denudation. Overall, our data suggest that empagliflozin stimulates smooth muscle cell KV channels KV1.5 and KV7, resulting in vasodilation in resistance-size mesenteric arteries. This study demonstrates for the first time a novel mechanism whereby empagliflozin regulates arterial contractility, resulting in vasodilation. Due to known antihypertensive properties, treatment with empagliflozin may complement conventional antihypertensive therapy.

Trehalose, a natural disaccharide, reduces stroke occurrence in the stroke-prone spontaneously hypertensive rat.

Cerebrovascular disease, a frequent complication of hypertension, is a major public health issue for which novel therapeutic and preventive approaches are needed. Autophagy activation is emerging as a potential therapeutic and preventive strategy toward stroke. Among usual activators of autophagy, the natural disaccharide trehalose (TRE) has been reported to be beneficial in preclinical models of neurodegenerative diseases, atherosclerosis and myocardial infarction. In this study, we tested for the first time the effects of TRE in the stroke-prone spontaneously hypertensive rat (SHRSP) fed with a high-salt stroke permissive diet (JD). We found that TRE reduced stroke occurrence and renal damage in high salt-fed SHRSP. TRE was also able to decrease systolic blood pressure. Through ex-vivo studies, we assessed the beneficial effect of TRE on the vascular function of high salt-fed SHRSP. At the molecular level, TRE restored brain autophagy and reduced mitochondrial mass, along with the improvement of mitochondrial function. The beneficial effects of TRE were associated with increased nuclear translocation of TFEB, a transcriptional activator of autophagy. Our results suggest that TRE may be considered as a natural compound efficacious for the prevention of hypertension-related target organ damage, with particular regard to stroke and renal damage.

Chronic 3D Vascular-Immune Interface Established by Coculturing Pressurized Resistance Arteries and Immune Cells.

[Figure: see text].

Allisartan ameliorates vascular remodeling through regulation of voltage-gated potassium channels in hypertensive rats.

BACKGROUND: The objective of the present study was to determine the effect of allisartan, a new angiotensin II type 1 receptor antagonist on vascular remodeling through voltage gated potassium channels (Kv7) in hypertensive rats. METHODS: The study included a total of 47 Sprague Dawley (SD) rats. The animals were randomized to sham operation (n = 14), untreated hypertensive control group (n = 18) and allisartan treatment group (n = 15). Using renal artery stenosis, hypertension was induced in animals. Single dose of allisartan was administered intra-gastrically to animals in the allisartan treatment group and match placebo in the other 2 groups. Wire myography was used to measure the muscle tension in isolated mesenteric arteries from the animals. Real-time polymerase chain reaction was used to quantify the expression of Kv7 channel mRNA subunits. RESULTS: After 4 weeks of treatment, a significant decrease in mean arterial, systolic and diastolic blood pressure (SBP and DBP) was observed in allisartan treatment group compared to hypertension control group. The median arterial wall thickness and area/diameter ratio reduced significantly in treatment group compared to untreated hypertension group (P < 0.05). Wire myography demonstrated increased relaxation of mesenteric artery with increase in concentration of ML213. A significant up-regulation in the expression of all Kv7 mRNA subunits was observed in allisartan group compared to untreated hypertension group. CONCLUSIONS: From the results, allisartan was found to lower BP and preserve vascular remodeling through Kv7 channels.