Expression of endothelin type B receptors (EDNRB) on smooth muscle cells is controlled by MKL2, ternary complex factors, and actin dynamics.

The endothelin type B receptor (ETB or EDNRB) is highly plastic and is upregulated in smooth muscle cells (SMCs) by arterial injury and following organ culture in vitro. We hypothesized that this transcriptional plasticity may arise, in part, because EDNRB is controlled by a balance of transcriptional inputs from myocardin-related transcription factors (MRTFs) and ternary complex factors (TCFs). We found significant positive correlations between the TCFs ELK3 and FLI1 versus EDNRB in human arteries. The MRTF MKL2 also correlated with EDNRB. Overexpression of ELK3, FLI1, and MKL2 in human coronary artery SMCs promoted expression of EDNRB, and the effect of MKL2 was antagonized by myocardin (MYOCD), which also correlated negatively with EDNRB at the tissue level. Silencing of MKL2 reduced basal EDNRB expression, but depolymerization of actin using latrunculin B (LatB) or overexpression of constitutively active cofilin, as well as treatment with the Rho-associated kinase (ROCK) inhibitor Y27632, increased EDNRB in a MEK/ERK-dependent fashion. Transcript-specific primers indicated that the second EDNRB transcript (EDNRB_2) was targeted, but this promoter was largely unresponsive to LatB and was inhibited rather than stimulated by MKL2 and FLI1, suggesting distant control elements or an indirect effect. LatB also reduced expression of endothelin-1, but supplementation experiments argued that this was not the cause of EDNRB induction. EDNRB finally changed in parallel with ELK3 and FLI1 in rat and human carotid artery lesions. These studies implicate the actin cytoskeleton and ELK3, FLI1, and MKL2 in the transcriptional control of EDNRB and increase our understanding of the plasticity of this receptor.

Vasomotor function in rat arteries after ex vivo and intragastric exposure to food-grade titanium dioxide and vegetable carbon particles.

BACKGROUND: Humans are continuously exposed to particles in the gastrointestinal tract. Exposure may occur directly through ingestion of particles via food or indirectly by removal of inhaled material from the airways by the mucociliary clearance system. We examined the effects of food-grade particle exposure on vasomotor function and systemic oxidative stress in an ex vivo study and intragastrically exposed rats. METHODS: In an ex vivo study, aorta rings from naïve Sprague-Dawley rats were exposed for 30 min to food-grade TiO2 (E171), benchmark TiO2 (Aeroxide P25), food-grade vegetable carbon (E153) or benchmark carbon black (Printex 90). Subsequently, the vasomotor function was assessed in wire myographs. In an in vivo study, lean Zucker rats were exposed intragastrically once a week for 10 weeks to vehicle, E171 or E153. Doses were comparable to human daily intake. Vasomotor function in the coronary arteries and aorta was assessed using wire myographs. Tetrahydrobiopterin, ascorbate, malondialdehyde and asymmetric dimethylarginine were measured in blood as markers of oxidative stress and vascular function. RESULTS: Direct exposure of E171 to aorta rings ex vivo increased the acetylcholine-induced vasorelaxation and 5-hydroxytryptamine-induced vasocontraction. E153 only increased acetylcholine-induced vasorelaxation, and Printex 90 increased the 5-hydroxytryptamine-induced vasocontraction, whereas Aeroxide P25 did not affect the vasomotor function. In vivo exposure showed similar results as ex vivo exposure; increased acetylcholine-induced vasorelaxation in coronary artery segments of E153 and E171 exposed rats, whereas E171 exposure altered 5-hydroxytryptamine-induced vasocontraction in distal coronary artery segments. Plasma levels of markers of oxidative stress and vascular function showed no differences between groups. CONCLUSION: Gastrointestinal tract exposure to E171 and E153 was associated with modest albeit statistically significant alterations in the vasocontraction and vasorelaxation responses. Direct particle exposure to aorta rings elicited a similar type of response. The vasomotor responses were not related to biomarkers of systemic oxidative stress.

Role of pannexin and adenosine triphosphate (ATP) following myocardial ischemia/reperfusion.

OBJECTIVES: The purinergic system has not been investigated in detail following ischemia/reperfusion (I/R) injury in the heart. In the present study, we focus on both release and response to extracellular adenosine triphosphate (ATP). Pannexin (Panx) channels have been shown to be involved in ATP release from myocytes and can activate P2X1 and P2Y2 receptors on the coronary artery. DESIGN: We applied a well-characterized I/R model in rats, with 24 hours of reperfusion. Panx expression in the myocardial tissue was measured with quantitative polymerase chain reaction (qPCR) and flow cytometry. ATP release was detected in situ using luminescence and the vascular response to nucleotides determined in a wire myograph. RESULTS: Here, we show that Panx expression is increased after experimental myocardial I/R, leading to an increase in extracellular ATP release, which could be inhibited by probenecid. Functional studies revealed that the P2Y2 receptor-dependent contraction is reduced in the coronary artery after I/R, which might be a response to the increased ATP levels. CONCLUSION: We, therefore, conclude that the regulation of the arterial purinergic system minimizes coronary contractions following ischemia.

Vitamin C Deficiency Exacerbates Dysfunction of Atherosclerotic Coronary Arteries in Guinea Pigs Fed a High-Fat Diet.

Vitamin C (vitC) deficiency has been associated with an increased risk of cardiovascular disease; while several putative mechanistic links have been proposed, functional evidence supporting a causal relationship is scarce. In this study, we investigated how vitC deficiency affects coronary artery vasomotor function and the development of coronary atherosclerotic plaques in guinea pigs subjected to chronic dyslipidemia by a high-fat diet regime. Female Hartley guinea pigs were fed either a control (low-fat diet and sufficient vitC) (N = 8) or a high-fat diet with either sufficient (N = 8) or deficient (N = 10) vitC for 32 weeks. Guinea pigs subjected to the high-fat diet developed significant atherosclerotic plaques in their coronary arteries, with no quantitative effect of vitC deficiency. In isolated coronary arteries, vasomotor responses to potassium, carbachol, nitric oxide, or bradykinin were studied in a wire myograph. Carbachol, bradykinin, and nitric oxide mediated relaxation in the coronary arteries of the control group. While vasorelaxation to carbachol and nitric oxide was preserved in the two high-fat diet groups, bradykinin-induced vasorelaxation was abolished. Interestingly, bradykinin induced a significant contraction in coronary arteries from vitC-deficient guinea pigs (p < 0.05). The bradykinin-induced contraction was unaffected by L-NAME but significantly inhibited by both indomethacin and vitC, suggesting that, during vitC deficiency, increased release of arachidonic acid metabolites and vascular oxidative stress are involved in the constrictor effects mediated by bradykinin. In conclusion, the present study shows supporting evidence that poor vitC status negatively affects coronary artery function.

Semaglutide treatment attenuates vessel remodelling in ApoE-/- mice following vascular injury and blood flow perturbation.

Background and aims: Randomized clinical studies have shown a reduction in cardiovascular outcomes with glucagon-like peptide 1 receptor agonist (GLP-1RA) treatment with the hypothesized mechanisms being an underlying effect on atherosclerosis. Here, we aimed to assess the pharmacological effects of semaglutide in an atheroprone murine model that recapitulates central mechanisms related to vascular smooth muscle cell (VSMC) phenotypic switching and endothelial dysfunction known to operate within the atherosclerotic plaque. Methods: In study A, we employed an electrical current to the carotid artery in ApoE-/- mice to induce severe VSMC injury and death, after which the arteries were allowed to heal for 4 weeks. In study B, a constrictive cuff was added for 6 h at the site of the healed segment to induce a disturbance in blood flow. Results: Compared to vehicle, semaglutide treatment reduced the intimal and medial area by ∼66% (p = 0.007) and ∼11% (p = 0.0002), respectively. Following cuff placement, expression of the pro-inflammatory marker osteopontin and macrophage marker Mac-2 was reduced (p < 0.05) in the semaglutide-treated group compared to vehicle. GLP-1R were not expressed in murine carotid artery and human coronary vessels with and without atherosclerotic plaques, and semaglutide treatment did not affect proliferation of cultured primary human VSMCs. Conclusions: Semaglutide treatment reduced vessel remodelling following electrical injury and blood flow perturbation in an atheroprone mouse model. This effect appears to be driven by anti-inflammatory and -proliferative mechanisms independent of GLP-1 receptor-mediated signalling in the resident vascular cells. This mechanism of action may be important for cardiovascular protection.

Activation of the renal GLP-1R leads to expression of Ren1 in the renal vascular tree.

The GLP-1 receptor (GLP-1R) in the kidney is expressed exclusively in vascular smooth muscle cells in arteries and arterioles. Downstream effects of the activation of the renal vascular GLP-1R are elusive but may involve regulation of the renin-angiotensin-aldosterone system (RAAS). The expression of Ren1 in the mouse renal vasculature was investigated by in situ hybridization after a single subcutaneous dose of liraglutide, semaglutide and after repeated injections of liraglutide. Single and repeated exposure to GLP-1R agonists induced expression of Ren1 in the renal vascular smooth muscle cell compartment compared with vehicle injected controls (p < .0001) for both semaglutide and liraglutide. The present data show a robust induction of Ren1 expression in the vascular smooth muscle cells of the kidney after single and repeated GLP-1R activation and this renin recruitment may be involved in the effects of GLP-1R agonist treatment on kidney disease.

Decreased expression of the GLP-1 receptor after segmental artery injury in mice.

The glucagon-like peptide-1 receptor (GLP1R) is expressed in the renal vasculature and known to be downregulated under hypertensive conditions in rats and humans. However, little is known about the regulation in other types of renal pathology involving vascular changes. This study investigates the expression of the GLP1R in renal vasculature after glomerular injury in the nephrotoxic nephritis mouse model, high cholesterol, and atherosclerosis in the Ldlr-/- mouse on Western diet, and ex vivo injury in an organ culture model. The immunohistochemical signal of the GLP1R was significantly decreased in arteries from mice with nephrotoxic nephritis after 42 days compared to 7 days and saline control (P < 0.05). Histological evaluation of kidneys from Ldlr-/- mice on Western diet showed a decreased GLP1R specific immunohistochemical signal (P < 0.05). The dilatory response to liraglutide was decreased in Western diet fed Ldlr-/- mice compared to C57Bl/6J controls (P < 0.05). Organ culture significantly decreased the immunohistochemical signal of the GLP1R (P <0.05) and the expression of Glp1r mRNA (P < 0.005) compared to fresh. Organ cultured vessels showed vascular smooth muscle cell remodelling as Acta2 expression was decreased (P < 0.005) and Ednrb was increased (P < 0.05). In conclusion, nephrotoxic nephritis and hypercholesterolaemia led to decreased GLP1R specific immunohistochemical signal. Ex vivo vascular injury in the organ culture model leads to a decrease in expression of GLP1R expressionand contractile VSMC specific markers and increase in expression of dedifferentiation markers suggestive of an inverse relationship between phenotypic switch of the VSMC and the expression of the GLP1R; however, the causal relationship remains elusive.

Liraglutide treatment improves endothelial function in the Ldlr-/- mouse model of atherosclerosis and affects genes involved in vascular remodelling and inflammation.

Recent clinical intervention studies have shown that the GLP1 analogue liraglutide lowers cardiovascular risk, but the underlying mechanism has not yet been fully elucidated. This study investigated the effects of liraglutide on endothelial function in the Ldlr-/- mouse model. Mice (n = 12/group) were fed Western diet (WD) or chow for 12 weeks followed by 4 weeks of treatment with liraglutide (1 mg/kg/day) or vehicle subcutaneously. Weight loss, blood lipid content, plaque burden, vasomotor function of the aorta and gene expression pattern in aorta and brachiocephalic artery were monitored. Liraglutide treatment significantly induced weight loss (P < .0001), decreased blood triglycerides (P < .0001) and total cholesterol (P < .0001) in WD-fed mice but did not decrease plaque burden. Liraglutide also improved endothelium-mediated dilation of the distal thoracis aorta (P = .0067), but it did not affect phenylephrine or sodium nitroprusside responses. Fluidigm analyses of 96 genes showed significantly altered expression of seven genes related to inflammation, vascular smooth muscle cells and extracellular matrix composition in liraglutide-treated animals. We conclude that treatment with liraglutide decreased endothelial dysfunction and that this could be linked to decreased inflammation or regulation of vascular remodelling.

Vitamin C Deficiency May Delay Diet-Induced NASH Regression in the Guinea Pig.

Oxidative stress is directly linked to non-alcoholic fatty liver disease (NAFLD) and the progression to steaotohepatitis (NASH). Thus, a beneficial role of antioxidants in delaying disease progression and/or accelerating recovery may be expected, as corroborated by recommendations of, e.g., vitamin E supplementation to patients. This study investigated the effect of vitamin C deficiency-often resulting from poor diets low in fruits and vegetables and high in fat-combined with/without a change to a low fat diet on NAFLD/NASH phenotype and hepatic transcriptome in the guinea pig NASH model. Vitamin C deficiency per se did not accelerate disease induction. However, the results showed an effect of the diet change on the resolution of hepatic histopathological hallmarks (steatosis, inflammation, and ballooning) (p < 0.05 or less) and indicated a positive effect of a high vitamin C intake when combined with a low fat diet. Our data show that a diet change is important in NASH regression and suggest that a poor vitamin C status delays the reversion towards a healthy hepatic transcriptome and phenotype. In conclusion, the findings support a beneficial role of adequate vitamin C intake in the regression of NASH and may indicate that vitamin C supplementation in addition to lifestyle modifications could accelerate recovery in NASH patients with poor vitamin C status.

Expression of endothelin type B receptors in uterine artery smooth muscle cells from pregnant Guinea pigs.

INTRODUCTION: It is well established that upregulation of endothelin type B (ETB) receptors in vascular smooth muscle cells plays a role in pathophysiological artery remodeling as response to ischemia and atherosclerosis. This study aimed to investigate the ETB receptors function and localization under normal physiological remodeling. Specifically, in the guinea pig uterine arteries during pregnancy. METHODS: Uterine artery contractility was assessed with sarafotoxin 6c and endothelin-1 in wire-myography in uterine arteries from non-pregnant and pregnant guinea pigs at gestational day 37 ±â€¯5. Localization of ETB receptors, proliferation marker Ki-67, and SMC differentiation marker SM22α in uterine arteries were investigated with immunohistochemistry. RESULTS: Uterine arteries from pregnant guinea pigs showed significantly increased ETB receptor-mediated vasoconstriction compared to uterine arteries from non-pregnant and to coronary arteries from pregnant guinea pigs (p < 0.001), suggesting that ETB-receptor upregulation in uterine artery SMCs is a normal physiological mechanism taking place during remodeling. Furthermore, uterine arteries from pregnant guinea pigs showed enhanced expression of ETB receptors, high density of Ki-67 positive SMCs and sparse SM22α staining in SMCs localized in the outer layer of the vessel wall. DISCUSSION: Our results suggest that ETB receptors are expressed in dedifferentiated proliferating SMCs of uterine arteries in pregnant guinea pigs. This study provides novel insight into the function and expression of ETB receptors in uterine vascular remodeling during pregnancy.

Vasomotor dysfunction in human subcutaneous arteries exposed ex vivo to food-grade titanium dioxide.

Animal studies have shown that titanium dioxide (TiO2) exposure affects arterial vasomotor function, whereas little is known about the effects in arteries from humans. This study investigated vasomotor responses after direct exposure of human subcutaneous arteries to food-grade TiO2 (E171) (14 or 140 µg/ml) for 30 min and 18 h. Vasomotor responses to bradykinin, 5-hydroxytryptamine (5-HT), sarafotoxin 6c (S6c) and nitroglycerin were recorded in wire-myographs. Vasoconstrictor responses to 5-HT were increased in arteries exposed to E171 for 18 h (P < 0.05). Furthermore, an increase in S6c responses was seen in low concentration E171 exposed arteries (30 min exposure; P < 0.05). The vasorelaxation response to nitroglycerin was increased in low concentration E171 exposed arteries (30 min exposure; P < 0.05). Vasorelaxation responses to bradykinin were unaffected after treatment with E171. There was no difference in gene expression levels of intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, 5-hydroxytryptamine receptor 1B, 5-hydroxytryptamine receptor 2A, endothelin receptor A and endothelin receptor B in E171 exposed arteries after exposure to TiO2 for 30 min or 18 h. In conclusion, this study shows that the same type of vasomotor dysfunction is found in artery segments of rats and humans following ex vivo exposure to E171.

Vitamin C Deficiency Reduces Muscarinic Receptor Coronary Artery Vasoconstriction and Plasma Tetrahydrobiopterin Concentration in Guinea Pigs.

Vitamin C (vitC) deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32) were randomized to high (1500 mg/kg diet) or low (0 to 50 mg/kg diet) vitC for 10-12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium, carbachol, sodium nitroprusside (SNP), U46619, sarafotoxin 6c (S6c) and endothelin-1 (ET-1) were recorded. Plasma vitC and tetrahydrobiopterin were measured by HPLC. Plasma vitC status reflected the diets with deficient animals displaying reduced tetrahydrobiopterin. Vasoconstrictor responses to carbachol were significantly decreased in vitC deficient coronary arteries independent of their general vasoconstrictor/vasodilator capacity (p < 0.001). Moreover, in vitC deficient animals, carbachol-induced vasodilator responses correlated with coronary artery diameter (p < 0.001). Inhibition of cyclooxygenases with indomethacin increased carbachol-induced vasoconstriction, suggesting an augmented carbachol-induced release of vasodilator prostanoids. Atropine abolished carbachol-induced vasomotion, supporting a specific muscarinic receptor effect. Arterial responses to SNP, potassium, S6c, U46619 and ET-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid metabolites and reduced muscarinic receptor expression/signaling.

Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat.

BACKGROUND: Coronary artery remodelling and vasospasm is a complication of acute myocardial ischemia and reperfusion. The underlying mechanisms are complex, but the vasoconstrictor peptide endothelin-1 is suggested to have an important role. This study aimed to determine whether the expression of endothelin-1 and its receptors are regulated in the myocardium and in coronary arteries after experimental ischemia-reperfusion. Furthermore, we evaluated whether treatment with a specific MEK1/2 inhibitor, U0126, modified the expression and function of these proteins. METHODS AND FINDINGS: Sprague-Dawley rats were randomly divided into three groups: sham-operated, ischemia-reperfusion with vehicle treatment and ischemia-reperfusion with U0126 treatment. Ischemia was induced by ligating the left anterior descending coronary artery for 30 minutes followed by reperfusion. U0126 was administered before ischemia and repeated 6 hours after start of reperfusion. The contractile properties of isolated coronary arteries to endothelin-1 and sarafotoxin 6c were evaluated using wire-myography. The gene expression of endothelin-1 and endothelin receptors were measured using qPCR. Distribution and localization of proteins (pERK1/2, prepro-endothelin-1, endothelin-1, and endothelin ETA and ETB receptors) were analysed by Western blot and immunohistochemistry. We found that pERK1/2 was significantly augmented in the ischemic area 3 hours after ischemia-reperfusion; this correlated with increased ETB receptor and ET-1 gene expressions in ischemic myocardium and in coronary arteries. ETB receptor-mediated vasoconstriction was observed to be increased in coronary arteries 24 hours after ischemia-reperfusion. Treatment with U0126 reduced pERK1/2, expression of ET-1 and ETB receptor, and ETB receptor-mediated vasoconstriction. CONCLUSIONS: These findings suggest that the MEK-ERK1/2 signaling pathway is important for regulating endothelin-1 and ETB receptors in myocardium and coronary arteries after ischemia-reperfusion in the ischemic region. Inhibition of the MEK-ERK1/2 pathway may provide a novel target for reducing ischemia-reperfusion damage in the heart.

Endothelin-1 and Endothelin-3 Regulate Endothelin Receptor Expression in Rat Coronary Arteries.

In ischaemic hearts, endothelin (ET) levels are increased, and vasoconstrictor responses to ET-1 are greatly enhanced. We previously reported that ETB receptors are up-regulated in the smooth muscle layer of coronary arteries after myocardial ischaemia-reperfusion and that the MEK-ERK1/2 signalling pathway is involved in ETB receptor up-regulation. Whether ETs are directly involved in receptor regulation has not been determined. We suggest that ET-1 and ET-3 alter the expression/activity of ET receptors in coronary vascular smooth muscle cells. Vasoconstrictor responses were studied in endothelium-denuded coronary artery segments from rats that were subjected to experimental ischaemia-reperfusion or in organ-cultured segments. Post-ischaemic and cultured coronary arteries exhibited similar increased sensitivity to ET-3. ETA receptor-mediated vasoconstriction was dominant in fresh and non-ischaemic arteries. Organ culture significantly up-regulated ETB receptors and down-regulated ETA receptor expression. Co-incubation with ET-1 (1 nM) or ET-3 (100 nM) induced further down-regulation of the ETA receptor mRNA, while the function and protein level of ETA remained unchanged. ET-3 (100 nM) further up-regulated ETB receptor mRNA and proteins but abolished ETB receptor-mediated vasoconstriction, suggesting a desensitization of ETB receptors that was not observed with ET-3 (1 nM). In conclusion, ET-1, which is the most prevalent isoform in the cardiovascular system, induces down-regulation of ETA receptor expression without changing ETA or ETB receptor function or protein levels. Intermediate concentrations of ET-3 had an effect that was similar to that of ET-1, such that high concentrations of ET-3 (100 nM) up-regulated the ETB receptor at the gene and protein levels but switched off the function of the ETB receptors via desensitization.

Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries.

AIMS: Endothelin ET(B) receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ET(B) receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ET(B) receptors. This study examines the early time course and mechanism behind upregulation of contractile ET(B) receptors in isolated rat coronary arteries during short-term organ culture. MAIN METHODS: Coronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ET(B) receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ET(B) and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry. KEY FINDINGS: Fresh arteries showed negligible vasoconstriction to S6c. However, incubation for only 4 and 7h increased S6c contractions two- and seven-fold, respectively. Furthermore, 7h incubation enhanced vasocontractile responses to endothelin-3 and increased ET(B) receptor density in vascular smooth muscle cells. ERK1/2 was activated rapidly after start of incubation. Moreover, incubation with either the transcriptional inhibitor actinomycin D or the mitogen-activated-protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 attenuated contractile ET(B) receptor upregulation. U0126 attenuated ET(B) receptor protein levels after 24 h of incubation. SIGNIFICANCE: Coronary arteries rapidly upregulate vasocontractile ET(B) receptors during organ culture via transcriptional mechanisms and MEK-ERK1/2 signalling. This model may mimic the mechanisms seen in ischemic conditions. Furthermore, these findings have important experimental implications in tissue bath experiments lasting for more than 4h.