Gry Freja Skovsted

Heart ischaemia-reperfusion induces local up-regulation of vasoconstrictor endothelin ETB receptors in rat coronary arteries downstream of occlusion.

Endothelins act via two receptor subtypes, ETA and ETB. Under physiological conditions in coronary arteries, ETA receptors expressed in smooth muscle cells mediate vasoconstriction whereas ETB receptors mainly found in endothelial cells mediate vasorelaxation. However, under pathophysiological conditions, ETB receptors may also be expressed in vascular smooth muscle cells mediating vasoconstriction. Here, we have investigated whether vasoconstrictor ETB receptors are up‐regulated in coronary arteries after experimental myocardial ischaemia in rats.

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.

Similar Adiponectin Levels in Obese Normotensive and Obese Hypertensive Men and No Vasorelaxant Effect of Adiponectin on Human Arteries.

Obesity is a strong risk factor for hypertension, but the mechanism linking obesity to hypertension is not fully elucidated. In obesity, circulating concentrations of adiponectin are decreased and hypoadiponectinaemia has in some but not all studies been associated with increased risk of hypertension. Due to this inconsistency, we decided to study adiponectin from two aspects in a cross‐sectional in vivo study and in an experimental in vitro study. In the cross‐sectional study, 103 men with body mass index (BMI) ≥ 30.0 kg/m2 were studied; 63 had 24‐hr ambulatory blood pressure (ABP) ≥ 130/80 mmHg (ObeseHT) and 40 had 24‐hr ABP < 130/80 mmHg (ObeseNT). As controls, we studied 27 men with BMI between 20.0 and 24.9 kg/m2 and 24‐hr ABP < 130/80 mmHg (LeanNT). Serum concentrations of adiponectin and body composition using dual‐energy X‐ray absorptiometry scanning were determined. In vitro, the direct vasomotor response of adiponectin was tested on subcutaneous resistance arteries from human abdominal adipose tissue. The two obese groups had lower adiponectin concentrations compared with LeanNT (p < 0.01) [median (interquartile range)]: ObeseHT 6.5 (5.1–8.3) mg/L; ObeseNT 6.6 (5.2–7.8) mg/L; and LeanNT 9.4 (6.7–12.4) mg/L, with no significant difference in adiponectin concentrations (or body composition) between ObeseHT and ObeseNT (p = 0.67). In vitro, adiponectin did not have any direct vasodilatory effect and adiponectin did not affect angiotensin II‐stimulated vasoconstriction. In conclusion, obese hypertensive men have similar serum concentrations of adiponectin as obese normotensive men. In combination with the in vitro data, these findings question a pathogenic role of adiponectin in human hypertension.

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.

Liraglutide Decreases Hepatic Inflammation and Injury in Advanced Lean Non-Alcoholic Steatohepatitis

Although commonly associated with obesity, non‐alcoholic fatty liver disease (NAFLD) is also present in the lean population representing a unique disease phenotype. Affecting 25% of the worlds population, NAFLD is associated with increased mortality especially when progressed to non‐alcoholic steatohepatitis (NASH). However, no approved pharmacological treatments exist. Current research focuses mainly on NASH associated with obesity, leaving the effectiveness of promising treatments in lean NASH virtually unknown. This study therefore aimed to evaluate the effect of liraglutide (glucagon‐like peptide 1 analogue) and dietary intervention, alone and in combination, in guinea pigs with non‐obese NASH. After 20 weeks of high‐fat feeding (20% fat, 15% sucrose, 0.35% cholesterol), 40 female guinea pigs were block‐randomized based on weight into four groups receiving one of four treatments for 4 weeks: continued high‐fat diet (HF, control), high‐fat diet and liraglutide treatment (HFL), chow diet (4% fat, 0% sucrose, 0% cholesterol; HFC) or chow diet and liraglutide treatment (HFCL). High‐fat feeding induced NASH with severe fibrosis. Liraglutide decreased inflammation (p < 0.05) and hepatocyte ballooning (p < 0.05), while increasing hepatic α‐tocopherol (p = 0.0154). Dietary intervention did not improve liver histopathology significantly, but decreased liver weight (p = 0.004), plasma total cholesterol (p = 0.0175), LDL‐cholesterol (p = 0.0063), VLDL‐cholesterol (p = 0.0034), hepatic cholesterol (p < 0.0001) and increased hepatic vitamin C (p = 0.0099). Combined liraglutide and dietary intervention induced a rapid weight loss, necessitating periodical liraglutide dose adjustment/discontinuation, limiting the strength of the findings from this group. Collectively, this pre‐clinical study supports the beneficial effect of liraglutide on NASH and extends this notion to lean NASH.

Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat.

Cerebral ischaemia results in enhanced endothelin B (ETB) receptor‐mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We suggest that rapid and sustained reduction in wall tension/stretch is a possible trigger mechanism for this vascular remodelling. Isolated rat middle cerebral artery (MCA) segments were incubated in a wire myograph with or without mechanical stretch, prior to assessment of their contractile response to the selective ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) was studied by their specific inhibitors U0126 and PF‐228, respectively. Compared with their stretched counterparts, unstretched MCA segments showed a significantly increased ETB receptor‐mediated contractile response after 12 hr of incubation, which was attenuated by either U0126 or PF‐228. The functionally increased ETB‐mediated contractility could be attributed to two different mechanisms: (i) a difference in ETB receptor localization from primarily endothelial expression to SMC expression and (ii) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor‐mediated contractility via triggering of an early mechanosensitive signalling pathway involving ERK1/2 and FAK signalling. A mechanism likely to be an initiating factor for the increased ETB receptor‐mediated contractility found after cerebral ischaemia.

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.

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.

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.

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.