Matthias Barton

Endothelins and Endothelin Receptor Antagonists Therapeutic Considerations for a Novel Class of Cardiovascular Drugs

The 21-amino acid peptide endothelin-1 (ET-1) is the predominant isoform of the endothelin peptide family, which includes ET-2, ET-3, and ET-4. It exerts various biological effects, including vasoconstriction and the stimulation of cell proliferation in tissues both within and outside of the cardiovascular system. ET-1 is synthesized by endothelin-converting enzymes (ECE), chymases, and non-ECE metalloproteases; it is regulated in an autocrine fashion in vascular and nonvascular cells. ET-1 acts through the activation of Gi-protein–coupled receptors. ETA receptors mediate vasoconstriction and cell proliferation, whereas ETB receptors are important for the clearance of ET-1, endothelial cell survival, the release of nitric oxide and prostacyclin, and the inhibition of ECE-1.ET is activated in hypertension, atherosclerosis, restenosis, heart failure, idiopathic cardiomyopathy, and renal failure. Tissue concentrations more reliably reflect the activation of the ET system because increased vascular ET-1 levels occur in the absence of changes in plasma. Experimental studies using molecular and pharmacological inhibition of the ET system and the first clinical trials have demonstrated that ET-1 takes part in normal cardiovascular homeostasis. Thus, ET-1 plays a major role in the functional and structural changes observed in arterial and pulmonary hypertension, glomerulosclerosis, atherosclerosis, and heart failure, mainly through pressure-independent mechanisms. ET antagonists are promising new agents in the treatment of cardiovascular diseases.

Angiotensin II Increases Tissue Endothelin and Induces Vascular Hypertrophy

BACKGROUND In vitro studies on vascular smooth muscle cells suggest that endothelin has a stimulating effect on cellular proliferation. This study was designed to determine the endogenous effect of endothelin on angiotensin II-induced hypertrophy of small arteries in vivo. METHODS AND RESULTS Two weeks of angiotensin II administration (200 ng x kg[-1] x min[-1]) increased media thickness, media/lumen ratio, and cross-sectional area of basilar and small mesenteric arteries, confirming the proliferative properties of angiotensin II. The tissue levels of endothelin-1 were elevated in mesenteric arteries after angiotensin II administration. The administration of the selective and specific ET(A)-receptor antagonist LU135252 (50 mg x kg[-1] x d[-1]) in combination with angiotensin II prevented the changes of vascular geometry and partially reduced the increase in blood pressure induced by angiotensin II. Indeed, part of the effect on the vascular structure of the endothelin-receptor antagonist seemed pressure-independent. CONCLUSIONS Our results therefore demonstrate that angiotensin II increases the production of endothelin in the blood vessel wall that, via ET(A) receptors, mediates changes in vascular structure of the cerebral and mesenteric circulation. Endothelin antagonists may therefore be of value to reduce blood pressure and to prevent vascular structural changes in conditions of increased activity of the renin-angiotensin system.

Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary artery.

Aging is an important determinant of vascular disease. Endothelium-derived nitric oxide (NO) is protective as a vasodilator and inhibitor of platelet function. This study was designed to directly measure effects of prolonged aging on endotheliai NO release in isolated blood vessels and to delineate differences between the systemic and pulmonary circulation. Aortas and pulmonary arteries from 5-6-mo-old (young), 18-19-mo-old (middle-aged), and 32-33-mo-old (old) normotensive female rats were used. Blood pressure and plasma estradiol-17beta (E2) remained unchanged. In isolated blood vessels, NO release was induced by the receptor-independent agonist calcium ionophore A23187 (10 micromol/liter) and measured in situ on the endothelial surface of vessels using a porphyrinic microsensor. In vessels suspended in organ chambers isometric tension was recorded. In the aorta, the initial rate of NO release and peak NO concentration were reduced in middle-aged and old rats (P < 0.0006 vs. young rats, n = 6). Furthermore, endothelium-dependent relaxations to calcium ionophore and acetylcholine (both 10(-10) - 10(-5) mol/liter) were also reduced in aortas from old as compared with young rats (n = 6, P < 0.05). The initial rate of NO release and peak NO concentration significantly correlated with maximal relaxation to calcium ionophore A23187 (correlation coefficients r - 0.916, P < 0.0018 and r = 0.961, P < 0.0001, respectively, n = 7). In pulmonary arteries, however, the initial rate of NO release as well as peak NO concentration did not decrease with age (n = 6 for each age group, NS). In both blood vessels, the NO release was unaffected by superoxide dismutase in all age groups (n = 6, NS). Thus, aging specifically reduces initial rate and peak concentrations of endothelial NO release from aorta but not pulmonary artery indicating reduced NO production. As arterial pressure did not change with aging, the chronic exposure of the aorta to higher pressure and/or pulsatility than in the pulmonary artery may be the cause. This appears important as NO plays a protective role by preventing vasoconstriction, thrombosis and atherosclerosis.

Regulatory Role of G Protein–Coupled Estrogen Receptor for Vascular Function and Obesity

We found that the selective stimulation of the intracellular, transmembrane G protein-coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Anatomic Heterogeneity of Vascular Aging: Role of Nitric Oxide and Endothelin

We investigated the effects of aging, a cardiovascular risk factor, on vascular function with regard to endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), and endothelin (ET-1) in aorta and femoral artery of the rat. Concentration-response curves to acetylcholine, calcium ionophore A23187, norepinephrine, ET-1, big endothelin, sodium nitroprusside, and exogenous SOD were obtained. Expression of eNOS mRNA was analyzed by reverse-transcription polymerase chain reaction, SOD activity was assessed using a chemiluminescence-based cytochrome c assay, and ET-1 plasma concentrations were measured by radioimmunoassay. In aorta of old rats, relaxations to acetylcholine and calcium ionophore A23187, basal NO release, and expression of eNOS mRNA in aortic endothelial cells were reduced (P<.05). In femoral arteries, relaxations to acetylcholine were preserved, whereas basal release of NO was attenuated (P<.05). Aging selectively increased contractions to norepinephrine and functional endothelin converting enzyme activity and attenuated contractions to ET-1 in aortas but not femoral arteries. Vascular SOD activity was higher in the femoral artery (P<.05) and unaffected by aging. Plasma ET-1 levels increased and plasma SOD activity decreased with age (P<.05). Aging was associated with an anatomic heterogeneity of endothelial dysfunction, functional endothelin converting enzyme activity, and vascular SOD activity. Vascular function was impaired in the aorta but not the femoral artery, which may be related to lower eNOS mRNA expression and SOD activity. These data suggest differential regulation of the vascular aging process that may contribute to the anatomic heterogeneity of atherosclerosis.

ETA Receptor Blockade Prevents Increased Tissue Endothelin-1, Vascular Hypertrophy, and Endothelial Dysfunction in Salt-Sensitive Hypertension

Sodium plays an important role in the pathogenesis and therapy of hypertension, a major risk factor for cardiovascular disease. This study investigated the involvement of endothelin in vascular alterations in salt-induced Dahl hypertension. Salt-sensitive (DS) and salt-resistant (DR) Dahl rats were treated with a high-sodium diet (NaCl 4%) with or without ET A receptor antagonist LU135252 for two months, and effects of treatments on systolic blood pressure, vascular endothelin-1 (ET-1) protein content, aortic hypertrophy, and vascular reactivity of isolated aortic rings were studied. In DS rats, a high-sodium diet increased systolic pressure (190±4 versus 152±2 mm Hg, P P P P P =.0011). ET-1 tissue levels were highly and inversely correlated with endothelium-dependent relaxations ( r =0.931, P r =0.77, P =.0007). LU135252 treatment reduced systolic blood pressure only in part (168±3 versus 190±4 mm Hg. P P A receptor antagonism may have therapeutic potential for lowering vascular ET-1 content, improving endothelial function, and preventing structural changes in salt-sensitive hypertension.

Effects of Chronic ETA-Receptor Blockade in Angiotensin II-Induced Hypertension

Angiotensin II, a constrictor and mitogen of vascular smooth muscle cells, affects the release of endothelium-derived factors such as nitric oxide or endothelin-1. This study investigated the influence of endothelin-1, using the selective endothelin A receptor antagonist LU135252, on blood pressure and endothelial function in angiotensin II-induced hypertension in the rat. Two weeks of angiotensin II administration (200 ng/kg per minute) increased systolic blood pressure (+35 +/- 5 mm Hg; tail-cuff method) compared with placebo (P < .05). LU135252 alone did not affect systolic pressure but lowered the angiotensin II-induced pressure increase (P < .05). In isolated aortic rings, endothelium-dependent relaxations to acetylcholine were reduced in the angiotensin II group (P < .05 versus placebo) and improved by concomitant chronic LU135252 treatment (P < .05 versus angiotensin II). Blood pressure elevation strongly correlated with impaired endothelium-dependent relaxations to acetylcholine (r = -.967). LU135252 did not affect endothelium-independent relaxations to sodium nitroprusside, which were diminished after angiotensin II treatment (P < .05). In quiescent rings, chronic angiotensin II administration enhanced endothelium-dependent contractions to acetylcholine, which were reduced by LU135252 (P < .05). Impaired contractions to endothelin-1 and norepinephrine in the angiotensin II group were normalized after treatment with LU135252 (P < .05). Thus, chronic therapy with LU135252 partially prevents angiotensin II-induced hypertension and the alternations of the endothelial function observed in this experimental model.

Estrogen biology: New insights into GPER function and clinical opportunities

Estrogens play an important role in the regulation of normal physiology, aging and many disease states. Although the nuclear estrogen receptors have classically been described to function as ligand-activated transcription factors mediating genomic effects in hormonally regulated tissues, more recent studies reveal that estrogens also mediate rapid signaling events traditionally associated with G protein-coupled receptors. The G protein-coupled estrogen receptor GPER (formerly GPR30) has now become recognized as a major mediator of estrogens rapid cellular effects throughout the body. With the discovery of selective synthetic ligands for GPER, both agonists and antagonists, as well as the use of GPER knockout mice, significant advances have been made in our understanding of GPER function at the cellular, tissue and organismal levels. In many instances, the protective/beneficial effects of estrogen are mimicked by selective GPER agonism and are absent or reduced in GPER knockout mice, suggesting an essential or at least parallel role for GPER in the actions of estrogen. In this review, we will discuss recent advances and our current understanding of the role of GPER and the activity of clinically used drugs, such as SERMs and SERDs, in physiology and disease. We will also highlight novel opportunities for clinical development towards GPER-targeted therapeutics, for molecular imaging, as well as for theranostic approaches and personalized medicine.

Obesity, insulin resistance and diabetes: sex differences and role of oestrogen receptors

Obesity increases the risk of coronary artery disease through insulin resistance, diabetes, arterial hypertension and dyslipidemia. The prevalence of obesity has increased worldwide and is particularly high among middle‐aged women and men. After menopause, women are at an increased risk to develop visceral obesity due to the loss of endogenous ovarian hormone production. Effects of oestrogens are classically mediated by the two nuclear oestrogen receptors (ERs) α and β. In addition, more recent research has shown that the intracellular transmembrane G‐protein‐coupled oestrogen receptor (GPER) originally designated as GPR30 also mediates some of the actions attributed to oestrogens. Oestrogen and its receptors are important regulators of body weight and insulin sensitivity not only in women but also in men as demonstrated by ER mutations in rodents and humans. This article reviews the role of sex hormones and ERs in the context of obesity, insulin sensitivity and diabetes as well as the related clinical issues in women and men.

Differential Effects of 17β-Estradiol on Function and Expression of Estrogen Receptor α, Estrogen Receptor β, and GPR30 in Arteries and Veins of Patients With Atherosclerosis

Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17&bgr;-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17&bgr;-Estradiol caused concentration-dependent, endothelium-independent relaxation in arteries (P<0.05 versus solvent control) but not in veins (P not significant). 17&bgr;-Estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor &bgr; was 10-fold higher than that of estrogen receptor &agr; or GPR30 (P<0.05). Expression of all 3 of the receptors was reduced after exposure to 17&bgr;-estradiol in arteries but not in veins (P<0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17&bgr;-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17&bgr;-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy.