Endothelin: 30 Years From Discovery to Therapy.

Discovered in 1987 as a potent endothelial cell-derived vasoconstrictor peptide, endothelin-1 (ET-1), the predominant member of the endothelin peptide family, is now recognized as a multifunctional peptide with cytokine-like activity contributing to almost all aspects of physiology and cell function. More than 30 000 scientific articles on endothelin were published over the past 3 decades, leading to the development and subsequent regulatory approval of a new class of therapeutics-the endothelin receptor antagonists (ERAs). This article reviews the history of the discovery of endothelin and its role in genetics, physiology, and disease. Here, we summarize the main clinical trials using ERAs and discuss the role of endothelin in cardiovascular diseases such as arterial hypertension, preecclampsia, coronary atherosclerosis, myocardial infarction in the absence of obstructive coronary artery disease (MINOCA) caused by spontaneous coronary artery dissection (SCAD), Takotsubo syndrome, and heart failure. We also discuss how endothelins contributes to diabetic kidney disease and focal segmental glomerulosclerosis, pulmonary arterial hypertension, as well as cancer, immune disorders, and allograft rejection (which all involve ETA autoantibodies), and neurological diseases. The application of ERAs, dual endothelin receptor/angiotensin receptor antagonists (DARAs), selective ETB agonists, novel biologics such as receptor-targeting antibodies, or immunization against ETA receptors holds the potential to slow the progression or even reverse chronic noncommunicable diseases. Future clinical studies will show whether targeting endothelin receptors can prevent or reduce disability from disease and improve clinical outcome, quality of life, and survival in patients.

Systemic inflammation impairs tissue reperfusion through endothelin-dependent mechanisms in cerebral ischemia.

BACKGROUND AND PURPOSE: Systemic inflammation contributes to diverse acute and chronic brain pathologies, and extensive evidence implicates inflammation in stroke susceptibility and poor outcome. Here we investigate whether systemic inflammation alters cerebral blood flow during reperfusion after experimental cerebral ischemia. METHODS: Serial diffusion and perfusion-weighted MRI was performed after reperfusion in Wistar rats given systemic (intraperitoneal) interleukin-1ß or vehicle before 60-minute transient middle cerebral artery occlusion. The expression and location of endothelin-1 was assessed by polymerase chain reaction, ELISA, and immunofluorescence. RESULTS: Systemic interleukin-1 caused a severe reduction in cerebral blood flow and increase in infarct volume compared with vehicle. Restriction in cerebral blood flow was observed alongside activation of the cerebral vasculature and upregulation of the vasoconstricting peptide endothelin-1 in the ischemic penumbra. A microthrombotic profile was also observed in the vasculature of rats receiving interleukin-1. Blockade of endothelin-1 receptors reversed this hypoperfusion, reduced tissue damage, and improved functional outcome. CONCLUSIONS: These data suggest patients with a raised inflammatory profile may have persistent deficits in perfusion after reopening of an occluded vessel. Future therapeutic strategies to interrupt the mechanism identified could lead to enhanced recovery of penumbra in patients with a heightened inflammatory burden and a better outcome after stroke.

The ocular endothelin system: a novel target for the treatment of endotoxin-induced uveitis with bosentan.

PURPOSE: We compared the anti-inflammatory effects of bosentan and dexamethasone in endotoxin-induced uveitis (EIU). METHODS: Endotoxin-induced uveitis was induced by subcutaneous injection of lipopolysaccharide (LPS, 200 µg) in Wistar rats. Rats were divided randomly into 10 groups (n = 6). Bosentan at doses of 50 and 100 mg/kg were administered orally 1 hour before and 12 hours after LPS injection, and dexamethasone was administered by intraperitoneally 30 minutes before and 30 minutes after LPS injection at a dose of 1 mg/kg. Data were collected at two time points for each control and treatment; animals were killed at either 3 or 24 hours after LPS injection. Histopathologic evaluation and aqueous humour measurements of TNF-α level were performed, and endothelin-1 (ET-1), inducible nitric oxide synthase (iNOS), and endothelin receptor A and B (EDNRA and B) expression were analyzed. RESULTS: The group treated with 100 mg/kg bosentan at 24 hours displayed significantly milder uveitis and fewer inflammatory cells compared to LPS-injected animals, and there were similar findings in the dexamethasone-treated group at 24 hours. The TNF-α levels in the dexamethasone treatment group were lower than those in the LPS-induced uveitis control group (P < 0.05); however, there was no difference between the dexamethasone and bosentan treatment groups at 3 and 24 hours after LPS administration. Bosentan treatment at doses of 50 and 100 mg/kg significantly decreased iNOS expression compared to LPS-injected animals (P < 0.001). The ET-1 expression was suppressed significantly by bosentan and dexamethasone at 3 and 24 hours after LPS administration (P < 0.001). The EDNRA expression in the bosentan treatment groups was statistically significantly lower than that in the LPS-induced uveitis control group at 3 and 24 hours after LPS administration (P < 0.05). CONCLUSIONS: Bosentan reduces intraocular inflammation and has similar effects as dexamethasone in a rat model of EIU.

Endothelin axis is upregulated in human and rat right ventricular hypertrophy.

RATIONALE: Right ventricular (RV) function is the most important determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Endothelin (ET)-1 receptor antagonists (ERAs) are approved therapies for PAH. It is not known whether ERAs have effects on the RV, in addition to their vasodilating/antiproliferative effects in pulmonary arteries. OBJECTIVE: We hypothesized that the ET axis is upregulated in RV hypertrophy (RVH) and that ERAs have direct effects on the RV myocardium. METHODS AND RESULTS: RV myocardial samples from 34 patients with RVH were compared with 16 nonhypertrophied RV samples, and from rats with normal RV versus RVH attributable to PAH. Confocal immunohistochemistry showed that RVH myocardial ET type A (but not type B) receptor and ET-1 protein levels were increased compared with the nonhypertrophied RVs and positively correlated with the degree of RVH (RV thickness/body surface area; r(2)=0.838 and r(2)=0.818, respectively; P<0.01). These results were recapitulated in the rat model. In modified Langendorff perfusions, ERAs (BQ-123 and bosentan 10(-7,-6,-5) mol/L) decreased contractility in the hypertrophied, but not normal RV, in a dose-dependent manner (P<0.01). CONCLUSIONS: Patients and rats with PAH have an upregulation of the myocardial ET axis in RVH. This might be a compensatory mechanism to preserve RV contractility, as the afterload increases. ERAs use might potentially worsen RV function, and this could explain some of the peripheral edema noted clinically with these agents. Further studies are required to evaluate the effects of ERAs on the RV in patients with RVH and PAH.

Expression of inflammatory and apoptosis factors following coronary stent implantation in coronary heart disease patients.

We investigated the changes in characteristics of neutrophil CD11b, monocyte CD11b, platelet CD62P, endothelin (ET), and neutrophil CD178 in patients with coronary heart disease (CHD) before and after primary coronary stenting. A total of 41 patients with CHD who underwent coronary stenting and 40 control subjects were enrolled in the study. In CHD patients, peripheral blood samples were taken 24 h before and 30 min, 24 h, and 72 h after successful coronary stenting. All markers were significantly elevated in patients with CHD compared with controls (P<0.05). Time-course studies revealed that the expressions of neutrophil CD11b, monocyte CD11b, platelet CD62P, and ET were lower at 30 min post-operation (PO) compared with that at 24 h before operation (BO) (P<0.05). All levels significantly increased from 30 min PO to 24 h PO (P<0.05) and decreased thereafter until 72 h PO (P>0.05). Time course changes in neutrophil CD11b levels after coronary stenting were significantly higher in patients with unstable angina pectoris than in patients with stable angina pectoris (P<0.05). CD11b levels were related to CD62P in patients with CHD (P<0.05). Neutrophil CD11b and monocyte CD11b levels were significantly increased in patients with CHD who underwent coronary stenting compared with controls (P<0.05). Results show that CD11b levels increased, meanwhile, the levels of CD62P and ET increased in CHD patients after coronary stenting. In addition, neutrophil CD178 levels of apoptosis factor in patients, which is important for regression of inflammation, remained high for a period of time after coronary stenting.

High production of endothelin after foam sclerotherapy: a new pathogenetic hypothesis for neurological and visual disturbances after sclerotherapy.

BACKGROUND: Visual and neurological disturbances have always been reported following liquid sclerotherapy (LS) for venous insufficiency. In 1993 Cabrera introduced foam sclerotherapy (FS) using a detergent sclerosant as Lauromacrogol 400 or sodium tetradecyl sulphate. Several authors have reported with FS an increased incidence of such transient visual disturbances and neurological complications. This has been associated with gas or air used to generate the sclerosing foam. The frequent association of the presence of a patent foramen ovale, a common condition in normal population, and such complications has led several authors to consider neurological and visual disturbances as paradoxical gas embolism. OBJECTIVE: We are introducing a new pathogenetic hypothesis for sclerotherapy complications. Medical literature shows evidence of a clear relationship among cerebral and retinal vasospasm, migraine and intimal irritation. We think that the irritating sclerosant agent may stimulate a significant release of vasoactive substances from the venous wall, specifically endothelin 1 (ET-1), the most powerful vasoconstricting agent. METHOD: We have studied systemic ET-1 levels after LS and FS with Lauromacrogol 400 in a group of 13 rats at one and five minutes after injection. RESULTS: While ET-1 levels did not change significantly in control and in the LS group, a significant increase was detected after FS at one and five minutes. CONCLUSION: We conclude that should the same results be found in patients treated using sclerosing foam (SF), ET-1 levels may closely correlate to the onset of visual or cerebral complications. Due to the bronchoconstrictor activity of ET-1, a relationship with post-treatment cough can be also postulated.

Modulators of the vascular endothelin receptor in blood pressure regulation and hypertension.

Endothelin (ET) is one of the most investigated molecules in vascular biology. Since its discovery two decades ago, several ET isoforms, receptors, signaling pathways, agonists and antagonists have been identified. ET functions as a potent endothelium-derived vasoconstrictor, but could also play a role in vascular relaxation. In endothelial cells, preproET and big ET are cleaved by ET converting enzymes into ET-1, -2, -3 and -4. These ET isoforms bind with different affinities to ET(A) and ET(B) receptors in vascular smooth muscle (VSM), and in turn increase [Ca(2+)](i), protein kinase C and mitogen-activated protein kinase and other signaling pathways of VSM contraction and cell proliferation. ET also binds to endothelial ET(B) receptors and stimulates the release of nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. ET, via endothelial ET(B) receptor, could also promote ET re-uptake and clearance. While the effects of ET on vascular reactivity and growth have been thoroughly examined, its role in the regulation of blood pressure and the pathogenesis of hypertension is not clearly established. Elevated plasma and vascular tissue levels of ET have been identified in salt-sensitive hypertension and in moderate to severe hypertension, and ET receptor antagonists have been shown to reduce blood pressure to variable extents in these forms of hypertension. The development of new pharmacological and genetic tools could lead to more effective and specific modulators of the vascular ET system for treatment of hypertension and related cardiovascular disease.

Obesity and aging: determinants of endothelial cell dysfunction and atherosclerosis.

Endothelial cells are both the source and target of factors contributing to atherosclerosis. After the discovery of the endothelium-derived relaxing factor (EDRF) by Robert F. Furchgott in 1980 it soon became clear that endothelial cells also release vasoactive factors distinct from nitric oxide (NO) namely, endothelium-derived contracting factors (EDCF) as well as hyperpolarizing factors (EDHF). Vasoactive factors derived from endothelial cells include NO/EDRF, reactive oxygen species, endothelins and angiotensins which have either EDRF or EDCF functions, cyclooxygenase-derived EDCFs and EDRFs, and EDHFs. Endothelial factors are formed by enzymes such as NO synthase, cyclooxygenase, converting enyzmes, NADPH oxidases, and epoxigenases, among others, and participate in the regulation of vascular homeostasis under physiological conditions; however, their abnormal regulation due to endothelial cell dysfunction contributes to disease processes such as atherosclerosis, arterial hypertension, and renal disease. Because of recent changes in world demographics and the declining health status of the world's population, both aging and obesity as independent risk factors for atherosclerosis-related diseases such as coronary artery disease and stroke, will continue to increase in the years to come. Obesity and associated conditions such as arterial hypertension and diabetes are now also some of the primary health concerns among children and adolescents. The similarities of pathomechanisms activated in obesity and aging suggest that obesity--at least in the vasculature--can be considered to have effects consistent with accelerated, "premature" aging. Pathomechanisms as well as the clinical issues of obesity- and aging-associated vascular changes important for atherosclerosis development and prevention are discussed.

Endothelin--biology and disease.

Endothelins are important mediators of physiological and pathophysiologic processes including cardiovascular disorders, pulmonary disease, renal diseases and many others. Additionally, endothelins are involved in many other important processes such as development, cancer biology, wound healing, and even neurotransmission. Here, we review the cell and molecular biology as well as the prominent pathophysiological aspects of the endothelin system.

Intradialytic hypertension: it is time to act.

Intradialytic hypertension (IDH) is defined by blood pressure (BP) values during and at the end of the dialysis session exceeding BP values at dialysis onset. It occurs in around 10% of hemodialysis (HD) patients. It is associated with HD patients' hospitalization and increased risk of death. Many hypotheses have been proposed to explain this phenomenon. Recent studies and reports highlight the important role of fluid overload, hemodynamic changes, and increased endothelin level. The importance of other hypotheses such as the renin-angiotensin system activation, sympathetic overactivity and ionic variations seems secondary but it deserves to be confirmed. Fluid removal remains the key point for treating IDH. Several important unanswered questions remain and the need for further research is highlighted.

Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation Novel mechanism of cardiac stress modulation by PDE5 inhibition.

Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.

Non-invasive approaches to visualize the endothelin axis in vivo using state-of-the-art molecular imaging modalities.

The endothelin axis plays a major role in cardiovascular diseases and a number of human cancers. This review summarizes the work that has been published in the past ten years using labeled endothelin receptor ligands for the visualization of endothelin receptor expression in vivo.

[Influnce of Tinglizi on some neuroendocrine factors and type I and III collagen in ventricular remodeling induced by abdominal aortic banding in rats].

OBJECTIVE: To approach the influnce of Tinglizi on some neuroendocrine factors and type I and III collagen in ventricular remodeling induced by Abdominal Aortic Banding (AAB) in rats. METHODS: Myocardial hypertrophy ventricular remodeling model was induced by abdominal aortic banding (AAB) in rats. After 30 day treatment, the systolic blood pressure (SBP) diastolic blood pressure (DBP) were measured; Then the ratios of LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) were calculated; The Angiotensin II (Ang II) and Endothelin (ET-1) content in heart tissue and the Aldosterone (ALD) concentration of blood plasma were determined by radioimmunoassay. The content of type I and III collagen in myocardium were determined using immunohistochemical analysis. Hydroxy proline content in heart tissue was measured by hydrolysis method. RESULTS: The experimental data demonstrated that Tinglizi could decrease SBP, DBP and the cardiac indexes of LVW/BW and HW/BW, significantly reduce the content of ANg II, ALD and ET, decrease the total collagen content and type I and III collagen (P<0.05). CONCLUSION: Tiglizi can significantly attenuate the experimental ventricular remodeling; the mechanism is related with its ability to inhibit the activation of rennin-angiontensin-aldosterone system (RAAS) and systema nervosum sympatheticum (SNS), decrease the content of neuroendocrine factors (Ang II , ET, ALD).

Apoptotic cardiomyocyte hypertrophy during sepsis and septic shock results from prolonged exposure to endothelin precursor.

Septic shock is a complex cardiovascular dysfunction which leads to regional circulatory alterations and multi-organ dysfunction in humans and animal models. To elucidate the role of stress-activated signaling molecules in the regulation of myocardial dysfunction, we have developed and standardized isolated ventricular myocyte techniques. These techniques allow the assessment of cardiodynamics at cellular (ventricular myocyte) level. These studies are carried out in a well defined model of systemic inflammatory response syndrome following polymicrobial sepsis in the rat. Evidence is provided that sepsis-induced myocardial dysfunction produces indications (signs) of early stages of heart failure. This evidence correlates with upregulation of stress-activated protein kinase cascade. These findings suggest that prolonged exposure to endothelin precursor causes decompensatory hypertrophy in adult rat ventricular myocytes (ARVMs) during sepsis. The decompensatory hypertrophy could, in turn, results in increased cytosolic caspases-3 activity in ARVMs.

[Effect of tetrandrine on endothelin expression in the lungs and clinical significance thereof: experiment with rat models with nitrofen-induced congenital diaphragmatic hernia].

OBJECTIVE: To investigate the effects of tetrandrine on endothelin expression in the lungs and its clinical significance. METHODS: 25 pregnant female SD rats were randomly divided into 5 equal groups: Groups A to D were fed with nitrofen to cause congenital diaphragmatic hernia (CDH) in the fetuses. Group A was injected with normal saline, Group B with dexamethasone (DXM), Group C with tetrandrine, and Group D with DXM + tetrandrine. Group E was control Group. On day 21.5 of pregnancy the fetuses were delivered by cesarean section and killed. Microscopy was used to observe the CDH formation, and the relative wall thickness (RWA) and relative wall area (RWA) of pulmonary arterioles. The lung/body weight ratio, and relative integrated optical density (IOD) of pulmonary arteriole and bronchiole were observed. The expression of endothelin in the lung tissues was detected by immunohistochemistry. RESULTS: 9 rats of Group A-D produced 57 fetuses with CDH with a CDH arte of 45.2%. The lung/body weight ratios, and RWA values of Group A-D were all significantly lower than that of Group E (all P < 0.05). The RWT of pulmonary arteriole was significantly lower in Groups B and C compared with Group E (both P < 0.05). The RWT and RWA of Group A were significantly lower than those of Group B-D (all P < 0.05). The values of relative IOD of pulmonary tissues and of pulmonary arteriole of Group A-D were all significantly lower than those of Group E (all P < 0.05). A positive correlation existed between the relative IOD of endothelin in pulmonary arteriole and in bronchiole (P < 0.01), and among the RWT and RWA of pulmonary arteriole, and relative IOD (all P < 0.01). CONCLUSION: Tetrandrine improves the pulmonary hypoplasia and degrades the pulmonary hypertension.

Expression of the Endothelin-axis in the different histologic subtypes of renal cell carcinoma: a tissue microarray analysis.

Endothelin-1 and its receptors ETAR and ETBR, commonly referred to as the Endothelin-axis, are emerging to play a role in cancer. The Endothelin-axis has been shown to be involved in proliferation, angiogenesis and metastasis in various human tumours. To assess the role of the Endothelin-axis in renal cell carcinoma, we analysed its expression in archival tumour tissue of 183 patients. Representative tumour blocks were selected for constructing a tissue microarray. Paraffin sections were assessed immunohistochemically using monoclonal and polyclonal antibodies for Endothelin-1, ETAR and ETBR. Staining intensities were analysed semiquantitatively and the results were correlated with various histopathologic factors. Overexpression of Endothelin-1, ETAR and ETBR was identified in 12.8%, 84.1% and 93.3% of cases, respectively. No association with pathological tumour stage and histologic grading was found. Papillary renal cell carcinomas expressed highly significantly more Endothelin-1 than clear cell renal cell carcinomas (34.5% vs. 6.7%, p<0.001), while there was no difference between ETAR- and ETBR-expression in these histologic subtypes. However, ETAR tended to be overexpressed in the subgroup of G3-tumours (p=0.044). Studies are underway assessing the role of the Endothelin-axis and its potential use as a molecular target in renal cell carcinoma.