Overexpression Of ET-1 Research Articles

At the heart of tissue: endothelin system and end-organ damage

ET (endothelin)-1 was first described as a potent vasoconstrictor. Since then, many other deleterious properties mediated via its two receptors, ETA and ETB, have been described, such as inflammation, fibrosis and hyperplasia. These effects, combined with a wide tissue distribution of the ET system, its up-regulation in pathological situations and a local autocrine/paracrine activity due to a high tissue receptor binding, make the tissue ET system a key local player in end-organ damage. Furthermore, ET-1 interacts in tissues with other systems such as the RAAS (renin-angiotensin-aldosterone system) to exert its effects. In numerous genetically modified animal models, non-specific or organ-targeted ET-1 overexpression causes intense organ damage, especially hypertrophy and fibrosis, in the absence of haemodynamic changes, confirming a local activity of the ET system. ET receptor antagonists have been shown to prevent and sometimes reverse these tissue alterations in an organ-specific manner, leading to long-term benefits and an improvement in survival in different animal models. Potential for such benefits going beyond a pure haemodynamic effect have also been suggested by clinical trial results in which ET receptor antagonism decreased the occurrence of new digital ulcers in patients with systemic sclerosis and delayed the time to clinical worsening in patients with PAH (pulmonary arterial hypertension). The tissue ET system allows therapeutic interventions to provide organ selectivity and beneficial effects in diseases associated with tissue inflammation, hypertrophy or fibrosis.

Renal phenotype of Et-1 transgenic mice is modulated by androgens

IntroductionActivation of the endothelin (ET) system promotes inflammation and fibrosis in various tissues including the kidney. Male ET-1 transgenic mice are characterized by chronic kidney inflammation and renal scarring. We hypothesized that this renal phenotype might be modulated by androgens. Thus the aim of our study was to elucidate the impact of gonadectomy in ET-1 transgenic mice on kidney function and morphology.MethodsMale ET-1 transgenic mice at the age of 10 weeks were randomly allocated to the following groups: normal ET transgenic mice (ET; n = 17) and ET transgenic mice that underwent castration (ET+cas; n = 12). Study duration was 9 months. Creatinine clearance and protein excretion was monitored. At study end animals were sacrificed and kidneys were harvested for histology/immunhistochemistry.ResultsCastration significantly ameliorated glomerulosclerosis in ET-1 transgenic mice (ET glomerulosclerosis-score: 3.0 ± 0.17 vs ET+cas: 2.4 ± 0.17; p < 0.05) as well as renal perivascular fibrosis (ET fibrosis-score: 3.0 ± 0.14 vs ET+cas: 2.2 ± 0.14; p < 0.05). However, interstitial fibrosis and media/lumenratio of renal arteries remained unaffected by castration. Regarding inflammation, castration significantly reduced the number of CD4-positive cells in renal tissue of ET-1 transgenic mice (ET CD4-positive cells/10000 cells: 355 ± 72 vs ET+cas: 147 ± 28; p < 0.05). Renal tissue contents of CD8 positive cells as well as of macrophages were not affected by castration. Regarding kidney function castration significantly reduced proteinuria in ET-1 transgenic mice whereas creatinine clearance did not differ between study groups.ConclusionOur study demonstrates that the renal histopathological phenotype in male ET-1 transgenic mice with regard to glomerulosclerosis, proteinuria, perivascular fibrosis and immune cell immigration is ameliorated by castration. We thus conclude that the effects of ET-1 overexpression on renal tissue injury are modulated by androgens.

Endothelin, sex and hypertension

The ETs (endothelins) comprise a family of three 21-amino-acid peptides (ET-1, ET-2 and ET-3) and 31-amino-acid ETs (ET-1(1-31), ET-2(1-31) and ET-3(1-31)). ET-1 is synthesized from a biologically inactive precursor, big ET-1, by ECEs (ET-converting enzymes). The actions of ET-1 are mediated through activation of the G-protein-coupled ET(A) and ET(B) receptors, which are found in a variety of cells in the cardiovascular and renal systems. ET-1 has potent vasoconstrictor, mitogenic, pro-inflammatory and antinatriuretic properties, which have been implicated in the pathophysiology of a number of cardiovascular diseases. Overexpression of ET-1 has been consistently described in salt-sensitive models of hypertension and in models of renal failure, and has been associated with disease progression. Sex differences are observed in many aspects of mammalian cardiovascular function and pathology. Hypertension, as well as other cardiovascular diseases, is more common in men than in women of similar age. In experimental models of hypertension, males develop an earlier and more severe form of hypertension than do females. Although the reasons for these differences are not well established, the effects of gonadal hormones on arterial, neural and renal mechanisms that control blood pressure are considered contributing factors. Sex differences in the ET-1 pathway, with males displaying higher ET-1 levels, greater ET-1-mediated vasoconstrictor and enhanced pressor responses in comparison with females, are addressed in the present review. Sex-associated differences in the number and function of ET(B) receptors appear to be particularly important in the specific characteristics of hypertension between females and males. Although the gonadal hormones modulate some of the differences in the ET pathway in the cardiovascular system, a better understanding of the exact mechanisms involved in sex-related differences in this peptidergic system is needed. With further insights into these differences, we may learn that men and women could require different antihypertensive regimens.

Leptin increases endothelin type A receptor levels in vascular smooth muscle cells

Leptin, one of the adipocyte-secreted peptides, is involved in the control of appetite and body weight. Several studies have demonstrated that plasma leptin levels are elevated in obese subjects and are positively correlated with body weight. The arterial endothelin (ET) system plays an important role in the regulation of vascular tone, and ET-1 overexpression may be involved in the pathogenesis of the hypertension associated with insulin resistance. This study was performed to explore the regulatory effects of leptin on ET receptor expression and ET binding in A10 vascular smooth muscle cells (VSMCs) by use of Northern blotting, immunoblotting, and a (125)I-labeled ET-1 binding assay. The effect of leptin on ET receptor-mediated cell proliferation was also tested. The results showed that leptin caused a significant increase in [(125)I]-ET-1 binding, which was time- and dose-dependent. Immunoblotting showed that expression of the ET type A receptor (ET(A)R) in leptin (10(-7) M)-treated cells was increased by up to 2.3-fold compared with controls. Levels of ET(A)R mRNA measured by Northern blotting were also increased by up to 2.2-fold in leptin (10(-7) M)-treated cells. Pretreatment with an ERK inhibitor, PD-98059 (2.5 x 10(-5) M), blocked the leptin-induced increase in (125)I-ET-1 binding. Finally, ET-1 (10(-7) M)-stimulated cell proliferation was enhanced by leptin (10(-7) M) pretreatment, with a maximal increase of twofold compared with controls. In conclusion, leptin increases ET(A)R expression in VSMCs in a time- and dose-dependent manner. This effect is ERK dependent and is associated with increased ET-1-stimulated cell proliferation. These findings provide support for roles for leptin and the ET system in the pathogenesis of obesity-associated hypertension.

Sitaxsentan for the Prevention of Experimental Shunt-Induced Pulmonary Hypertension

We previously reported on the partial prevention of experimental shunt-induced pulmonary arterial hypertension (PAH) by the nonselective endothelin (ET) ET-A/ET-B receptor antagonist bosentan. As the respective roles of the ET-A and ET-B receptor signaling in the pathobiology of the disease remain undefined, we investigated the effects of selective ET-A receptor blockade by sitaxsentan in the same early stage PAH model. Twenty-one 3-wk-old piglets were randomized to placebo or sitaxsentan therapy (1.5 mg/kg/d), after anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation, followed by pulmonary tissue sampling for morphometry and real-time-quantitative-PCR for ET-1, angiopoietin-1, and bone morphogenetic receptor (BMPR) signaling molecules. Three months of left to right shunting induced an increase in pulmonary vascular resistance (PVR) and medial thickness, an overexpression of ET-1, ET-B receptor, and angiopoietin-1, and a decreased expression of BMPR-2 and BMPR-1A. Pretreatment with sitaxsentan prevented shunt-induced increase in PVR and decreased medial thickness by 64%. Sitaxsentan therapy completely prevented the decreased expression of BMPR-2 and limited the overexpression of ET-1, ET-B and angiopoietin-1, and the decreased expression of BMPR-1A. In conclusion, selective ET-A receptor blockade partially prevents shunt-induced PAH.

Neutralization of transforming growth factor-β attenuates hypertension and prevents renal injury in uremic rats

We investigate the role of transforming growth factor-beta (TGF-beta) in hypertension and renal failure progression in uremic rats, and whether it modulates the endothelin (ET) system. Following renal mass reduction, uremic rats (Nx) received the pan-specific TGF-beta neutralizing antibody 1D11 (0.5 mg/kg, three times/week), the isotype control antibody 13C4 or the AT1 antagonist losartan (10 mg/kg per day) for 6 weeks. Before treatment, the blood pressure was higher in Nx rats and increased further over time in Nx+13C4 rats. At the end of the study, Nx+13C4 rats exhibited increased serum creatinine, proteinuria and renal expression and excretion of TGF-beta1 and ET-1. ET-1 concentrations were greater in vascular and renal tissues, whereas the ETB receptor expression was reduced. Renal injuries were comprised of blood vessel hypertrophy, glomerular sclerosis, tubular atrophy and interstitial fibrosis, which was associated with increased alpha-smooth muscle actin expression. Treatment of uremic rats with the 1D11 antibody attenuated the increase in blood pressure and the decline in renal function. Losartan normalized the blood pressure and significantly attenuated the increase in serum creatinine and proteinuria. However, both treatments prevented renal TGF-beta1 and ET-1 overexpression, and prevented all renal histological injuries. The 1D11 antibody only improved ETB receptor expression. Neutralization of TGF-beta attenuates hypertension and renal failure progression in uremic animals, in part, by preventing renal injury processes. These effects may be related to the modulation of the ET system, preventing renal ET-1 overproduction and the reduction of ETB receptor expression. Our data also suggest that TGF-beta1 is involved, at least in part, in the pathological effects related to angiotensin II in chronic renal failure.

Chronic heart failure is associated with vascular remodeling of the brachial artery

Endothelial dysfunction has been shown to correlate with severity of congestive heart failure (CHF) and recent data suggest morphological changes of peripheral vasculature to be associated with the syndrome. We therefore investigated the hypothesis that vascular remodeling is associated with functional changes in peripheral conduit arteries and with systemic overexpression of ET-1 in patients suffering from CHF. 57 consecutive patients referred to the Innsbruck Heart Failure and Transplantation Program (EF=23+/-7%) and 16 matched controls (EF=60+/-5%) were studied. Flow-mediated vasodilation (FMD), nitroglycerin-mediated vasodilation (NMD), wall thickness (WT), and incremental elastic modulus (Einc) were assessed by high-resolution ultrasound of the brachial artery. FMD (P=0.004) and NMD (P=0.02) were significantly higher in controls as compared to moderate and severe CHF patients. In contrast, brachial artery-wall thickness (BA-WT) was increased in severe CHF patients (P=0.038). BA-WT was significantly correlated with both FMD (r=-0.28; P=0.049) and NMD (r=-0.38; P=0.003), and with the Einc (r=0.45, P=0.001). Lumen diameter was not different among groups. In patients with BA-WT>0.31 mm, bigET-1 was higher compared to BA-WT<0.31 mm (P<0.05). CHF is associated with remodeling of the brachial artery, which is characterized by morphological, mechanical and functional changes of the vessel wall. Endothelin-1 may play a role in the vascular remodeling process.

Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice.

The pulmonary endothelin (ET) system has been implicated in the pathogenesis of chronic lung diseases such as pulmonary hypertension, asthma, chronic obstructive lung disease, idiopathic pulmonary fibrosis, and bronchiolitis obliterans. However, the etiologic role of ET-1 in these diseases has not yet been established. We recently demonstrated that ET-1 transgenic mice, generated using the human prepro-ET-1 expression cassette including the cis-acting transcriptional regulatory elements, had predominant transgene expression in lung, brain, and kidney. We used these mice in the present study to analyze the pathophysiologic consequences of long-term pulmonary overexpression of ET-1. We found that ET-1 overexpression in the lungs did not result in significant pulmonary hypertension, but did result in development of a progressive pulmonary fibrosis and recruitment of inflammatory cells (predominantly CD4-positive cells). Our study provides evidence that a long-term activated pulmonary ET system, without any other stimuli, produces chronic lymphocytic inflammation and lung fibrosis. This suggests that overexpression of ET-1 may be a central event in the pathogenesis of lung diseases associated with fibrosis and chronic inflammation, such as pulmonary fibrosis and bronchiolitis.

Increased expression of endothelin B receptor mRNA following subarachnoid hemorrhage in monkeys.

These studies tested the hypothesis that the cerebral vasospasm that follows subarachnoid hemorrhage (SAH) is due to alterations in endothelin (ET) and ET receptor expression. Eight monkeys underwent cerebral angiography and induction of SAH. Angiography was repeated 7 days later to confirm the presence of cerebral vasospasm, and animals were killed. RNA was isolated from right (vasospastic) and left (control) side middle cerebral arteries and surrounding cerebral cortex. The levels of prepro (PP) ET-1 (ppET-1) and ppET-3 and ETA and ETB receptor MRNAs were determined using a quantitative reverse transcriptase polymerase chain reaction-based assay. ET-1 peptide was also measured in CSF at baseline and after 7 days. Specific agonist binding to ETA and ETB receptors in both middle cerebral arteries and in surrounding brain cortex was measured in three animals by autoradiographic binding assays. Levels of ETB receptor mRNA were 3.4 +/- 2.2-fold higher in the right than in the left cerebral arteries (p < 0.01). There were no significant differences in the levels of ppET-1, ppET-3, or ETA receptor mRNA in cerebral arteries. ET-1 peptide was not elevated in CSF. Levels of ETA and ETB receptor mRNAs were 2.6 +/ 1.1- and 2.1 +/ 1.3-fold higher, respectively, in the right than in the left cerebral cortex, while the level of ppET-3 mRNA was 2.1 +/- 1.0-fold lower. There were no differences in ppET-1 mRNA levels between right and left cerebral cortex. Binding to ETA and ETB receptors in cerebral arteries and cortex did not differ significantly between right and left sides. These results do not support the hypothesis that overexpression of ET-1 is principal cause of vasospasm, but rather they suggest that SAH causes complex changes in the ET system that together are responsible for the cellular response to SAH.

Enhanced expression of the endothelin-1 gene in blood vessels of DOCA-salt hypertensive rats: correlation with vascular structure.

Endothelin (ET)-1 is a potent vasoconstrictor peptide which is also endowed with hypertrophic and mitogenic properties. Enhanced ET-1 gene expression in blood vessels of rats with hypertension induced by deoxycorticosterone acetate (DOCA)-salt has been previously demonstrated. The objective of this study was: (1) to investigate the individual effects of salt and DOCA, and of the development of hypertension in DOCA-salt-hypertensive rats, on ET-1 gene expression in blood vessels; and (2) to correlate the presence of enhanced ET expression and the severity of vascular hypertrophy. By providing salt and/or DOCA to rats which were or were not unilaterally nephrectomized, groups of rats with variable degrees of blood pressure elevation were generated. Increased abundance of ET-1 mRNA and a greater content of immunoreactive ET-1 were found with progression of hypertension in aorta and the mesenteric arterial bed only in unilaterally nephrectomized DOCA-salt-treated rats (DOCA-salt-hypertensive rats). Vascular expression of ET-1 was not enhanced in other DOCA-or salt-treated rats, even when blood pressure rose to a mean systolic pressure of 180 mm Hg. The wet weight of aorta and the mesenteric arterial bed, the media thickness, the media cross-sectional area, and the media-to-lumen ratio of mesenteric small arteries of all groups of rats exhibited a close correlation with systolic blood pressure. In DOCA-salt-hypertensive rats after 5 weeks, in which overexpression of ET-1 was maximal, the vascular morphometric parameters were excessive for the level of systolic blood pressure. However, in DOCA-salt-hypertensive rats treated with the combined ETA/ETBET receptor antagonist bosentan, vascular morphometry correlated closely with blood pressure, even though blood pressure was only slightly lower than than of untreated DOCA-salt-hypertensive rats. These data support the hypothesis that ET-1 gene overexpression in blood vessels may accentuate vascular hypertrophy in some forms of hypertension.

Measurement of C-Terminal Fragment of Big Endothelin-1

We developed a procedure for individual measurement of big endothelin-1 (big ET-1) and the two products of big ET-1 conversion (ET-1 and C-terminal fragment (CTF) of big ET-1 (big ET-1(22-38)), using selective solid-phase extraction and specific radioimmunoassays. These techniques were used to measure the levels of these peptides in extracts of human plasma after brachial artery infusions of big ET-1. Infusion of big ET-1 (50 pmol/min, n = 6) caused a decrease in forearm blood flow from 3.03 to 1.55 ml/dl/min after 60 min (p < 0.05). In agreement, the levels of plasma immunoreactive (IR) big ET-1, ET, and CTF were significantly increased from basal levels in the infused arm (from undetectable to 386 pM, 2.2-7.5 pM, and 0.17-37 pM, respectively; p < 0.05). In the presence of the metalloprotease inhibitor phosphoramidon (30 nmol/min), the increase in IR-ET and the associated vasoconstriction were abolished. However, IR-CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive endothelin-converting enzyme (ECE) was occurring and/or that CTF was being preserved from further proteolysis by phosphoramidon. These data confirm that exogenous big ET-1 is locally converted to ET-1 and CTF in the human forearm, at least in part by a phosphoramidon-sensitive ECE. Furthermore, because measurable levels of newly synthesized ET-1 are probably rapidly reduced as a result of receptor binding, the assay of IR-CTF may be a more sensitive measure of the overexpression of ET-1 in disease.