Global Overexpression of ET-1 Decreases Blood Pressure - A Systematic Review and Meta-Analysis of ET-1 Transgenic Mice.

BACKGROUND/AIMS: ET-1 has independent effects on blood pressure regulation in vivo, it is involved in tubular water and salt excretion, promotes constriction of smooth muscle cells, modulates sympathetic nerve activity, and activates the liberation of nitric oxide. To determine the net effect of these partially counteracting mechanisms on blood pressure, a systematic meta-analysis was performed. METHODS: Based on the principles of Cochrane systematic reviews, we searched in major literature databases - MEDLINE (PubMed), Embase, Google Scholar, and the China Biological Medicine Database (CBM-disc) - for articles relevant to the topic of the blood pressure phenotype of endothelin-1 transgenic (ET-1+/+) mice from January 1, 1988 to March 31, 2016. Review Manager Version 5.0 (Rev-Man 5.0) software was applied for statistical analysis. In total thirteen studies reported blood pressure data. RESULTS: The meta-analysis of blood pressure data showed that homozygous ET-1 transgenic mice (ET-1+/+ mice) had a significantly lower blood pressure as compared to WT mice (mean difference: -2.57 mmHg, 95% CI: -4.98∼ -0.16, P = 0.04), with minimal heterogeneity (P = 0.86). A subgroup analysis of mice older than 6 months revealed that the blood pressure difference between ET-1+/+ mice and WT mice was even more pronounced (mean difference: -6.19 mmHg, 95% CI: -10.76∼ -1.62, P = 0.008), with minimal heterogeneity (P = 0.91). CONCLUSION: This meta-analysis provides robust evidence that global ET-1 overexpression in mice lowers blood pressure in an age-dependent manner. Older ET-1+/+ mice have a somewhat more pronounced reduction of blood pressure.

25 years of endothelin research: the next generation.

In the past three decades, endothelin and endothelin receptor antagonists have received great scientific and clinical interest, leading to the publication of more than 27,000 scientific articles since its discovery. The Thirteenth International Conference on Endothelin (ET-13) was held on September 8-11, 2013, at Tokyo Campus of the University of Tsukuba in Japan. Close to 300 scientists from 25 countries from around the world came to Tokyo to celebrate the anniversary of the discovery of the endothelin peptide discovered 25 years ago at the University of Tsukuba. This article summarizes some of the highlights of the conference, the anniversary celebration ceremony, and particularly the participation of next generation of endothelin researchers in endothelin science and the anniversary celebration. As a particular highlight, next generation endothelin researchers wrote a haiku (a traditional form of Japanese poetry originating from consisting of no more than three short verses and 27 on, or Japanese phonetic units) to describe the magic of endothelin science which they presented to the conference audience at the anniversary ceremony. The text of each haiku - both in its original language together with the English translation - is part of this article providing in an exemplary fashion how poetry can be bridged with science. Finally, we give an outlook towards the next 25 years of endothelin research.

Vascular endothelium derived endothelin-1 is required for normal heart function after chronic pressure overload in mice.

BACKGROUND: Endothelin-1 participates in the pathophysiology of heart failure. The reasons for the lack of beneficial effect of endothelin antagonists in heart failure patients remain however speculative. The anti-apoptotic properties of ET-1 on cardiomyocytes could be a reasonable explanation. We therefore hypothesized that blocking the pro-apoptotic TNF-α pathway using pentoxifylline could prevent the deleterious effect of the lack of ET-1 in a model for heart failure. METHODS: We performed transaortic constriction (TAC) in vascular endothelial cells specific ET-1 deficient (VEETKO) and wild type (WT) mice (n = 5-9) and treated them with pentoxifylline for twelve weeks. RESULTS: TAC induced a cardiac hypertrophy in VEETKO and WT mice but a reduction of fractional shortening could be detected by echocardiography in VEETKO mice only. Cardiomyocyte diameter was significantly increased by TAC in VEETKO mice only. Pentoxifylline treatment prevented cardiac hypertrophy and reduction of fractional shortening in VEETKO mice but decreased fractional shortening in WT mice. Collagen deposition and number of apoptotic cells remained stable between the groups as did TNF-α, caspase-3 and caspase-8 messenger RNA expression levels. TAC surgery enhanced ANP, BNP and bcl2 expression. Pentoxifylline treatment reduced expression levels of BNP, bcl2 and bax. CONCLUSIONS: Lack of endothelial ET-1 worsened the impact of TAC-induced pressure overload on cardiac function, indicating the crucial role of ET-1 for normal cardiac function under stress. Moreover, we put in light a TNF-α-independent beneficial effect of pentoxifylline in the VEETKO mice suggesting a therapeutic potential for pentoxifylline in a subpopulation of heart failure patients at higher risk.

Inhibition of vascular endothelial growth factor receptor under hypoxia causes severe, human-like pulmonary arterial hypertension in mice: potential roles of interleukin-6 and endothelin.

AIMS: Severe pulmonary arterial hypertension (PAH) is an incurable disease whose exact mechanisms remain unknown. However, growing evidence highlights the role of inflammation and endothelin (ET) signaling. The lack of reliable models makes it difficult to investigate the pathophysiology of this disease. Our aim was therefore to develop a mouse model of severe PAH closely mimicking the human condition to explore the role of interleukin-6 (IL-6), and ET signaling in advanced PAH progression. MAIN METHODS: Young male SV129 mice received vascular endothelial growth factor receptor inhibitor (SU5416) three times a week and were exposed to hypoxia (10% O2) for three weeks. Molecular analysis and histological assessment were examined using real-time PCR, Western blot and immunostaining, respectively. KEY FINDINGS: The developed murine model presented important characteristics of severe PAH in human: concentric neointimal wall thickening, plexogenic lesions, recruitment of macrophages, and distal arteriolar wall muscularization. We detected an increase of IL-6 production and a stronger macrophage recruitment in adventitia of remodeled arterioles developing plexogenic lesions. Moreover, ET-1 and ET receptor A were up-regulated in lung lysates and media of remodeled arterioles. Recombinant IL-6 stimulated the proliferation and regulated endothelial cells in increasing ET-1 and decreasing ET receptor B. SIGNIFICANCE: These data describe a murine model, which displays the most important features of human severe PAH. We assume that inflammation, particularly IL-6 regulating ET signaling, plays a crucial role in forming plexogenic lesions. This model is thus reliable and might be used for a better understanding of severe PAH progression and treatment.

Chronic hyperaldosteronism in cryptochrome-null mice induces high-salt- and blood pressure-independent kidney damage in mice.

Although aldosterone has an essential role in controlling electrolyte and body fluid homeostasis, aldosterone also exerts certain pathological effects on the kidney. Several previous studies have attempted to examine these deleterious effects. However, the majority of these studies were performed using various injury models, including high-salt treatment and/or mineralocorticoid administration, by which the kidney changes observed were not only due to aldosterone but also due to prior injury caused by salt and hypertension. In the present study, we investigated aldosterone's pathological effect on the kidney using a mouse model with a high level of endogenous aldosterone. We used cryptochrome-null (Cry 1, 2 DKO) mice characterized by high aldosterone levels and low plasma renin activity and observed that even under normal salt exposure conditions, these mice showed increased albumin excretion and kidney tubular injury, decreased nephrin expression and increased reactive oxygen species production in the absence of hypertension. Exposure to high salt levels exacerbated the kidney damage observed in these mice. Moreover, we noted that decreasing blood pressure without blocking aldosterone action did not provide beneficial effects to the kidney in high-salt-treated Cry 1, 2 DKO mice. Thus, our findings support the hypothesis that aldosterone has deleterious effects on the kidney independent of high-salt exposure and high blood pressure.

Endothelin-1 overexpression and endothelial nitric oxide synthase knock-out induce different pathological responses in the heart of male and female mice.

AIMS: The nitric oxide and endothelin systems are key components of a local paracrine hormone network in the heart. We previously reported that diastolic dysfunction observed in mice lacking the endothelial nitric oxide synthase (eNOS-/-) can be prevented by a genetic overexpression of ET-1. Sexual dimorphisms have been reported in both ET-1 and NO systems. Particularly, eNOS-/- mice present sex related phenotypic differences. MAIN METHODS: We used the ET-1 transgenic (ET+/+), eNOS-/-, and crossbred ET+/+eNOS-/- mice, and wild type controls. We measured cardiac function by heart catheterization. Cardiac ventricles were collected for histological and molecular profiling. KEY FINDINGS: We report here that (i) the level of ET-1 expression in eNOS-/- mice was elevated in males but not in females. (ii) Left ventricular end-diastolic blood pressure was higher in male eNOS-/- mice than in females. (ii) eNOS-/- males but not females developed cardiomyocyte hypertrophy. (iv) Perivascular fibrosis of intracardiac arteries developed in female ET+/+ and eNOS-/- mice but not in males. Additionally, (v) the cardiac expression of metalloprotease-9 was higher in eNOS-/- males compared to females. Finally, (vi) cardiac proteome analysis revealed that the protein abundance of the oxidative stress related enzyme superoxide dismutase presented with sexual dimorphism in eNOS-/- and ET+/+ mice. SIGNIFICANCE: These results indicate that the cardiac phenotypes of ET-1 transgenic mice and eNOS knockout mice are sex specific. Since both systems are key players in the pathogenesis of cardiovascular diseases, our findings might be important in the context of gender differences in patients with such diseases.

Rhodamine-labeled LDL as a tool to monitor the lipoprotein traffic in experimental model of early atherosclerosis in mice.

Modifications of proteoglycans, subendothelial retention of low-density lipoproteins (LDL) and their subsequent oxidation initiate the development of atherosclerosis. Therefore, detection of lipoprotein entrapment in the arterial wall is an important feature for the analysis of the mechanisms of atherosclerosis. The administration of fluorescent-labeled LDL in vivo is a breakthrough way to assess the traffic of LDL in the arterial wall. The present study demonstrated the feasibility of visualizing LDL in carotid ligation-induced intimal thickening of arterial wall after intravenous rhodamine-labeled LDL injection in mice. Kinetics of rhodamine-labeled LDL showed similar characteristics as native LDL and labeled-LDL could be detected both by spectrophotometric and microscopic analysis. Kinetics analysis of rhodamine-labeled LDL revealed that the labeled LDL was present in almost all tissue, predominantly in the liver, 6 hours after injection. Rhodamine-labeled LDL was visualized in intimal thickening of carotid 6 to 18 hours after injection, indicating that the LDL was actively trapped in the arterial wall. In conclusion, rhodamine-labeled LDL would be a useful tool to investigate the development of atherosclerosis.

Endothelin-converting enzyme-1 gene ablation attenuates pulmonary fibrosis via CGRP-cAMP/EPAC1 pathway.

Endothelin-1 (ET-1) has been shown to be involved in human pulmonary fibrosis. However, recent clinical trials targeting the ET-1 pathway with ET-1 receptor antagonists failed to achieve beneficial outcomes. Another strategy opposing the actions of ET-1 involves the inhibition of endothelin-converting enzyme-1 (ECE-1). We hypothesize that ECE-1 inhibition exerts beneficial effects on pulmonary fibrosis. Pulmonary fibrosis was induced by instilling bleomycin intratracheally into ECE-1 heterozygous knockout mice (ECE-1(+/-)) and their wild-type control mice (ECE-1(+/+)). Lung inflammation and fibrosis were assessed on Days 7, 14, and 28 after bleomycin instillation. The activity of ECE-1 and the concentrations of its related peptides, ET-1, bradykinin, atrial natriuretic peptide (ANP), and calcitonin gene-related peptide (CGRP), were determined. ECE-1(+/-) mice demonstrated less lung inflammation and limited fibrosis compared with control mice. ECE-1 activity was half-reduced in ECE-1(+/-) mice, and this activity also altered ET-1 and CGRP concentrations, but not concentrations of bradykinin and ANP. ET-1 concentrations were found to be lower in ECE-1(+/-) mice after the development of fibrosis, in contrast to the unaltered concentrations during inflammation. Reduced ECE-1 activity resulted in higher CGRP concentrations, which altered the pathological functionality of the lung, indicating the activation of the CGRP pathway involving cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP and cAMP/protein kinase A in ECE-1(+/-) mice. Bleomycin instillation on Day 14 induced the accumulation of M2 macrophages expressing CGRP receptors in ECE-1(+/-) mice. Our results emphasize that the in vivo ECE-1-mediated degradation of CGRP promotes the transition from lung inflammation to fibrosis. Further, our study identified M2 macrophages as the target cells of CGRP action during this transition.

Localization and characterization of a novel secreted protein, SCUBE2, in the development and progression of atherosclerosis.

Inhibition of atherosclerosis progression has long been the subject of intensive pathophysiologic investigations. The identification of a novel target molecule to redeem the cellular processes remains a major challenge in cardiology. Signal peptide CUB domain EGF-like repeat protein (SCUBE) family has been detected on human tissues and cultured cells. Two members of the SCUBE family, SCUBE1 and SCUBE3 are reported to play a role in cardiovascular diseases. The other member, SCUBE2 has been reported to mediate Hedgehog (Hh) protein signaling and is expressed in major blood vessels during mouse embryogenesis. However its involvement in cardiovascular diseases is not known yet. The aim of this study was to investigate SCUBE2 expression and localization in diffuse intimal thickening (DIT), an early event of atherosclerosis and in advanced lesion of atherosclerotic plaque. Carotid artery ligation in C57BL/6J mice was performed to induce intimal thickening, mimicking DIT in human. After 2 weeks of ligation, mRNA level of SCUBE2 increased significantly, while in LDLr-/- mice fed with high fat diet, a human atherosclerosis model, mRNA level of SCUBE2 expression markedly increased 8 weeks after start of the high fat diet. Our findings were confirmed by the observation of SCUBE2 expression in human coronary artery with DIT and advanced lesion of atherosclerotic plaque. Previous investigations described that SCUBE2 mediates Hh signaling pathway. We have observed that SCUBE2 expression is associated with Sonic Hedgehog (Shh) and its receptor, Patched (Ptc), both in DIT and advanced plaque lesion. Our results suggested that SCUBE2 is a new target molecule in atherosclerosis and might play an important role in atherosclerotic plaque progression via Hh signal transduction.

Analysis of cardiac and renal endothelin receptors by in situ hybridization in mice.

BACKGROUND: Endothelin-1 (ET-1) is a multifunctional peptide, which is implicated in the renal and cardiac physicology as well as in many pathologies of these systems. ET-1's actions take place after the activation of two receptors: ET(A) and ET(B). The expression of these receptors may be modulated during the pathologic process. The analysis of the distribution and level of expression of the receptors in animal models is therefore crucial. METHODS: We developed a protocol for non-radioactive in situ hybridization for the mRNA of the two endothelin receptors on paraffin-embedded tissue using digoxigenin-labeled RNA probes. RESULTS: In heart and kidney, the staining was reliable and specific. In a mouse model for endothelin/nitric oxide imbalance, cardiac ET(B) expression was reduced. The distribution of the receptors was in accordance with the actual knowledge. Differences in cell specific expression are discussed. CONCLUSIONS: We developed a protocol for the in situ hybridization of the endothelin receptors in mice. Given that the endothelin system is implicated in the development of many diseases, we believe that this protocol may be useful for a number of future preclinical studies..

Physiological relevance of hydrolysis of atrial natriuretic peptide by endothelin-converting enzyme-1.

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that cleaves biologically inactive big endothelin-1 (ET-1) into active ET-1. ET-1 is involved in the cardiovascular homeostasis and the development of cardiovascular diseases including pulmonary arterial hypertension and heart failure. Atrial natriuretic peptide (ANP) is an endogenous hormone that is released from the heart in response to myocardial stretch and overload. ANP was shown to be hydrolyzed by neutral endopeptidase 24.11 (NEP) which shares important structural features with ECE-1. Previous in vitro studies using recombinant soluble ECE-1 suggested that ECE-1 cleaved several biologically active peptides including ANP in addition to big ET-1. However, physiological relevance of ANP-degrading activity by ECE-1 has stayed unclear. Here, we aimed to investigate whether endogenous ECE-1 is able to hydrolyze ANP using live-cell based assay and ECE-1-deficient mice. Chinese hamster ovary (CHO) cells, which lack detectable levels of ECE activity, degraded ANP in the medium efficiently when transfected with ECE-1 cDNA. ANP peptide contents in the E14-15 embryos were significantly higher in ECE-1+/- mice compared with ECE-1+/+ mice. These observations strongly suggest that ECE-1 is involved in the physiological degradation of ANP in vivo. Thus, pharmacological inhibition of ECE-1 may provide a novel strategy to treat various cardiovascular diseases by suppressing and potentiating the ET and ANP pathway, respectively.

ET-1 deletion from endothelial cells protects the kidney during the extension phase of ischemia/reperfusion injury.

BACKGROUND: The prognosis of patients after acute kidney injury (AKI) is poor and treatment is limited. AKI is mainly caused by renal ischemia/reperfusion injury (IRI). During the extension phase of IRI, endothelial damage may participate in ischemia and inflammation. Endothelin-1 (ET-1) which is mostly secreted by endothelial cells is an important actor of IRI, particularly through its strong vasoconstrictive properties. We aimed to analyze the specific role of ET-1 from the endothelial cells in AKI. METHODS: We used mice lacking ET-1 in the vascular endothelial cells (VEETKO). We induced IRI in VEETKO mice and wild type controls by clamping both kidneys for 30min. Sham operated mice were used as controls. Mice were sacrificed one day after IRI in order to investigate the extension phase of IRI. Kidney function was assessed based on serum creatinine concentration. Levels of expression of ET-1, its receptor ET(A), protein kinase C, eNOS, E-Cadherin and inflammation markers were evaluated by real time PCR or western blot. Tubular injury was scored on periodic acid Schiff stained kidney preparations. Lumen and wall area of small intrarenal arteries were measured on kidney slices stained for alpha smooth muscle cell actin. Oxidative stress, macrophage infiltration and cell proliferation was evaluated on slices stained for 8-hydroxy-2'-deoxyguanosine, F4/80 and PCNA, respectively. RESULTS: IRI induced kidney failure and increased ET-1 and ET(A) receptor expression. This was accompanied by tubular injury, wall thickening and reduction of lumen area/wall area ratio of small renal arteries, increased oxidative stress and inflammation. These parameters were attenuated in VEETKO mice. CONCLUSION: Our results suggest that suppression of ET-1 from the endothelial cells attenuates IRI kidney injury. Blocking ET-1 effects may represent a therapeutic strategy in the management of AKI.

Endothelin and endothelin receptors in the renal and cardiovascular systems.

Endothelin-1 (ET-1) is a multifunctional hormone which regulates the physiology of the cardiovascular and renal systems. ET-1 modulates cardiac contractility, systemic and renal vascular resistance, salt and water renal reabsorption, and glomerular function. ET-1 is responsible for a variety of cellular events: contraction, proliferation, apoptosis, etc. These effects take place after the activation of the two endothelin receptors ET(A) and ET(B), which are present - among others - on cardiomyocytes, fibroblasts, smooth muscle and endothelial cells, glomerular and tubular cells of the kidney. The complex and numerous intracellular pathways, which can be contradictory in term of functional response depending on the receptor type, cell type and physiological situation, are described in this review. Many diseases share an enhanced ET-1 expression as part of the pathophysiology. However, the use of endothelin blockers is currently restricted to pulmonary arterial hypertension, and more recently to digital ulcer. The complexity of the endothelin system does not facilitate the translation of the molecular knowledge to clinical applications. Endothelin antagonists can prevent disease development but secondary undesirable effects limit their usage. Nevertheless, the increasing understanding of the effects of ET-1 on the cardiac and renal physiology maintains the endothelin system as a promising therapeutic target.

ET-1 from endothelial cells is required for complete angiotensin II-induced cardiac fibrosis and hypertrophy.

AIMS: Hypertensive patients develop cardiac hypertrophy and fibrosis with increased stiffness, contractile deficit and altered perfusion. Angiotensin II (AngII) is an important factor in the promotion of this pathology. The effects of AngII are partly mediated by endothelin-1 (ET-1) and transforming growth factor-ß. The exact feature of these pathways and the intercellular communications involved remain unclear. In this study, we explored the role of endothelial cell-derived ET-1 in the development of AngII-induced cardiac fibrosis and hypertrophy. MAIN METHODS: We used mice with vascular endothelial cell specific ET-1 deficiency (VEETKO) and their wild type littermates (WT). Mice were infused for one week with AngII (3.2mg/kg/day, n=12) or vehicle (0.15 mol/L NaCl and 1 mmol/L acetic acid, n=5), using subcutaneous mini-pumps. Hearts were stained with hematoxylin-eosin and masson's trichrome for histology. Cardiac gene expression and protein abundance were measured by Northern Blot, real time PCR and Western Blot. KEY FINDINGS: AngII-induced cardiac hypertrophy, interstitial and perivascular fibrosis were less pronounced in VEETKO mice compared to WT. Blood pressure increased similarly in both genotypes. Expression of connective tissue growth factor, tumor growth factor-ß, collagen I and III in response to AngII required endothelial ET-1. Endothelial ET-1 was also necessary to the elevation in protein kinase C δ abundance and ERK1/2 activation. AngII-induced elevation in PKCε abundance was however ET-1 independent. SIGNIFICANCE: This study underscores the significance of ET-1 from the vasculature in the process of AngII-induced cardiac hypertrophy and fibrosis, independently from blood pressure. Endothelial ET-1 represents therefore a possible pharmacological target.

Renal function and blood pressure: molecular insights into the biology of endothelin-1.

The therapeutic implications of the actions of endothelin (ET)-1 upon renal and cardiovascular function are evident. Among other diseases, ET-1 is recognized to be involved in hypertension and renal failure and, in a rush to develop novel treatments, has been extensively studied. However, given the broad localization of the two receptors (ET(A) and ET(B)) and the diverse effects resulting from their activation, analysis of the role of ET-1 in kidney-regulated blood pressure remains complicated. Moreover, the actions of ET-1 depend upon the cell type and physiological situation. To add to the complexity, both receptors often activate opposing signaling pathways within a single cell. Thus, until recently, reliable insights into the respective involvement of both receptors in the physiology and pathology of the kidney were eagerly awaited. These have been obtained using mice that are genetically modified for different members of the ET system. In this article, the molecular biology of ET-1 and its receptors in the control of renal vasculature tonicity, glomerular function, and management of water and salt reabsorption is discussed. The role of renal ET-1 in the context of blood pressure regulation will be discussed, and the potential of utilizing ET receptor antagonism in the treatment and prevention of glomerular and proteinuric diseases is also outlined.

Dietary phytoestrogen supplementation induces sex differences in the myocardial protein pattern of mice: a comparative proteomics study.

Elevated cardiovascular risk in postmenopausal women and beneficial actions of estrogen replacement in animal models have been related to protective effects of estrogens. However, randomized trials of hormone replacement therapy with synthetic estrogens in humans failed confirmation and phytoestrogens, natural plant hormones with agonistic properties for estrogen receptors, could represent potential alternatives. The aim of the present study is to characterize an animal model for alternative hormone replacement with genistein as a natural estrogenic compound. We performed a 2-DE/ESI-LC-MS approach in order to identify protein species varying with genistein receipt and sex in their relative abundance in the healthy murine heart (http://www.mpiib-berlin.mpg.de/2D-PAGE). Oral genistein treatment revealed a substantial effect on the relative abundance of both estrogen receptors. Several enzymes of the fatty acid metabolism and their transcriptional regulators varied differentially in male and in female animals, at the transcript and/or the protein species level. Increased levels of enzyme species involved in the oxidative phosphorylation and generation of ROS were accompanied by decreased amounts of antioxidants in male mice receiving genistein compared with control males, which have been previously associated with various pathological conditions. Exposure of female animals to genistein provoked an increased abundance of two species of LIM domain-binding protein and one species of desmin. These proteins have been associated with cardiac hypertrophy and our data warrant caution for the use of them as molecular markers, since the animals did not exhibit any histological signs of cardiac hypertrophy.

Endothelin-1 overexpression restores diastolic function in eNOS knockout mice.

BACKGROUND: The cardiac nitric oxide and endothelin-1 (ET-1) systems are closely linked and play a critical role in cardiac physiology. The balance between both systems is often disturbed in cardiovascular diseases. To define the cardiac effect of excessive ET-1 in a status of nitric oxide deficiency, we compared left ventricular function and morphology in wild-type mice, ET-1 transgenic (ET(+/+)) mice, endothelial nitric oxide synthase knockout (eNOS(-/-)) mice, and ET(+/+)eNOS(-/-) mice. METHODS AND RESULTS: eNOS(-/-) and ET(+/+)eNOS(-/-) mice developed high blood pressure compared with wild-type and ET(+/+) mice. Left ventricular catheterization showed that eNOS(-/-) mice, but not ET(+/+)eNOS(-/-) , developed diastolic dysfunction characterized by increased end-diastolic pressure and relaxation constant tau. To elucidate the causal molecular mechanisms driving the rescue of diastolic function in ET(+/+)eNOS(-/-) mice, the cardiac proteome was analyzed. Two-dimensional gel electrophoresis coupled to mass spectrometry offers an appropriate hypothesis-free approach. ET-1 overexpression on an eNOS(-/-) background led to an elevated abundance and change in posttranslational state of antioxidant enzymes (e.g., peroxiredoxin-6, glutathione S-transferase mu 2, and heat shock protein beta 7). In contrast to ET(+/+)eNOS(-/-) mice, eNOS(-/-) mice showed an elevated abundance of proteins responsible for sarcomere disassembly (e.g., cofilin-1 and cofilin-2). In ET(+/+)eNOS(-/-) mice, glycolysis was favored at the expense of fatty acid oxidation. CONCLUSION: eNOS(-/-) mice developed diastolic dysfunction; this was rescued by ET-1 transgenic overexpression. This study furthermore suggests that cardiac ET-1 overexpression in case of eNOS deficiency causes specifically the regulation of proteins playing a role in oxidative stress, myocytes contractility, and energy metabolism.

Lack of endothelial nitric oxide synthase promotes endothelin-induced hypertension: lessons from endothelin-1 transgenic/endothelial nitric oxide synthase knockout mice.

Endothelin-1 (ET-1) is one of the most potent biologic vasoconstrictors. Nevertheless, transgenic mice that overexpress ET-1 exhibit normal BP. It was hypothesized that vascular effects of ET-1 may be antagonized by an increase of the endothelial counterpart of ET-1, nitric oxide (NO), which is produced by the endothelial NO synthase (eNOS). Therefore, cross-bred animals of ET transgenic mice (ET+/+) and eNOS knockout (eNOS-/-) mice and were generated, and BP and endothelial function were evaluated in these animals. Endothelium-dependent and -independent vascular function was assessed as relaxation/contraction of isolated preconstricted aortic rings. The tissue ET and NO system was determined in aortic rings by quantitative real-time PCR and Western blotting. Systolic BP was similar in ET+/+ and wild-type (WT) mice but was significantly elevated in eNOS-/- mice (117 +/- 4 mmHg versus 94 +/- 6 mmHg in WT mice; P < 0.001) and even more elevated in ET+/+ eNOS-/- cross-bred mice (130 +/- 4 mmHg; P < 0.05 versus eNOS-/-). Maximum endothelium-dependent relaxation was enhanced in ET+/+ mice (103 +/- 6 versus 87 +/- 4% of preconstriction in WT littermates; P < 0.05) and was completely blunted in eNOS-/- (-3 +/- 4%) and ET+/+ eNOS-/- mice (-4 +/- 4%), respectively. Endothelium-independent relaxation was comparable among all groups. Quantitative real-time PCR as well as Western blotting revealed an upregulation of the aortic ET(A) and ET(B) receptors in ET+/+ eNOS-/-, whereas eNOS was absent in aortic rings of eNOS-/- and ET+/+ eNOS-/- mice. ET-1 aortic tissue concentrations were similar in WT mice and ET+/+ eNOS-/- mice most probably as a result of an enhanced clearance of ET-1 by the upregulated ET(B) receptor. These data show for the first time that in transgenic mice that overexpress human ET-1, additional knockout of eNOS results in a further enhancement of BP as compared with eNOS-/- mice. The human ET+/+ eNOS-/- mice therefore represent a novel model of hypertension as a result of an imbalance between the vascular ET-1 and NO systems.