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

AbstractAimsThe 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 methodsWe 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 findingsWe 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.SignificanceThese 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

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

Analyse von Endothelin-1 transgenen und eNOS knockout Mäusen

The hormone Endothelin‐1 (ET‐1) exerts vasoconstrictive, inotropic, fibrotic and mitogenic effects on the cardiovascular system through the activation of ET receptor A. On the other hand, activation of ET receptor B stimulates the endothelial nitric oxide synthase (eNOS); NO presents strong vasodilative and cardioprotective properties and represses ET‐1 expression. The clinical relevance of this delicate interplay has been acknowledged because of its implication in many cardiovascular diseases, such as pulmonary arterial hypertension, systemic hypertension, and coronary artery disease. However, the underlying molecular mechanisms remain to be fully clarified. ET‐1 transgenic (ET+/+) mice develop pulmonary, cardiac and renal fibrosis, glomerulosclerosis and decreased glomerular filtration rate. However, in spite of the strong vasoconstrictive feature of ET‐1, ET+/+ mice remain normotensive. The natural functional antagonist of ET‐1, NO, was assumed to counteract the ET‐1 effect on blood pressure in ET+/+ mice. To test this hypothesis in vivo, ET+/+ mice were crossbred with eNOS knock‐out (eNOS‐/‐) mice. Similar to the eNOS‐/‐ model, the ET+/+eNOS‐/‐ mice develop high blood pressure compared to wild type (WT) and ET+/+ animals. However, at the age of nine months, the eNOS‐/‐, but not ET+/+eNOS‐/‐ mice, are characterized by diastolic dysfunction. These findings suggested that transgenic overexpression of ET‐1 on an eNOS‐/‐ background could be beneficial for diastolic functions. In this thesis, it is shown that cardiac ET‐1 gene expression was elevated in both eNOS‐/‐ and ET+/+eNOS‐/‐ mice compared to WT at the age of nine months with no significant difference between these groups. This suggests that the functional differences observed between eNOS‐/‐ and eNOS‐/‐ET+/+ animals are due to the chronic overexpression of ET‐1 mediated by the transgene, which may have conditioned ET+/+eNOS‐/‐ animals over time to prevent the development of diastolic dysfunction. In line with this, eNOS‐/‐ animals displayed normal cardiomyocyte diameters at the age of three months, but developed cardiac hypertrophy at the age of nine months, whereas ET+/+eNOS‐/‐ mice showed enlarged cardiomyocytes at the age of three months that were not detectable in animals nine months of age. Histological analysis showed that cardiac arterioles were dilated in both ET+/+ and ET+/+eNOS‐/‐ mice compared to WT and eNOS‐/‐ mice. By enhancing blood flow, this could be beneficial for cardiac functions. In order to dissect the molecular changes underlying this phenomenon, the cardiac proteome of the different genotypes were compared to WT using two‐dimensional electrophoresis coupled to mass spectrometry. Because these changes may take place early in the life of the animals, three months old animals were analyzed. The proteomics study revealed that transgenic overexpression of ET‐1, with or without eNOS, led to a higher abundance of proteins regulating oxidative stress indicating that, in contrast to eNOS‐/‐ animals, ET+/+ and ET+/+eNOS‐/‐ mice developed molecular mechanisms limiting oxidative damages. Moreover, diastolic dysfunction observed in eNOS‐/‐ mice may be explained by the differential abundance of proteins involved in the contractile machinery. Overexpression of ET‐1 in eNOS‐/‐ mice restored these changes and may have thereby benefited the cardiac functions. Finally, this study indicated that a shift from fatty acid to glucose metabolism, considered as cardioprotective, may have occurred to a greater extent in crossbred animals than in eNOS‐/‐ mice. Taken together, this study showed that transgenic overexpression of ET‐1 in mice can have beneficial effects on cardiac function, even in the absence of eNOS, by modulating various systems (oxidative stress, contractile machinery, and energy metabolism). The clinical relevance of these findings should be confirmed by analyzing the impact of a pharmacological interference with these systems on cardiac function. Finally, the fact that additional overexpression of ET‐1 restored the deleterious effect of eNOS deficiency underlines the importance of the interplay between the two systems in the heart of mice.Endothelin‐1 (ET‐1) ist ein Peptidhormon, das durch Aktivierung des ET‐1 Rezeptors A vasokonstriktorische, inotrope, fibrotische und mitogene Effekte im kardiovaskulären System vermittelt. Gleichzeitig stimuliert Aktivierung des ET‐1 Rezeptors B die endotheliale Stickstoffmonoxid‐Synthase (eNOS), wobei NO starke vasodilatative and kardioprotektive Eigenschaften aufweist und die ET‐1 Expression hemmt. Aufgrund seines wesentlichen Einflusses auf zahlreiche Erkrankungen des kardiovaskulären Systems wie zum Beispiel die pulmonal‐arterielle und systemische Hypertonie oder die koronare Herzerkrankung ist dieses fein abgestimmte Gleichgewicht von erheblicher klinischer Relevanz; die zugrundeliegenden molekularen Mechanismen konnten bislang jedoch nur unvollständig identifiziert werden. In vorangegangenen Arbeiten konnte gezeigt werden, dass transgene Mäuse mit einer ET‐1 Überexpression eine pulmonale, kardiale und renale Fibrose sowie eine Glomerulosklerose mit verminderter glomerulärer Filtrationsrate entwickeln. Interessanterweise zeigen diese Tiere jedoch trotz der bekannten vasokonstriktorischen Effekte von ET‐1 keine Hypertonie. Um die Hypothese, dass dies auf eine Antagonisierung der ET‐1 Wirkung durch NO zurückzuführen ist, in einem in vivo System zu überprüfen, wurden ET+/+ Mäuse mit eNOS knock‐out Tieren (eNOS‐/‐) gekreuzt. Analog zum Phänotyp der eNOS‐/‐ Tiere zeigte sich in diesem kombinierten ET+/+eNOS‐/‐ Modell eine arterielle Hypertonie. Während eNOS‐/‐ Mäuse jedoch im Alter von neun Monaten eine diastolische Dysfunktion entwickelten, waren diese Veränderungen im kombinierten ET+/+eNOS‐/‐ Modell nicht nachzuweisen, so dass sich damit Hinweise für einen möglichen protektiven Effekt der ET‐1 Überexpression auf die diastolische Funktion ergaben. In der vorliegenden Arbeit wird gezeigt, dass sowohl eNOS‐/‐ als auch ET+/+eNOS‐/‐ Tiere im Alter von neun Monaten eine im Vergleich zu Wildtypkontrollen erhöhte ET‐1 Expression zeigen, die sich zwischen diesen Gruppen nicht signifikant unterscheidet. Dies deutet darauf hin, dass die funktionellen Unterschiede zwischen beiden Modellen darauf zurückzuführen sein könnten, dass die Expression des ET‐1 Transgens in ET+/+eNOS‐/‐ Tieren zu einer bereits früh beginnenden ET‐1 Überexpression führt, während sich dies in eNOS‐/‐ Mäusen erst im Verlauf entwickelt. Eine chronische ET‐1 Überexpression könnte so im kombinierten ET+/+eNOS‐/‐ Modell zu einer Konditionierung der Tiere führen, die im weiteren Verlauf die Entwicklung einer diastolischen Dysfunktion verhindert. Dies wird durch die Beobachtung unterstützt, dass eNOS-/‐ Tiere im Alter von drei Monaten normale Kardiomyozytendurchmesser aufweisen, im Alter von neun Monaten jedoch eine kardiale Hypertrophie entwickeln, während eine im Alter von drei Monaten in ET+/+eNOS‐/‐ Mäusen nachweisbare Vergrößerung der Kardiomyozytendurchmesser in neun Monate alten Tieren nicht mehr nachweisbar ist. Histologisch zeigte sich darüber hinaus im Vergleich zu Wildtyp‐Kontrollen und eNOS‐/‐ Tieren sowohl in ET+/+ als auch in ET+/+eNOS‐/‐ Mäusen eine Dilatation kardialer Arteriolen, so dass eine vermehrte myokardiale Durchblutung zu den beobachteten funktionellen Unterschiede beitragen könnte. Um die molekularen Mechanismen, die diesen Beobachtungen zugrunde liegen, zu analysieren, erfolgte ein Vergleich des kardialen Proteoms jeder Genotypen mit Wildtyp Kontrollen durch zweidimensionale Elektrophorese in Kombination mit Massenspektrometrie. Hier zeigte sich, dass die Überexpression von ET‐1 sowohl im Wildtyp als auch im eNOS ‐/‐ Hintergrund zu einer höheren Expression von Proteinen der oxidativen Stressantwort führt. Dies deutet darauf hin, dass ET+/+ and ET+/+eNOS‐/‐ Tiere im Gegensatz zu eNOS‐/‐ Mäusen molekulare Mechanismen entwickelt haben, um Schäden durch oxidativen Stress zu limitieren. Darüberhinaus war die beobachtete diastolische Dysfunktion in eNOS‐/‐ Mäusen mit Änderungen in der Expression von Proteinen, die bei der Regulation kontraktiler Prozesse eine Rolle spielen, assoziiert. ET‐1 Überexpression führte in eNOS‐/‐ Tieren zu einer Antagonisierung dieser Veränderungen. Schließlich zeigte sich in diesen Untersuchungen, dass im ET+/+eNOS‐/‐ Modell im Gegensatz zu eNOS‐/‐ Tieren in höherem Maße eine Verschiebung vom Fettsäure‐Metabolismus zur vermehrten Glukose‐Utilisation erfolgt, die als kardioprotektiv betrachtet wird. Zusammenfassend wird in der vorliegenden Arbeit damit gezeigt, dass eine chronische Überexpression von ET‐1 in Mäusen durch die Modulation verschiedener Systeme (oxidativer Stress, Kontraktionsprozesse, Energiemetabolismus) auch in Abwesenheit der eNOS protektive Effekte auf die kardiale Funktion haben kann. Untersuchungen zum Einfluss einer pharmakologischen Modulation dieser Systeme auf kardiale Funktionsparameter könnten dazu beitragen, die klinische Relevanz dieser Beobachtungen zu überprüfen

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

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

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

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

AbstractAimsHypertensive 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-β. 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 methodsWe 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 (0.15mol/L NaCl and 1mmol/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 findingsAngII-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-β, 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.SignificanceThis 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