Blood pressure and cardiac phenotype of eNOS-knockout mice with additional overexpression of human endothelin-1

Endothelin-1 (ET-1) ist ein autokrines und parakrines Peptid-Hormon, welches hauptsächlich in den Endothelzellen der Gefäße gebildet wird1. Es ist eines der stärksten Vasokonstriktoren, die bis heute bekannt sind, und wirkt darüber hinaus profibrotisch, mitogen und proinflammatorisch1. Es wird zu 80 Prozent basolateral in Richtung der Gefäßmedia ausgeschieden1, wo es an G-Protein- gekoppelte Rezeptoren, den Endothelin A-Rezeptor (ETA-Rezeptor) und den Endothelin B-Rezeptor (ETB-Rezeptor), bindet und im endoplasmatischen Retikulum über die Freisetzung von Kalzium (Ca2+) eine Kontraktion der glatten Gefäßmuskelzellen auslöst1, 25-29. Überraschenderweise zeigten Tiermodelle mit erhöhter Endothelin-Expression wie human-Endothelin-1-transgene Mäuse (ET-1) und Endothelin-2-transgene (ET-2) Ratten keine Erhöhung des Blutdrucks76, 150, 151. Die Expression und/oder Wirkung von Endothelin (ET) muss unter physiologischen Bedingungen gegenreguliert werden. Einer der wichtigsten bisher bekannten Gegenspieler des Endothelins ist Stickstoffmonoxid (NO). Die vasokonstriktorische Wirkung des Endothelins wird direkt in den Gefäßen durch NO reduziert1, 3, und NO reguliert außerdem die prepro-ET-1-mRNA-Expression herunter1, 159. Wir haben daher human-ET-1-transgene Mäuse (ET+/+) mit Mäusen gekreuzt, die einen Knockout der endothelialen Stickstoffmonoxid-Synthase aufweisen (eNOS-knockout-Mäuse, eNOS-/-). Auf diesem Weg haben wir homozygote human-ET-1-transgene Mäuse mit gleichzeitigem homozygoten eNOS-knockout rückgezüchtet (eNOS-/- ET+/+). Ziel dieser Arbeit war es zu untersuchen, ob eine ET-Überexpression bei gleichzeitiger eingeschränkter NO-Produktion zu einer Erhöhung des Blutdrucks führt. Darüber hinaus sollte untersucht werden, ob diese ET-NO-Imbalance kardiale und vaskuläre Schäden am Herzen verursacht bzw. diese verstärkt werden und ob dies zu einer Einschränkung der Herzfunktion führt. Wir haben daher bei eNOS-/- ET+/+ -Mäusen und den entsprechenden Kontrollgruppen (ET+/+ Mäusen, eNOS-/- -Mäusen und WT -Mäusen) den Blutdruck gemessen, die Herzfunktion mittels Herzkatheter untersucht und die Herzen histologisch analysiert. Der direkte Vergleich mit nur human- ET-1-überexprimierenden Tieren und nur eNOS-knockout-Tieren erlaubte eine Analyse der Veränderungen, die durch einen eNOS-Knockout oder einen ET-1-Überschuss allein entstehen, oder durch Kombination beider Faktoren. Mäuse mit eNOS-knockout allein (eNOS-/- -Mäuse) entwickelten einen erhöhten Blutdruck, der zu einer kardialen Hypertrophie führte. Die Hypertrophie wiederum verursachte eine kardiale diastolische Dysfunktion. Dieser Phänotyp war deutlicher bei den männlichen Tieren ausgeprägt. Eine ET-1-Überexpression (in ET+/+-Mäusen) führte wiederum zu einer veränderten Gefäßgeometrie mit Lumenvergößerung und perivaskulärer Fibrose. Die Kombination von eNOS-Knockout und ET-1-Überexpression (in eNOS-/- ET+/+-Mäusen) führte zu einer signifikant höheren kardialen interstitiellen Fibrose im Vergleich zu allen anderen Gruppen. Diese fibrotischen Veränderungen deuten auf eine chronische Inflammation hin. Die eNOS-/- ET+/+-Mäuse zeigten die selben Veränderungen der Gefäßgeometrie wie die Tiere mit alleiniger ET-1-Überexpression (Lumenvergößerung und perivaskuläre Fibrose). Wie bei den eNOS-/- auch, kam es bei den eNOS-/-ET+/+ zu einer Erhöhung des systolischen Blutdrucks. Dennoch war die Tendenz zur Entstehung einer kardialen Hypertrophie deutlich geringer ausgeprägt als bei den eNOS-/-, und die eNOS-/- ET+/+ wiesen kaum eine Einschränkung der Herzfunktion im Herzkatheter auf. Vermutlich wurden bei ihnen kompensatorische Stoffwechselwege aktiv, so dass dieses Tiermodell geeignet ist, um derartige Mechanismen zu untersuchen. Fibrotische Veränderungen und chronische Inflammation können aber auch Spätfolgen haben. Auch wenn sich bei den eNOS-/-ET+/+-Tieren im Alter von neun Monaten noch keine kardiale Funktionseinschränkung gezeigt hat, kann dies in einem höheren Alter noch eintreten. Daher können an diesem Tiermodell die Spätfolgen der kardialen Fibrose (verursacht durch ET-NO-Imbalance) untersucht und neue therapeutische Ansätze entwickelt werden.Endothelin-1 (ET-1) is an autocrine and paracrine acting peptide hormone, which is mainly produced by endothelial cells of the vasculature.1 It is one of the most potent vasoconstrictors known so far and acts furthermore as a antiapoptotic, mitogenic, profibrotic and inflammatory agent. ET-1 is released to 80% onto the smooth musculature, where it binds on two G-protein-coupled receptors: the Endothelin-A receptor (ETA-receptor) and the Endothelin-B receptor (ETB-receptor)1. ETA-receptor-activation induces an increase of cytoplasmatic Ca2+, which leads to contraction of the smooth muscle cells.25-29 Surprisingly, animal models with ET-overexpression did not show an increase of arterial blood-pressure, neither ET-1-transgenic mice nor ET-2 -transgenic-rats76, 150, 151. Therefore there must be an activation of antagonistic mechanisms which are able to lower the expression and/or the effects of ET-1. One possible antagonist to ET-activity might be nitric oxide, which is mainly produced in the vasculature by the endothelial nitric oxide synthase (eNOS). NO is a strong vasodilator and is known to downregulate the expression of prepro-ET-mRNA1, 3, 159. To investigate, if eNOS-derived NO is the dominant antagonist of ET-action in the vasculature, we crossbred human- ET-1-transgenic mice with eNOS-knockout-mice. After multiple rebreeding steps, we received four groups: eNOS-/- ET+/+-mice, only eNOS-/-, only ET+/+ and WT (wildtypes) (mice were all homozygous for ET-transgen and/or eNOS-knockout). We wanted to examine, if the imbalance between ET-1 and NO leads to elevated blood pressure, to cardiac and vascular damage and hence to cardiac dysfunction. Blood pressure was assessed via tail-cuff plethysmography, cardiac function by catheterisation of the left ventricle (monitoring of left ventricular pressure during systole and diastole), and the morphology of cardiac tissue and coronary vasculature by histological analysis. Mice with eNOS-knockout (eNOS-/-) only showed an increase in systolic blood pressure and left ventricular hypertrophy with cardiac left ventricular diastolic dysfunction. This phenotype was more promounced in male eNOS-/-. ET-1-overexpressing mice (ET+/+ and eNOS-/-ET+/+) developed a dilated vessel- morphology with a normal media-area but a larger lumen-area and perivascular fibrosis. The combination of and eNOS-knockout and ET-1-overexpression (eNOS-/- ET+/+-mice) led to interstitial cardiac fibrosis, which indicates that there might be a chronic inflammation in the cardiac tissue. eNOS-/- ET+/+-mice furthermore showed significantly higher values for systolic blood pressure compared to eNOS-/- in early generations. In later generations the systolic blood pressure on the same elevated level like eNOS-/-, but was not significantly higher anymore. Although the blood pressure was as elevated as in eNOS-/-, eNOS-/- ET+/+ animals did not develop such pronounced signs of cardiac hypertrophy and a restored cardiac diastolic function. Only the left ventricular end-diastolic pressure was increased, which indicates that there is a loss of ventricular elasticity due to interstitial matrix accumulation. We suggest, that these animals developed other compensatory mechanisms (over multiple generations), which protected them from cardiac damage and lowered the blood pressure on eNOS-/--level. Therefore these animals can be used to determine possible protecting pathways against NO-deficiency and ET- overexpression itself. Nevertheless, in this work we were able to show, that eNOS-derived NO counterregulates some effects of ET-activity, like interstitial matrix-protein accumulation and blood pressure elevation (only in early generations, this effect should be checked by other experiments, like pharmacological selective eNOS-inhibition in ET+/+). eNOS-/- ET+/+-mice might represent a novel animal-model of cardiac fibrosis which can be used to develop new strategies against cardiac diseases connected with excessive matrix-protein accumulation

Endothelin-1 overexpression improves renal function in eNOS knockout mice

BACKGROUND/AIMS: To investigate the renal phenotype under conditions of an activated renal ET-1 system in the status of nitric oxide deficiency, we compared kidney function and morphology in wild-type, ET-1 transgenic (ET+/+), endothelial nitric oxide synthase knockout (eNOS-/-) and ET+/+eNOS-/- mice. METHODS: We assessed blood pressure, parameters of renal morphology, plasma cystatin C, urinary protein excretion, expression of genes associated with glomerular filtration barrier and tissue remodeling, and plasma metabolites using metabolomics. RESULTS: eNOS-/- and ET+/+eNOS-/- mice developed hypertension. Osteopontin, albumin and protein excretion were increased in eNOS-/- and restored in ET+/+eNOS-/- animals. All genetically modified mice developed renal interstitial fibrosis and glomerulosclerosis. Genes involved in tissue remodeling (serpine1, TIMP1, Col1a1, CCL2) were up-regulated in eNOS-/-, but not in ET+/+eNOS-/- mice. Plasma levels of free carnitine and acylcarnitines, amino acids, diacyl phosphatidylcholines, lysophosphatidylcholines and hexoses were descreased in eNOS-/- and were in the normal range in ET+/+eNOS-/- mice. CONCLUSION: eNOS-/- mice developed renal dysfunction, which was partially rescued by ET-1 overexpression in eNOS-/- mice. The metabolomics results suggest that ET-1 overexpression on top of eNOS knockout is associated with a functional recovery of mitochondria (rescue effect in β-oxidation of fatty acids) and an increase in antioxidative properties (normalization of monounsaturated fatty acids levels)

Relaxin-2 Does Not Ameliorate Nephropathy in an Experimental Model of Type-1 Diabetes

Background/Aims: In diabetic nephropathy (DN), the current angiotensin-II-blocking pharmacotherapy is frequently failing. For diabetic cardiomyopathy (DC), there is no specific remedy available. Relaxin-2 (Rlx) - an anti-fibrotic, anti-inflammatory, and vasoprotecting peptide - is a candidate drug for both. Methods: Low-dose (32 µg/kg/day) and high-dose (320 µg/kg/day) Rlx were tested against vehicle (n = 20 each) and non-diabetic controls (n = 14) for 12 weeks in a model of type-1 diabetes induced in endothelial nitric oxide synthase knock-out (eNOS-KO) mice by intraperitoneal injection of streptozotocin. Results: Diabetic animals showed normal plasma creatinine, markedly increased albuminuria and urinary malonyldialdehyde, elevated relative kidney weight, glomerulosclerosis, and increased glomerular size, but no relevant interstitial fibrosis. Neither dose of Rlx affected these changes although the drug was active and targeted plasma levels were achieved. Of note, we found no activation of the renal TGF-β pathway in this model. In the hearts of diabetic animals, no fibrotic alterations indicative of DC could be determined which precluded testing of the initial hypothesis. Conclusions: We investigated a model showing early DN without overt tubulo-interstitial fibrosis and activation of the TGF-β-Smad-2/3 pathway. In this model, Rlx proved ineffective; however, the same may not apply to other models and types of diabetes

Significant reduction of MDA- positive renal tissue by combination therapy: Immunhistochemical detection of MDA in non-diabetic (A) and diabetic (B) untreated eNOS knockout mice.

<p>Percent volume of MDA-positive area in renal cortex (F). Values are given as means ± SEM. For comparisons, student's t test was used. ^** p<0.01; ^***p<0.001 vs. diabetic vehicle and ^#p<0.05; ^##p<0.01 vs. non-diabetic vehicle. Abbreviations used: <b>MDA</b>, malondialdehyde.</p

Experimental progress of blood pressure and blood glucose in diabetic eNOS knockout mice treated with riociguat (3 mg/kg/d), telmisartan (1 mg/kg/d), both (3 mg/kg/d and 1 mg/kg/d) or vehicle.

<p>Values are given as means ± SEM. For comparisons, student's t test was used.</p>*<p>p<0.05 vs. diabetic vehicle.</p><p>Additionally non-diabetic eNOS knockout mice treated with vehicle were examined.</p

Final urinary albumin as well as organ weights and histology of diabetic mice after 11 weeks of treatment with riociguat (3 mg/kg/d), telmisartan (1 mg/kg/d), riociguat and telmisartan (3 mg/kg/d and 1 mg/kg/d) or vehicle and of non-diabetic control mice, respectively.

<p>Values are given as means ± SEM. For comparisons, student's t test and Mann-Whitney u test, respectively, were used, what appropriate.</p>*<p>p<0.05 vs. diabetic vehicle.</p