Winfried Goettsch

Obesity Is Associated With Tissue-Specific Activation of Renal Angiotensin-Converting Enzyme In Vivo: Evidence for a Regulatory Role of Endothelin

In the C57BL/6J mice model, we investigated whether obesity affects the function or expression of components of the tissue renin-angiotensin system and whether endothelin (ET)-1 contributes to these changes. ACE activity (nmol. L His-Leu. mg protein(-1)) was measured in lung, kidney, and liver in control (receiving standard chow) and obese animals treated for 30 weeks with a high-fat, low cholesterol diet alone or in combination with LU135252, an orally active ET(A) receptor antagonist. ACE mRNA expression was measured in the kidney, and the effects of LU135252 on purified human ACE were determined. Aortic and renal tissue ET-1 protein content was measured, and the vascular contractility to angiotensin II was assessed. Obesity was associated with a tissue-specific increase in ACE activity in the kidney (55+/-4 versus 33+/-3 nmol/L) but not in the lung (34+/-2 versus 32+/-2 nmol/L). Long-term LU135252 treatment completely prevented this activation (13.3+/-0.3 versus 55+/-4 nmol/L, P<0.05) independent of ACE mRNA expression, body weight, or renal ET-1 protein but did not affect pulmonary or hepatic ACE activity. Obesity potentiated contractions in response to angiotensin II in the aorta (from 6+/-2% to 33+/-5% KCl) but not in the carotid artery (4+/-1% to 3.6+/-1% KCl), an effect that was completely prevented with LU135252 treatment (6+/-0.4% versus 33+/-5% KCl). No effect of LU135252 on purified ACE was observed. Thus, obesity is associated with the activation of renal ACE in vivo independent of its mRNA expression and enhanced vascular contractility to angiotensin II. These effects are regulated by ET in an organ-specific manner, providing novel mechanisms by which ET antagonists may exert organ protection.

Obesity increases prostanoid-mediated vasoconstriction and vascular thromboxane receptor gene expression.

Objectives Vasoconstrictor prostanoids have been implicated in abnormal vasomotion in atherosclerosis and hypertension. Method Using lean and diet-induced obese mice, we investigated whether obesity affects vascular function or expression of genes involved in prostanoid action. Results In lean C57BL/6J mice, at high concentrations acetylcholine caused endothelium-dependent contractions in the carotid artery but not in the aorta. Endothelium-dependent contractions to acetylcholine were blocked by the non-selective cyclooxygenase (COX) inhibitors indomethacin and meclofenamate, or a prostaglandin H2/thromboxane A2 receptor antagonist, but not by inhibitors of COX-2, thromboxane synthase or cytochrome P450 monooxygenase. Obesity increased endothelium-dependent contractions to acetylcholine in the carotid artery, and prostanoid-mediated vasoconstriction was now present in the aorta. Similarly, contractions to endothelin-1 were largely blocked by meclofenamate and were increased in the aorta of obese mice. Real-time quantitative polymerase chain reaction analysis of the thromboxane receptor gene in the carotid artery revealed a robust upregulation in obese animals (18-fold, P < 0.05); in comparison, obesity had a less pronounced effect on thromboxane synthase (2.1-fold increase, P < 0.05), or preproendothelin-1 gene expression (4.2-fold increase, P < 0.05). Conclusions These data demonstrate that obesity augments prostanoid-dependent vasoconstriction and markedly increases vascular thromboxane receptor gene expression. These changes are likely to promote the development of vascular disease, hypertension and thrombosis associated with obesity.

Flow-dependent regulation of angiopoietin-2.

Endothelial cells are constantly exposed to high or low shear stress in arteries and veins by the flowing blood. Angiopoietin‐2 (Ang‐2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. In this study, flow‐dependent regulation of Ang‐2 was analyzed in vitro and in vivo. Ang‐2 mRNA, protein expression and release was upregulated by 24 h of low (1 dyne/cm2), but downregulated by high flow (30 dyne/cm2) in human endothelial cells. Increased endothelial NO synthase expression and NO formation was not affecting regulation of Ang‐2 by low or high flow. Low and high flow increased VEGF‐A expression. Inhibition of VEGFR‐2 prevented upregulation of Ang‐2 by low flow, but not downregulation of Ang‐2 by high flow. Furthermore, upregulation of Ang‐2 by VEGF was reduced by application of high flow. Forkhead box O (FOXO) transcription factor FOXO1 has been shown to regulate Ang‐2 expression in endothelial cells. FOXO1 binding activity was reduced by high flow. Nuclear localization of transcription factor FOXO1 was not changed by low flow, but reduced by high flow. In vivo, Ang‐2 was higher expressed in veins compared to arteries. Arterial ligation augmented Ang‐2 expression in distal arterial low flow areas. Our results support a VEGF‐dependent induction of Ang‐2 in low flow areas, and FOXO1‐dependent downregulation of Ang‐2 in high flow areas. These data suggest a new mechanism of flow‐dependent regulation of vessel stability and differentiation. J. Cell. Physiol. 214: 491–503, 2008.

Erythropoietin-induced excessive erythrocytosis activates the tissue endothelin system in mice.

The endothelium controls blood flow and pressure by releasing several vasoactive factors, among them the vasodilator nitric oxide (NO) and the potent vasoconstrictor endothelin‐1 (ET‐1). Although increased NO levels have been found in excessive erythrocytosis, little is known concerning ET‐1 expression in this condition. Thus, we examined the endothelin system in transgenic mice that due to constitutive overexpression of erythropoietin (Epo) reached hematocrit levels of ~80%. Surprisingly, despite generalized vasodilatation, polycythemic mice exhibited a two‐ to fivefold elevation in ET‐1 mRNA levels in aorta, liver, heart, and kidney. In line with this, increased expression of ET‐1 protein was detected in the pulmonary artery by immunohistochemical analysis. Compared with their wild‐type littermates, aortic rings of Epo transgenic animals exhibited a marked reduction in vascular reactivity to ET‐1 and big ET‐1, but this effect was abrogated upon preincubation with the NO synthase inhibitor N‐nitro‐l‐arginine methyl ester (L‐NAME). Pretreatment of polycythemic mice with the ETA receptor antagonist darusentan for 3 wk significantly prolonged their survival upon acute exposure to L‐NAME. Taken together, these results demonstrate for the first time that excessive erythrocytosis induces a marked activation of the tissue endothelin system that results in increased mortality upon blockade of NO‐mediated vasodilatation. Because ETA antagonism prolonged survival after acute blockade of NO synthesis, endothelin may be regarded as a contributor to the adverse cardiovascular effects of erythrocytosis and may thus represent a new target in the treatment of cardiovascular disease associated with erythrocytosis.

Endothelial Protection, AT1 Blockade and Cholesterol-Dependent Oxidative Stress The EPAS Trial

Background— Statins and angiotensin type 1 (AT1) receptor blockers reduce cardiovascular mortality and morbidity. In the Endothelial Protection, AT1 blockade and Cholesterol-Dependent Oxidative Stress (EPAS) trial, impact of independent or combined statin and AT1 receptor blocker therapy on endothelial expression of anti-atherosclerotic and proatherosclerotic genes and endothelial function in arteries of patients with coronary artery disease were tested. Methods and Results— Sixty patients with stable coronary artery disease undergoing elective coronary artery bypass grafting (CABG) surgery were randomized 4 weeks before surgery to: (A) control without inhibition of renin-angiotensin system or statin; (B) statin (pravastatin 40 mg/d); (C) AT1 blockade (irbesartan 150 mg/d); or (D) combination of statin and AT1 blocker in same dosages. Primary end point was a priori therapy-dependent regulation of an anti-atherosclerotic endothelial expression quotient Q including mRNA expression (in arbitrary units measured by real-time polymerase chain reaction) of endothelial nitric oxide synthase and C-type natriuretic peptide, divided by expression of oxidized low-density lipoprotein receptor LOX-1 and NAD(P)H oxidase subunit gp91phox in left internal mammary arteries biopsies obtained by CABG surgery; 49 patients completed the study. Statin therapy increased lnQ from 3.2±0.4 to 4.4±0.4 significantly versus control. AT1 blockade showed a trend to increase lnQ to 4.2±0.5. Combination of statin and AT1 blocker further increased lnQ to 5.1±0.6, but a putative interaction of both therapies in lnQ was not significant. Furthermore, preoperative therapy with statin, AT1 blocker and their combination improved endothelial function in internal mammary artery rings. Conclusions— Statin and AT1 blocker therapy independently and in combination improve an anti-atherosclerotic endothelial expression quotient and endothelial function.

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22(phox) expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-beta no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22(phox). The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.

Endothelial EphrinB2 Is Controlled by Microenvironmental Determinants and Associates Context-Dependently With CD31

Objective—The EphB ligand ephrinB2 has been identified as a critical determinant of arterial endothelial differentiation and as a positive regulator of invading endothelial cells during angiogenesis. This study was aimed at identifying determinants of endothelial cell ephrinB2 expression. Methods and Results—Arteriovenous asymmetrical endothelial cell ephrinB2 expression in vivo is lost on transfer into culture with aortic endothelial cells becoming partially ephrinB2-negative and saphenous vein endothelial cells becoming partially ephrinB2-positive. Contact with smooth muscle cells and angiogenic stimulation by vascular endothelial growth factor lead to an increased endothelial cell ephrinB2 expression. Quiescent, smooth muscle-contacting endothelial cells express ephrinB2 uniformly on their luminal surface. In contrast, monolayer endothelial cells translocate ephrinB2 to interendothelial cell junctions, which is strongly enhanced by EphB4-Fc-mediated receptor body activation. Junctional ephrinB2 colocalizes and coimmunoprecipitates with CD31. Conclusions—This study identifies distinct regulatory mechanisms of endothelial ephrinB2 expression and cellular distribution in quiescent and activated endothelial cells. The data demonstrate that endothelial cell ephrinB2 expression is controlled by microenvironmental determinants rather than being an intrinsic endothelial cell differentiation marker.

Down-Regulation of Endothelial EphrinB2 Expression by Laminar Shear Stress

The EphB receptors and their ephrinB ligands are involved in vascular assembly and differentiation. In this study, the authors analyzed the regulation of ephrinB2 and EphB4 in response to laminar shear stress in human endothelial cells. In order to simulate different flow conditions in vitro, human endothelial cells were exposed to laminar shear stress (1 to 50 dyn/cm2 for up to 24 h) in a cone-and-plate viscometer. EphrinB2 mRNA expression is down-regulated by arterial, but not by venous, laminar shear stress in a dose-dependent manner in primary cultures of human umbilical vein endothelial cells (HUVECs) (maximum at 30 dyn/cm2, 24 h: 46% +/- 4%of internal control without shear stress, n = 16, p < .05). The down-regulation of ephrinB2 by arterial shear stress is blocked by the protein kinase C inhibitor RO-31-8220. A similar shear stress-dependent down-regulation of ephrin-B2 can be found in human coronary artery endothelial cells (HCAECs). Chronic application of laminar shear stress does not affect EphB4 expression in venous and arterial endothelial cells. The down-regulation of ephrinB2 in response to laminar shear stress may contribute to the differentiation of endothelial cells into a nonactivated phenotype.

Angiotensin-converting enzyme inhibitor therapy prevents upregulation of endothelin-converting enzyme-1 in failing human myocardium

In this study, we investigated the role of the renin-angiotensin system in expression of the endothelin system in atrial myocardium of patients with congestive heart failure. Atrial myocardium of control patients without angiotensin-converting enzyme (ACE) inhibitor therapy and heart failure patients without or with ACE inhibitor therapy undergoing aorto-coronary bypass surgery was studied. Endothelin-converting enzyme-1 (ECE-1) expression and endothelin-1 peptide level was upregulated in myocardium of heart failure patients without ACE inhibition. ACE inhibitor therapy prevented upregulation of ECE-1 and endothelin-1 in failing myocardium. Prepro-endothelin-1 and endothelin receptor A expression were not affected by heart failure. Endothelin receptor B was downregulated in heart failure patients. Our data demonstrate an upregulation of ECE-1 mRNA expression in failing human myocardium. Inhibition of the renin-angiotensin system by ACE inhibitor treatment prevents upregulation of ECE-1, suggesting that angiotensin II regulates ECE-1 expression in vivo.

Shear stress mediates tyrosylprotein sulfotransferase isoform shift in human endothelial cells

In this study, we examined expression of tyrosylprotein sulfotransferase (TPST) isoforms TPST1 and TPST2 in primary cultures of human umbilical vein endothelial cells. For the first time coexpression of both isoforms is shown in primary human cells. Application of physiological levels of shear stress regulates expression of TPST isoforms in a time- and dose-dependent manner. Sustained application of arterial laminar shear stress causes downregulation of TPST1 mRNA and protein expression, while TPST2 is upregulated. This TPST isoform shift is mediated by different signaling pathways. Shear stress-dependent downregulation of TPST1 involves tyrosine kinase, while upregulation of TPST2 is mediated by a protein kinase C-dependent pathway [corrected].