Henning Morawietz

Dose-Dependent Regulation of NAD(P)H Oxidase Expression by Angiotensin II in Human Endothelial Cells Protective Effect of Angiotensin II Type 1 Receptor Blockade in Patients With Coronary Artery Disease

Objective—Angiotensin II (Ang II)–mediated induction of vascular superoxide anion formation could contribute to the development of endothelial dysfunction, hypertension, and atherosclerosis. An NAD(P)H oxidase has been identified as a major endothelial source of superoxide anions. However, the molecular mechanism underlying the regulation of NAD(P)H oxidase activity in response to Ang II is not well understood. Methods and Results—We investigated the dose-dependent regulation of superoxide anion formation and of NAD(P)H oxidase subunit expression in response to Ang II in human endothelial cells. Ang II regulates superoxide anion formation and the limiting subunit of endothelial NAD(P)H oxidase, gp91-phox, in a dose-dependent manner via Ang II type 1 (AT1) receptor–mediated induction and Ang II type 2 receptor–mediated partial inhibition at higher Ang II concentrations. Furthermore, AT1 receptor blocker therapy before coronary bypass surgery downregulates the gp91-phox expression in internal mammary artery biopsies of patients with coronary artery disease. Conclusions—Our data support a dose-dependent induction of proatherosclerotic oxidative stress in human endothelial cells in response to Ang II. The expression of NAD(P)H oxidase subunit gp91-phox is critical for endothelial superoxide anion formation. AT1 receptor blockade has an antiatherosclerotic and antioxidative potential by the reduction of oxidative stress in the vessel wall.

Regulation of the endothelin system by shear stress in human endothelial cells

1 In this study, the effect of shear stress on the expression of genes of the human endothelin‐1 system was examined. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress of 1, 15 or 30 dyn cm−2 (i.e. 0.1, 1.5 or 3 N m−2) (venous and two different arterial levels of shear stress) in a cone‐and‐plate viscometer. 2 Laminar shear stress transiently upregulates preproendothelin‐1 (ppET‐1) mRNA, reaching its maximum after 30 min (approx 1.7‐fold increase). In contrast, long‐term application of shear stress (24 h) causes downregulation of ppET‐1 mRNA in a dose‐dependent manner. 3 Arterial levels of shear stress result in downregulation of endothelin‐converting enzyme‐1 isoform ECE‐1a (predominating in HUVEC) to 36.2 ± 8.5%, and isoform ECE‐1b mRNA to 72.3 ± 1.9% of static control level. 4 The endothelin‐1 (ET‐1) release is downregulated by laminar shear stress in a dose‐dependent manner. 5 This downregulation of ppET‐1 mRNA and ET‐1 release is not affected by inhibition of protein kinase C (PKC), or tyrosine kinase. Inhibition of endothelial NO synthase (L‐NAME, 500 μm) prevents downregulation of ppET‐1 mRNA by shear stress. 6 In contrast, increasing degrees of long‐term shear stress upregulate endothelin receptor type B (ETB) mRNA by a NO‐ and PKC‐, but not tyrosine kinase‐dependent mechanism. 7 In conclusion, our data suggest the downregulation of human endothelin synthesis, and an upregulation of the ETB receptor by long‐term arterial laminar shear stress. These effects might contribute to the vasoprotective and anti‐arteriosclerotic potential of arterial laminar shear stress.

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.

Effects of obesity on endothelium-dependent reactivity during acute nitric oxide synthase inhibition: modulatory role of endothelin

This study investigated vascular reactivity in response to acetylcholine, in the presence of acute inhibition of nitric oxide synthase, in the carotid artery and aorta of obese C57Bl6/J mice fed on a high-fat diet for 30 weeks, and of control mice. A subgroup of obese animals was also treated with the ET(A) receptor antagonist darusentan (50 mg x kg(-1) x day(-1)). In vascular rings from control animals, acetylcholine caused endothelium-dependent contractions in the carotid artery, but not in the aorta. In vascular rings from obese mice, contractility to acetylcholine was also evident in the aorta, and that in the carotid artery was increased compared with control mice. ET(A) receptor blockade by darusentan treatment of the obese mice prevented enhanced vasoconstriction to acetylcholine, resulting in mild vasodilatation. Thus obesity increases endothelium-dependent vasoconstriction in the absence of endothelial nitric oxide. This effect can be completely prevented by chronic ET(A) receptor blockade, suggesting that endothelin modulates increased endothelium-dependent vasoconstriction in obesity.

Identification of an essential Drosophila gene that is homologous to the translation initiation factor eIF-4A of yeast and mouse

SummaryA gene encoding a protein homologous to the translation initiation factor eIF-4A in mouse has been identified in Drosophila melanogaster. The predicted amino acid sequence shows 73% identity with the mouse gene and 67% identity with a homologous protein from yeast. The single-copy Drosophila gene is located on chromosome arm 2L at 26A7-9. Several recessive lethal mutations have been isolated and genetically characterized. Northern blot hybridization shows two abundant transcripts of 1.75 kb and 1.9 kb throughout all developmental stages. Both transcripts are maternally provided to the oocyte.

Shear Stress–Dependent Regulation of the Human β-Tubulin Folding Cofactor D Gene

Abstract— The flowing blood generates shear stress at the endothelial cell surface. The endothelial cells modify their phenotype by alterations in gene expression in response to different levels of fluid shear stress. To identify genes involved in this process, human umbilical vein endothelial cells were exposed to laminar shear stress (venous or arterial levels) in a cone-and-plate apparatus for 24 hours. Using the method of RNA arbitrarily primed polymerase chain reaction, we cloned a polymerase chain reaction fragment representing an mRNA species downregulated by arterial compared with venous shear stress (shear stress downregulated gene-1, SSD-1). According to Northern blot analysis, corresponding SSD-1 cDNA clones revealed a similar, time-dependent downregulation after 24 hours of arterial shear stress compared with venous shear stress or static controls. Three SSD-1 mRNA species of 2.8, 4.1, and 4.6 kb were expressed in a tissue-specific manner. The encoded amino acid sequence of the human endothelial SSD-1 isoform (4.1-kb mRNA species) revealed 80.4% identity and 90.9% homology to the bovine &bgr;-tubulin folding cofactor D (tfcD) gene. Downregulation of tfcD mRNA expression by shear stress was defined at the level of transcription by nuclear run-on assays. The tfcD protein was downregulated by arterial shear stress. The shear stress–dependent downregulation of tfcD mRNA and protein was attenuated by the NO synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester. Furthermore, the NO donor DETA-NO downregulated tfcD mRNA. Because tfcD was shown to be a microtubule-destabilizing protein, our data suggest a shear stress–dependent regulation of the microtubular dynamics in human endothelial cells.

Augmented endothelial uptake of oxidized low-density lipoprotein in response to endothelin-1

Endothelin-1 (ET-1) may be involved in the development and progression of atherosclerosis. Furthermore, endothelin receptor blockade was shown to reduce the formation of atherosclerotic lesions in experimental studies. Another potent pro-atherosclerotic risk factor is oxidized low-density lipoprotein (oxLDL). Endothelial cells mediate the uptake of oxLDL by the recently identified lectin-like oxLDL receptor-1 (LOX-1), which accumulates in atherosclerotic lesions. In the present study, we analysed the effects of ET-1 on oxLDL uptake and LOX-1 expression in primary cultures of human umbilical vein endothelial cells (HUVEC). ET-1 stimulated uptake of oxLDL in HUVEC, which reached a maximum after 1 h. In further studies, we found a similar induction of LOX-1 mRNA and protein expression in response to ET-1. The augmented oxLDL uptake and the increased LOX-1 expression in response to ET-1 are mediated by the endothelin receptor B. Our data support a new pathophysiological mechanism by which locally and systemically increased ET-1 levels, e.g. in hypertensive patients, could promote LOX-1-mediated oxLDL uptake in human endothelial cells. This mechanism could promote the development and progression of endothelial dysfunction and atherosclerosis. In addition, endothelin receptor blockade could be considered as a new anti-atherosclerotic therapeutic principle.

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.

Mechanism of ETA-receptor stimulation-induced increases in intracellular Ca2+ in SK-N-MC cells

1 The mechanism underlying endothelin‐1 (ET‐1)‐induced increases in intracellular Ca2+ concentrations in the human neuroblastoma cell‐line SK‐N‐MC was investigated. 2 ET‐receptor agonists increased inositol phosphate (IP)‐formation (assessed as accumulation of total [3H]‐IPs in [3H]‐myo‐inositol prelabelled cells) and intracellular Ca2+ (assessed by the FURA‐2 method) with an order of potency: ET‐1>sarafotoxinu20036b (S6b)>ET‐3=S6c; the ETA‐receptor antagonist BQ‐123 inhibited both responses with apparent pKi‐values of 8.3 and 8.6, respectively, while the ETB‐receptor antagonist BQ‐788 did not. 3 Pretreatment of the cells with pertussis toxin (PTX, 500u2003ngu2003ml−1 overnight) reduced ET‐1‐induced Ca2+ increases by 46±5%, but rather enhanced ET‐1‐induced IP‐formation. 4 Chelation of extracellular Ca2+ by 5u2003mm EGTA did not affect ET‐1‐induced IP‐formation. However, in the presence of 5u2003mm EGTA or SKFu200396365, an inhibitor of receptor mediated Ca2+ influx (1.0–3.0×10−5u2003m) ET‐1‐induced Ca2+ increases were inhibited in normal, but not in PTX‐treated cells. 5 [125I]‐ET‐1 binding studies as well as mRNA expression studies (by RT–PCR) detected only ETA‐receptors whereas expression of ETB‐receptor mRNA was marginal. 6 ET‐1 (10−8u2003m) inhibited isoprenaline‐evoked cyclic AMP increases; this was antagonized by BQ‐123, not affected by BQ‐788 and abolished by PTX‐treatment. 7 We conclude that SK‐N‐MC cells contain a homogeneous population of ETA‐receptors that couple to IP‐formation and inhibition of cyclic AMP formation. Stimulation of these ETA‐receptors increases intracellular Ca2+ by at least two mechanisms: a PTX‐insensitive IP‐mediated Ca2+ mobilization from intracellular stores and a PTX‐sensitive influx of extracellular Ca2+.

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].