Volker Breu

The endothelin ETB receptor mediates both vasodilation and vasoconstriction in vivo

It has been suggested that the endothelin (ET) ETB receptor could mediate endothelium-dependent vasodilation to ET-1 or ET-3, but its in vivo role is still largely unknown. We used sarafotoxin S6C, a selective agonist of the ETB receptor, to study the in vivo effects of ETB stimulation. SRTX S6C induced a transient decrease in blood pressure, followed by a long-lasting pressor response accompanied by a marked renal and mesenteric vasoconstriction. No constriction was observed in isolated mesenteric arteries in vitro, indicating that the in vivo vasoconstrictor effect is most likely indirect. The pressor effect of SRTX S6C was not dependent on central stimulation of ETB receptors and was not mediated by catecholamines from the adrenal medulla, prostanoids or ET-1.

NF-κB Inhibition Ameliorates Angiotensin II–Induced Inflammatory Damage in Rats

We recently reported that the activation of nuclear factor-kappaB (NF-kappaB) promotes inflammation in rats harboring both human renin and angiotensinogen genes (double-transgenic rats [dTGR]). We tested the hypothesis that the antioxidant pyrrolidine dithiocarbamate (PDTC) inhibits NF-kappaB and ameliorates renal and cardiac end-organ damage. dTGR feature hypertension, severe renal and cardiac damage, and a 40% mortality rate at 7 weeks. Electrophoretic mobility shift assay showed increased NF-kappaB DNA binding activity in hearts and kidneys of dTGR. Chronic PDTC (200 mg/kg SC) treatment decreased blood pressure (162+/-8 versus 190+/-7 mm Hg; P=0.02) in dTGR compared with dTGR controls. The cardiac hypertrophy index was also significantly reduced (4.90+/-0.1 versus 5.77+/-0.1 mg/g; P 95% (2.5+/-0.8 versus 57. 1+/-8.7 mg/d; P<0.001) and prevented death. Vascular injury was ameliorated in small renal and cardiac vessels. Electrophoretic mobility shift assay showed that PDTC inhibited NF-kappaB binding activity in heart and kidney, whereas AP-1 activity in the kidney was not decreased. dTGR exhibited increased left ventricular c-fos and c-jun mRNA expression. PDTC treatment reduced c-fos but not c-jun mRNA. Immunohistochemistry showed increased p65 NF-kappaB subunit expression in the endothelium and smooth muscle cells of damaged small vessels, as well as infiltrating cells in glomeruli, tubules, and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65. PDTC also prevented the NF-kappaB-dependent transactivation of the intercellular adhesion molecule ICAM-1 and inducible nitric oxide synthase. Monocyte infiltration was markedly increased in dTGR kidneys and hearts. Chronic treatment reduced monocyte/macrophage infiltration by 72% and 64%, respectively. Thus, these results demonstrate that PDTC inhibits NF-kappaB activity, ameliorates inflammation, and protects against angiotensin II-induced end-organ damage.

Monocyte Infiltration and Adhesion Molecules in a Rat Model of High Human Renin Hypertension

Hypertension and kidney damage in the double transgenic rat (dTGR) harboring both human renin and human angiotensinogen genes are dependent on the human components of the renin angiotensin system. We tested the hypothesis that monocyte infiltration and increased adhesion molecule expression are involved in the pathogenesis of kidney damage in dTGR. We also evaluated the effects of long-term angiotensin-converting enzyme (ACE) inhibition, AT1 blockade, and human renin inhibition on monocyte recruitment and inflammatory response in dTGR. Systolic blood pressure and 24-hour albuminuria were markedly increased in 7-week-old dTGR as compared with age-matched normotensive Sprague Dawley rats. We found a significant monocyte/macrophage infiltration in the renal perivascular space and increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the interstitium, intima, and adventitia of the small renal vessels. alphaLbeta2 integrin and alpha4beta1 integrin, the corresponding ligands for ICAM-1 and VCAM-1, were also found on infiltrating monocytes/macrophages. The expression of plasminogen activator inhibitor-1 and fibronectin in the kidneys of dTGR were increased and distributed similarly to ICAM-1. In 4-week-old dTGR, long-term treatment with ACE inhibition (cilazapril), AT1 receptor blockade (valsartan), and human renin inhibition (RO 65-7219) (each drug 10 mg/kg by gavage once a day for 3 weeks) completely prevented the development of albuminuria. However, only cilazapril and valsartan were able to decrease blood pressure to normotensive levels. Interestingly, the drugs were all equally effective in preventing monocyte/macrophage infiltration and the overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin in the kidney. Our findings indicate that angiotensin II causes monocyte recruitment and vascular inflammatory response in the kidney by blood pressure-dependent and blood pressure-independent mechanisms. ACE inhibition, AT1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR.

Hypertension-Induced End-Organ Damage: A New Transgenic Approach to an Old Problem

Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-beta and deposition of extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure, pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or indirectly via endothelin 1 and that NFkappaB is upregulated in this model. We speculate that the transcription factors NFkappaB and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique model and our pharmacological probes will enable us to test these hypotheses.

Mineralocorticoid Receptor Affects AP-1 and Nuclear Factor-κB Activation in Angiotensin II–Induced Cardiac Injury

Aldosterone is implicated in cardiac hypertrophy and fibrosis. We tested the role of the mineralocorticoid receptor in a model of angiotensin II–induced cardiac injury. We administered spironolactone (SPIRO; 20 mg · kg−1 · d−1), valsartan (VAL; 10 mg · kg−1 · d−1), or vehicle to rats double transgenic for the human renin and angiotensinogen genes (dTGR). We investigated basic fibroblast growth factor (bFGF), platelet-derived growth factor, transforming growth factor-&bgr;1, and the transcription factors AP-1 and nuclear factor (NF)-&kgr;B. We used immunohistochemistry, electrophoretic mobility shift assays, and TaqMan RT-PCR. Untreated dTGR developed hypertension, cardiac hypertrophy, vasculopathy, and fibrosis with a 50% mortality rates at 7 weeks. SPIRO and VAL prevented death and reversed cardiac hypertrophy, while only VAL normalized blood pressure. Both drugs prevented vasculopathy. bFGF was markedly upregulated in dTGR, whereas platelet-derived growth factor-B and transforming growth factor-&bgr;1 were little changed. VAL and SPIRO suppressed this upregulation. Both AP-1 and NF-&kgr;B were activated in dTGR compared with controls. VAL and SPIRO reduced both transcription factors and reduced bFGF, collagen I, fibronectin, and laminin in the interstitium. These findings show that aldosterone promotes hypertrophy, cardiac remodeling, and fibrosis, independent of blood pressure. The effects involve AP-1, NF-&kgr;B, and bFGF. Mineralocorticoid receptor blockade downregulates these effectors and reduces angiotensin II–induced cardiac damage.

Up-regulation of endothelin-B receptors in atherosclerotic human coronary arteries.

Both endothelin-A (ETA) and endothelin-B (ETB) receptors are known to be present in human coronary arteries. However, their absolute and relative amounts, functional roles, and the influence of pathology are uncertain. The goal of the present study was to characterize endothelin receptors mediating constriction in human coronary arteries and to assess the influence of cardiomyopathy (CMP) and coronary artery disease (CAD) on ET receptors in human tissue. For comparison, porcine coronary arteries were evaluated in parallel. Competition binding experiments using [125I]ET-1 and different selective and nonselective ETA- and ETB-receptor agonists or antagonists revealed similar relative densities (relative Bmax) of ETA and ETB receptors in coronary arteries from human cardiomyopathic hearts (83% ETA and 17% ETB; n = 5) and porcine hearts (78% ETA and 22% ETB; n = 5). In marked contrast, the relative Bmax of ETB receptors were significantly higher in coronary arteries from human atherosclerotic hearts (51% ETA and 49% ETB; n = 3). Total receptor density (Bmax; fmol/mg protein) was highest in porcine (385 +/- 29) arteries, followed by human CAD (253 +/- 41) and CMP (174 +/- 20) coronary arteries. The relative and absolute Bmax values for ETA and ETB receptors in coronary arteries from a donor heart were similar to those obtained in CMP hearts. There were no significant differences in affinity constants (KD) values for ET-1, ET-3, Sarafotoxin S6c (SRTX S6c), BQ-123, and bosentan (Ro 47-0203) between tissues. In human coronary arteries from CMP hearts, ET-induced constriction seemed to be solely mediated via ETA receptors. In contrast, in porcine coronary arteries 20% of the maximal effect mediated by ET-1 could be attributed to ETB receptors, in agreement with the binding data. The functional role of ETB receptors in CAD tissue could not be evaluated because of the occurrence of spontaneous phasic contractions. We conclude that ETB receptors are up-regulated in human atherosclerotic coronary arteries. Further studies are needed to determine the pathophysiological importance of these receptors.

Effect of Bosentan on NF-κB, Inflammation, and Tissue Factor in Angiotensin II–Induced End-Organ Damage

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.

Endothelin antagonism with bosentan: a review of potential applications

Endothelin receptor antagonists have been proposed for the treatment of a variety of disorders in which the endothelins may act as pathogenic mediators, such as congestive heart failure, systemic and pulmonary hypertension, and cerebral vasospasm. Bosentan (Ro 47-0203) is a nonpeptide competitive antagonist, which can be a good tool for studying the endothelin system because it may be administered either acutely or chronically. It is specific for the endothelin system and blocks the actions of endothelin at both mammalian receptors (A and B). In experimental models of heart failure bosentan acts as a vasodilator and neurohormonal blocker that improves overall left ventricular performance and reduces renal dysfunction. Furthermore, in chronic studies, bosentan attenuates cardiac remodeling and significantly improves survival. In patients with chronic heart failure bosentan produces pulmonary and systemic vasodilation and may enhance conventional treatment with angiotensin-converting enzyme inhibitors. Long-term studies are being conducted to characterize the full therapeutic potential of bosentan in chronic heart failure. In experimental models bosentan reverses established pulmonary hypertension. Preclinical efficacy has also been demonstrated in essential hypertension, where bosentan can reduce blood pressure and end-organ damage. Clinical trials in hypertensive patients indicate that bosentan reduces blood pressure without heart rate increase or neurohumoral stimulation. Finally, bosentan is being considered for the treatment of cerebral vasospasm following subarachnoid hemorrhage. Bosentan reverses experimentally induced vasospasm of the basilar artery, and preliminary trials indicate that it can increase cerebral blood flow after aneurysmal subarachnoid hemorrhage.

Potent vasoconstriction mediated by endothelin ETB receptors in canine coronary arteries.

Endothelin (ET) 1 is a powerful vasoconstrictor of coronary arteries and may play a role in coronary spasm, atherosclerosis, and myocardial infarction. Previous studies have demonstrated that intracoronary ET caused marked vasoconstriction of the coronary circulation; however, it remains unclear which ET receptor types are present and which of these receptors mediate this vasoconstriction. To characterize the ET receptors present in dog coronary arteries, competition binding assays with radiolabeled ET-1 using ET-1, ET-3, ETA receptor antagonist BQ-123, and sarafotoxin S6c were performed. Three binding sites were apparent in the left circumflex coronary artery: an ETA receptor, a high-affinity ETB receptor, and a lower-affinity ETB receptor. To investigate the in vivo effects of ETB receptor stimulation, intracoronary sarafotoxin S6c, a highly selective ETB agonist, was administered in anesthetized open-chest dogs in a constant-pressure coronary artery perfusion model. Sarafotoxin S6c doses of 0.1 and 0.3 microgram caused a transient pronounced decrease in coronary resistance. Doses of 1.0 and 3.0 micrograms caused marked decreases in coronary diameter and blood flow, as well as myocardial segmental shortening. These effects of sarafotoxin S6c were not inhibited by constant infusion of BQ-123. The present study demonstrates the presence of ETB receptors in the canine coronary circulation that can mediate both vasodilation and vasoconstriction. These findings have important implications for an understanding of the pathophysiological function of ET in the coronary vasculature and for the development of therapeutically effective ET antagonists.

Blood Pressure–Independent Effects in Rats With Human Renin and Angiotensinogen Genes

The blood pressure-independent effects of angiotensin II (Ang II) were examined in double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes, in which the end-organ damage is due to the human components of the renin angiotensin system. Triple-drug therapy (hydralazine 80 mg/L, reserpine 5 mg/L, and hydrochlorothiazide 25 mg/L in drinking water) was started immediately after weaning. Triple-drug therapy normalized blood pressure and coronary resistance, only partially prevented cardiac hypertrophy, and had no effect on ratio of renal weight to body weight. Although triple-drug therapy delayed the onset of renal damage, severe albuminuria nevertheless occurred. Semiquantitative scoring of ED-1-positive and MIB-5-positive (nuclear cell proliferation-associated antigen Ki-67) cells showed profound perivascular monocyte/macrophage infiltration and cell proliferation in kidneys and hearts of untreated dTGR. Triple-drug therapy had only a minimal effect on local inflammatory response or vascular cell proliferation. In contrast, a novel orally active human renin inhibitor (HRI), 30 mg/kg by gavage for 4 weeks, normalized blood pressure and coronary resistance and also prevented cardiac hypertrophy and albuminuria. ED-1-positive cells and MIB-5-positive cells were decreased by HRI in hearts and kidneys almost to levels observed in normotensive Sprague-Dawley rats. The renoprotective effects of HRI were at least in part due to improved renal hemodynamics and distal tubular function, since HRI shifted renal pressure-diuresis/natriuresis curves leftward by approximately 35 mm Hg, increased glomerular filtration rate and renal blood flow, and shifted the fractional water and sodium excretion curves leftward. In untreated dTGR, plasma Ang II was increased by 400% and renal Ang II level was increased by 300% compared with Sprague-Dawley rats. HRI decreased plasma human renin activity by 95% and normalized Ang II levels in both plasma and kidney compared with triple-drug therapy. Our findings indicate that in dTGR harboring human renin and angiotensinogen genes, Ang II causes end-organ damage and promotes inflammatory response and cellular growth largely independent of blood pressure.