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Abstract —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)-κB and AP-1 expression in the kidney and heart; the p65 NF-κB 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-κB 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-κB and AP-1 activation and the NF-κB– 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-κB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET A/B receptors.

Abstract —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 ≈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.

Abstract —We recently reported that the activation of nuclear factor-κB (NF-κB) 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-κB 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-κB 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 fos and c- jun mRNA expression. PDTC treatment reduced c- fos but not c- jun mRNA. Immunohistochemistry showed increased p65 NF-κB 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-κB–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-κB activity, ameliorates inflammation, and protects against angiotensin II-induced end-organ damage.

Activation of the endothelin (ET) ET B receptor can mediate opposite effects, endothelium-dependent vasodilation but also direct vasoconstriction. So far one gene encoding an ET B receptor has been identified and associated with endothelium-dependent relaxation. It has been suspected that the presence of another ET B gene could explain ET B - mediated contraction. The goal of the present study was to evaluate in Piebald-lethal (s 1 ) mice, a naturally occurring mutant with deletion of the known ET B receptor gene, whether ET B receptor-mediated constriction is lost. Piebald-lethal (s 1 ) mice, in contrast to control mice, completely lacked ET B specific ligand binding. The pressor effect of the ET B receptor selective agonist sarafotoxin S6c was completely absent. In vitro, contraction of stomach strips induced by sarafotoxin S6c was also abolished in Piebald-lethal (s 1 ) mice. These results demonstrate the responsibility of the known ET B receptor gene in ET B -mediated constriction.

We have identified the human counterpart of the corticotropin-releasing factor receptor subtype 2beta. Its functional response to human urocortin was demonstrated after stable expression in HEK-293 cells. The receptor was also shown to bind sauvagine, corticotropin-releasing factor and urocortin. In contrast to rodents, the human CRF(2beta) receptor is only weakly expressed in heart and skeletal tissues, where the CRF(2alpha) isoform is predominant. Moreover, we have identified additional mRNAs of the CRF(2beta) type which are probably a consequence of aberrant splicing events.

In addition to their potent vasoconstrictor properties, the endothelins (endothelin-1 and -3) may possess neurotransmitter/neuromediator and neuroendocrine actions. The aim of the present study was to evaluate the role of endothelins (ET) in mediating neurogenic inflammation of cephalic tissues in the rat. For this purpose, bosentan, a specific non-peptide mixed antagonist of ET receptors, was tested in rat models of neurogenic and non-neurogenic plasma extravasation in the dura mater and extracranial tissues (eyelid, conjunctiva, lip, tongue). Bosentan was effective for preventing neurogenic inflammation in the dura mater induced by unilateral electrical stimulation of the trigeminal ganglion or intravenous injection of capsaicin, whereas it was ineffective in extracranial tissues or after injection of substance P (non-neurogenic inflammation). The effect of nerve fiber stimulation on ET plasma concentrations in superior sagittal sinus was measured using selective radioimmunoassays for ET-1 and -3. Endothelin-3 concentration significantly increased after intravenous injection of capsaicin, whereas ET-1 levels remained unchanged. Competition binding assays on microsomal membranes from the trigeminal ganglion revealed a single class of binding sites with equal affinity for ET-1 and ET-3, suggesting a homogenous population of ETB receptors. The role of ETB receptors in mediating inflammation was evidenced by the lack of efficacy of a selective ETA receptor antagonist, in contrast to the full efficacy of a selective ETB receptor antagonist, for preventing neurogenic inflammation induced by unilateral stimulation of the trigeminal ganglion. The role of ETB receptors was finally confirmed by the observation that exogenous administration of the ETB receptor agonist sarafotoxin S6c also induced plasma protein extravasation in the dura mater. This extravasation was not a direct effect of ETB receptor stimulation, because it was inhibited by spantide, a selective tachykinin receptor antagonist. These data strongly suggest that ET, acting through ETB receptors, may play an important role in mediating neurogenic inflammation in the meninges of rats. Since the profile of activity of bosentan is similar to that of the 5-HT1D/B agonists, sumatriptan and ergot alkaloids, one may speculate that ET receptor antagonists might be potentially effective in the treatment of acute migraine attacks.

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.

Isolated human coronary arteries are known to develop spontaneous phasic contractions (SPCs). The goal of the present study was to assess the role of endothelin (ET) in these contractions. Endothelium-denuded rings from left anterior descending (LAD) coronary arteries of patients with coronary artery disease (CAD; n = 3) undergoing cardiac transplantation developed SPCs within 1-2 h. The contractions were abolished by the ETA receptor antagonist BQ-123 or the mixed ET receptor antagonist bosentan (1 and 3 microM), whereas the contractions persisted for several hours in control rings. Exogenous ET-1 (10 pM) increased the amplitude of SPCs and initiated SPCs in rings that did not exhibit SPCs. Higher concentrations of ET-1 (> or = 1 nM) were able to re-induce SPC in rings in which SPCs had been abolished by BQ-123 or bosentan. These results suggest that ET plays an important role in spontaneous phasic contractions of isolated human coronary artery rings.

ACE inhibition has been shown to prevent neointima formation after vascular injury. However, it is not known if this effect is due to a specific inhibition of the renin angiotensin system or to another mechanism such as the accumulation of bradykinin. In order to answer this question we compared the effects of maximal effective doses of cilazapril, an angiotensin converting enzyme (ACE) inhibitor, and ciprokiren, a new renin inhibitor, in guinea pigs. Vascular injury was induced by endothelial denudation of the right carotid artery of guinea pigs treated either by saline (control group), cilazapril (30 mg/kg/day) or ciprokiren (24 mg/kg/day). Twelve days after the ballooning, the guinea pigs were sacrificed, the carotid arteries were perfused fixed and neointima formation was evaluated by quantitative morphometry. Both, ciprokiren and cilazapril prevented neointima formation to the same extent (inhibition by 42 and 49%, respectively, p These results suggest that, in guinea pigs, renin inhibition prevents neointima formation to a similar extent as ACE inhibition. Therefore, ACE inhibitors seem to act in this model by inhibiting the renin angiotensin system and not by other effects such as accumulation of bradykinin.

SPECIFIC AIMSAspirin inhibits IKKβ in vitro; however, the in vivo relevance of the phenomenon is unclear. We have developed a model of severe angiotensin (Ang) II-induced vascular injury and now tested the hypothesis that chronic aspirin treatment of transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) protects from Ang II-induced end organ damage by inhibiting NF-κB activation in vivo.PRINCIPAL FINDINGSHigh-dose aspirin reduces mortality and renal damage independent of blood pressureRenal glomerulus of untreated 7-wk-old dTGR shows mesangial expansion and podocyte damage. Renal dTGR arterioles exhibit severe changes in wall structure, including proliferation of smooth muscle cells and deposition of dense vacuoles. In contrast, nontransgenic Sprague-Dawley (SD) rats showed no vascular or glomerular damage. Untreated dTGR show signs of glomerular sclerosis and vascular damage with mortality > 50% at wk 7. High-dose aspirin reduced mortality to < 10% whereas low-dose aspirin produ...

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.

The goal of our study was to evaluate and compare the effects of receptor blockade with different endothelin (ET) receptor antagonists on plasma concentrations of ET-1, big ET-1 and ET-3 in conscious rats. Ro 46-2005 (10 mgkg, i.v.), a novel non-peptide antagonist of both ETA and ETB receptors, increased the concentrations of ET-1 in plasma to 200 ± 13% of basal levels (P < 0.001). This effect was dose- and time-dependent and reached a maximum at 15 min. Ro 46-2005 had no effect on plasma concentrations of big ET-1 and only a minor effect on those of ET-3. In contrast to Ro 46-2005, the selective peptide ETA antagonists BQ-123 and FR-139317 had no effect on plasma ET-1 concentrations. The increase in plasma ET-1 concentrations by Ro 46-2005 was most likely not due to de novo synthesis, since big ET-1 levels were not increased and peak levels were reached early after compound injection, but perhaps to displacement of ET-1 from the ETB receptors.

Abstract —Leukocyte infiltration and adhesion molecule activation play a central role in the pathogenesis of angiotensin II (Ang II)–induced end-organ damage in double transgenic rats (dTGR) harboring human renin and angiotensinogen genes. We tested the hypothesis that the immunosuppressive agent cyclosporine (CsA) protects against the Ang II–induced myocardial and renal damage in dTGR. Furthermore, we investigated the influence of CsA on interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) expression and the DNA binding activity of transcription factor necrosis factor-κB (NF-κB). The 4-week-old rats were divided into 4 groups: (1) control dTGR (n=20), (2) dTGR plus CsA (5 mg/kg SC for 3 weeks, n=15), (3) normotensive Sprague-Dawley (SD) rats (n=10), and (4) SD rats plus CsA (n=8). In dTGR, CsA completely prevented cardiovascular death (0 of 15 versus 9 of 20), decreased 24-hour albuminuria by 90% and systolic blood pressure by 35 mm Hg, and protected against the development of cardiac hypertrophy. Whole blood CsA concentrations 24 hours after the last drug treatment were 850±15 ng/mL. Semiquantitative ED-1 and Ki-67 (a nuclear cell proliferation–associated antigen) scoring showed that CsA prevented perivascular monocyte/macrophage infiltration and prevented cell proliferation in the kidneys and hearts of dTGR, respectively. The beneficial effects of CsA were, at least in part, mediated by the suppression of IL-6 and iNOS expression. Electrophoretic mobility shift assay revealed that CsA regulated inflammatory response in part through the NF-κB transcriptional pathway. In contrast to dTGR, CsA increased blood pressure in normotensive SD rats by 10 mm Hg and had no effect on cardiac mass or 24-hour urinary albumin excretion. Perivascular monocyte/macrophage infiltration, IL-6, and iNOS expression or cell proliferation were not affected by CsA in SD rats. Our findings indicate that CsA protects against Ang II–induced end-organ damage and underscore the central role of vascular inflammatory response in the pathogenesis of myocardial and renal damage in dTGR. The beneficial effects of CsA in the kidney and heart are mediated, at least in part, by suppression of IL-6 and iNOS expression via NF-κB transcriptional pathway.

The poor interspecies conservation of the renin-angiotensin system prevents the use of nonprimate in vivo models to test renin inhibitors. Thus the small New-World monkey marmoset is used in many instances as a model. However, large differences between the potencies of renin inhibitors as measured in human and marmoset plasma were observed. To understand this phenomenon, we cloned marmoset renin and angiotensinogen. They were highly homologous to their human counterparts, except for a six-residue deletion in the marmoset renin propeptide. Human and marmoset recombinant renins were found in vitro to display comparable activities, suggesting that the observed differences in plasma apparent affinity of inhibitors could be due to different plasma protein binding of the inhibitors.

Introduction: Endothelin (ET) and nitric oxide (NO) act as opponents in the regulation of the hepatic microcirculation. During ischemia/reperfusion (I/R) ET levels are increased, whereas no rise in NO levels is observed. This imbalance may be responsible for microcirculatory disturbances. The aim of this study was to restore the delicate ET/NO balance to maintain the integrity of the hepatic microcirculation and to reduce I/R injury. Methods: Ischemia was induced by crossclamping of the hepatoduodenal ligament for 30 min with portal decompression using a splenocaval shunt (56 Wistar rats, 200–250 g). Sham operation, ischemia and treatment groups with the endothelin receptor antagonist (ERA) bosentan (1 mg/kg body weight i.v.) and the NO donor l-arginine (400 mg/kg body weight i.v.) were performed. For assessment of the microcirculation, sinusoidal perfusion rate, diameters of sinusoids and postsinusoidal venules, leukocyte endothelium interactions and velocity of free-flowing leukocytes were investigated by means of in vivo microscopy 30–90 min after reperfusion. Local hepatic tissue pO2 was measured prior to ischemia, 30 min and 60 min after reperfusion and aspartate aminotransferase (AST)/alanine aminotransferase (ALT) levels were investigated 2 h and 6 h after reperfusion. Results: After ischemia, sinusoidal and venular diameters were reduced to 76% and 85%, respectively, of sham operation group values (P

Background —An activated endothelin (ET) system may be of pathophysiological relevance in human heart failure. We characterized the functional effects of ET-1, ET receptors, and ET-1 peptide concentration in left ventricular myocardium from 10 nonfailing hearts (NF) and 27 hearts in end-stage failure due to idiopathic dilative cardiomyopathy (DCM). Methods and Results —Inotropic effects were characterized in isolated muscle strips (1 Hz; 37°C). ET-1 0.0001 to 0.3 μmol/L significantly ( P P A receptor antagonist BQ-123 shifted the concentration-response curves for ET-1 rightward. The ET B receptor agonist sarafotoxin S6c 0.001 to 0.3 μmol/L had no functional effects. The inotropic response to ET-1 was not associated with increased intracellular Ca 2+ transients, as assessed in aequorin-loaded muscle strips. ET receptor density (B max ; radioligand binding) was 62.5±12.5 fmol/mg protein in NF and 122.4±24.3 fmol/mg protein in DCM ( P max in DCM resulted from an increase in ET A receptors without change in ET B receptors. ET-1 peptide concentration (radioimmunoassay) was higher in DCM than in NF (14 447±2232 versus 4541±1340 pg/mg protein, P Conclusions —ET-1 exerts inotropic effects in human myocardium through ET A receptor–mediated increases in myofibrillar Ca 2+ responsiveness. In DCM, functional effects of ET-1 are attenuated, but ET A receptor density and ET-1 peptide concentration are increased, indicating an activated local cardiac ET system and possibly a reduced postreceptor signaling efficiency.

Abstract —The physiological role of prorenin is unknown; however, the possibility that prorenin inhibits renin locally has been suggested. We tested the hypothesis that prorenin may be an endogenous competitor for renin uptake in the tissue. We also investigated whether prorenin can be activated to active renin and affect mean arterial pressure (MAP). Isolated perfused hindquarters of rats transgenic for human angiotensinogen were infused with human renin and/or prorenin. The plateau phase of angiotensin (Ang) I release 15 minutes after cessation of infusions was used as a parameter for renin uptake. Renin (10 ng/mL for 15 minutes) caused sustained release of Ang I (153±16 fmol/mL). Coinfusion with a 15-fold excess of prorenin did not affect local Ang I formation (153±19 fmol/mL). Prorenin infusion alone showed no activation to active renin. In addition, we investigated MAP and plasma Ang II levels after injection of saline (ΔMAP, −1±2 mm Hg; 40±5 fmol/mL Ang II), 9 ng renin (ΔMAP, +37±3 mm Hg; 378±39 fmol/mL), and 144 ng prorenin (ΔMAP, +10±5 mm Hg; 61±5 fmol/mL) and the coinjection of renin and prorenin (ΔMAP, +41±4 mm Hg; 305±23 fmol/mL) in anesthetized rats. The data show that prorenin was not activated to active renin and did not affect MAP in short-term experiments. Renin-induced Ang formation was not affected by prorenin. Renin may have been taken up specifically because of its physical and chemical properties or because of nonspecific sequestration in the extravascular space. We conclude that prorenin does not act as an endogenous antagonist for the long-lasting effects of renin in the vascular wall. Moreover, prorenin does not affect acute renin-related effects on blood pressure.

Abstract —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-β 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 NFκB is upregulated in this model. We speculate that the transcription factors NFκB 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.

We assessed the role of endogenous endothelin (ET) in reperfusion injury using a novel nonpeptidic ET antagonist of ETA and ETB receptors: bosentan (Ro 47-0203). In preliminary experiments in nonischemic rat hearts, we confirmed that bosentan 10(-5) M could block the changes in coronary flow (CF) and release of prostacyclin induced by ET-1 and the ETB-selective agonist sarafotoxin S6c (SRTX S6c). Thereafter, we induced global ischemia in paced hearts by closing the perfusion line for 20 min; this was followed by reperfusion. In one group of rats, bosentan (10(-5) M) was added to the perfusion medium before ischemia. In preischemic conditions, bosentan slightly increased CF by 14% (p = 0.094) in nonpaced hearts and by 35% (p = 0.001) in paced hearts, but did not influence cardiac contractility. Global ischemia produced severe ischemic damage, as shown by electromechanical dissociation (EMD) and decreased cardiac contractility. Bosentan did not influence recovery of cardiac function and did not ameliorate postischemic hemodynamic variables as compared with those of the control group. We conclude that endogenous ET does not play a major role in induction of reperfusion injury in isolated perfused rat heart.

The present study characterizes the new transition-state renin inhibitor ciprokiren (Ro 44-9375) in squirrel monkeys. Arterial blood pressure was monitored by telemetry in freely moving, chronically instrumented conscious animals. In vitro at pH 7.4, ciprokiren inhibited human renin in buffer and human plasma with an IC50 of 0.07 and 0.65 nmol/L, respectively. It was equipotent against primate plasma renin and also inhibited plasma renin from dog and guinea pig in the nanomolar range (IC50, 29 and 65 nmol/L, respectively). After acute oral administration it reduced arterial blood pressure dose dependently in normotensive sodium-depleted and cyclosporin-induced hypertensive squirrel monkeys, starting with the minimal oral dose of 3 micrograms/kg. Daily oral doses of 1 microgram/kg showed a progressive blood pressure decrease, with a maximal response reached after 1 week. The drug could also be applied transdermally with similar hemodynamic effects without any decrease of plasma renin activity or plasma immunoreactive angiotensin II. Thus, ciprokiren is characterized in squirrel monkeys as a renin inhibitor with high in vivo potency that might act mainly in the tissular compartment.

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.

Since its discovery, endothelin-1 has attracted considerable scientific interest because of its extremely potent and long-lasting vasoconstrictor effect and its binding to G-protein-coupled receptors. Plasma concentrations of endothelin-1 are low and its release by endothelial cells is polarized towards the basolateral side, suggesting that it is a paracrine factor and not a hormone. Consequently, the effect of injected endothelin-1 may not reflect the effect of endogenous endothelin-1. In contrast, blockade of the action of endogenous endothelin-1 using receptor antagonists should be a valuable means of investigating its physiological and pathological effects. We report here evidence for the pathophysiological role of endothelin-1 as brought by the first synthetic orally active nonpeptide antagonist of endothelin receptors, Ro 46-2005.

The potent vasoconstrictor endothelin-1 (ET-1) is a paracrine, but also autocrine, factor for some types of cells. The goal of our study was to evaluate whether the receptor population in cells expressing endothelin receptor subtype A (rat mesangial cells) or endothelin receptor subtype B (human and rat endothelial cells) was affected by the autocrine production of ET-1. We therefore studied maximal binding capacity of 125I-labeled ET-1 in the presence or absence of the metalloprotease inhibitors phosphoramidon, which blocks the intracellular processing of Big ET-1 to ET-1, and thiorphan, which does not block this conversion. Phosphoramidon inhibited the release of ET-1 by human umbilical vein endothelial cells, rat aortic endothelial cells, and rat mesangial cells, and increased 1.4- to 17-fold the maximal binding capacity in the three types of cells. Thiorphan affected neither ET-1 release nor binding. The increase in receptor binding by phosphoramidon was associated with an increase in the functional effect of ET-1, as measured by arachidonic acid release in rat mesangial cells. We conclude that autocrine production of ET-1 decreases, either by binding or by downregulation, the number of binding sites available for ET-1 of paracrine or systemic sources. This aspect of modulation of the vasoconstrictor effect of endothelin should be considered in pathological situations or after endothelin-converting-enzyme inhibition.

Ro 46-2005 is a small-molecular-weight nonpeptide antagonist of the endothelin (ET), ETA and ETB receptors, chemically derived from a class of compounds identified by systematic screening of chemicals. In vivo, Ro 46-2005 inhibited the depressor and, only at high doses (100 mg/kg i.v.) the pressor effect of ET-1. However, much lower doses (1 to 10 mg/kg i.v.) were sufficient to inhibit the pressor effect of the precursor big ET-1. As intravenous big ET-1 injection potentially reproduces better than ET-1 injection the physiological pattern of release of ET-1, we propose that intravenous big ET-1 should be used to evaluate the effects of antagonists on the constricting endothelin receptors.

11 The balance between cell proliferation and apoptosis is essential for physiological function. We tested the hypothesis that angiotensin II (ANG II) has anti-apoptotic effects via the activation of AKT kinase in rats overexpressing the human renin and angiotensinogen genes (dTGR). dTGR are hypertensive (182±8 mm Hg) with severe renal damage (150-fold increased albuminuria), perivascular and interstitial fibrosis and vasculopathy, and die at 7 weeks. Renal NF-κB DNA binding activity is increased. We studied the time course of the pathogenesis from week 5 to week 7. Blood pressure, albuminuria, fibrosis, vasculopathy and monocyte infiltration increased over time. In contrast, KI-67 proliferating cells were more abundant in week 5 compared to week 7 as well as compared to non-transgenic rats. Phosphorylation of AKT kinase increased with the severity of the disease. Immunostaining of p-AKT and the activated NF-κB subunit p65 were observed in the glomeruli and endothelium. While Bcl-2 and phosphorylated Bad was predominantly expressed in the endothelium in week 7, the strongest bax immunostaining in the vessel wall media was observed in week 5. We then investigated the effect of chronic high dose aspirin (ASA; 600 mg/kg/d) on remodeling. While ASA did not change blood pressure, albuminuria and fibrosis were similar to non-transgenic controls. ASA completely normalized AKT protein synthesis as well as AKT phosphorylation and NF-κB DNA binding activity. Bax immunostaining at week 7 increased in the media, while Bcl-2 decreased. Western blot in dTGR kidney homogenates showed increased Bcl-2 levels compared ASA-treated and non-transgenic rats. We then confirmed the effect of ASA on p-AKT stimulating endothelial cells in vitro with ANG II (10-7 M; 10 min). Western blot showed reduced p-AKT in ASA pretreated cells. These results show that ANG II is anti-apoptotic by activating AKT kinase. ASA is able to restore the impaired balance of tissue remodeling. Finally, in addition to inhibiting I-κB kinase, ASA may exert a part of its ameliorating effects in this model by inhibiting AKT phosphorylation.

77 Aldosterone is a multifunctional mineralocorticoid affecting homeostasis of water and electrolytes, sympathetic activity, and tissue fibrosis. Aldosterone synthesis is induced by activation of the renin-angiotensin system. We tested whether or not the inhibition of aldosterone signaling can prevent fibrotic tissue remodeling in vivo. In a transgenic rat model overexpressing the human renin and angiotensinogen gens (dTGR), we investigated the effect of spironolactone (SPIRO 20 mg/kg/d) and the AT1 receptor blocker valsartan (VAL; 10 mg/kg/d) on growth factors and transcription factors leading chronic inflammation and tissue fibrosis. Untreated dTGR develop severe hypertension, cardiac hypertrophy, vasculopathy, and perivascular fibrosis. The hearts also show chronic inflammation and the animals have a 50% mortality at 7 weeks. VAL completely reversed these pathological features. SPIRO and VAL both prevented mortality, while only VAL normalized systolic blood pressure (VAL 121±9, SPIRO 161±11, dTGR 182±8, SD 109±2 mm Hg). Both reduced cardiac hypertrophy (SPIRO 4.2±0.1, VAL 3.6±0.1, dTGR 5.7±0.2, SD 3.6±0.1 mg/g) and reduced vasculopathy. Quantitative bFGF RT-PCR showed a complete mRNA reduction in the left ventricle in both treatment groups (SPIRO 11±4, VAL 5±1 dTGR 43±4, SD 4±1 arbitrary units). In contrast, RT-PCR of TGF beta and PDGF showed no upregulation in dTGR compared to non-transgenic hearts. Gel shift assay of the heart demonstrated a suppression of AP-1 and NF-κB DNA binding activity after SPIRO and VAL treatment. Immunohistology revealed reduced bFGF expression and less collagen I, fibronectin, and laminin in the interstitium of SPIRO and VAL-treated rats. These findings show that aldosterone promotes hypertrophy, cardiac remodeling, and fibrosis. In dTGR hearts this effect is at least partly mediated through the transcription factors AP-1 and NF-κB as well as bFGF. Blocking the aldosterone receptor downregulates these effector molecules and reduces angiotensin II-induced cardiac damage.

P4 In a recent report, aspirin (ASA) inhibited I-κB kinase beta in vitro; however, in vivo relevance has not been shown. We previously showed that NF-κB activation and inflammation are crucial role in the pathogenesis of Ang II-induced vasculopathy. We now tested the hypothesis that ASA inhibits NF-κB and ameliorates renal and cardiac end-organ damage. dTGR feature hypertension, renal and cardiac damage and die at 7 weeks. We treated rats chronically with ASA (600 and 25 mg/kg/d ip). Only ASA 600 prevented mortality, while untreated dTGR and ASA 25 showed mortality >50% at 7 weeks. ASA 600 reduced cardiac hypertrophy (4.0±0.2 vs. 5.7±0.2 mg/g, p

P168 Recently, clinical trials have demonstrated the efficacy of spironolactone (SPIRO) in men. Nevertheless the molecular mechanism of action not completely understood. Locally generated angiotensin II (ANG II) stimulates aldosterone. Therefore, we have tested the hypothesis that SPIRO ameliorates ANG II-induced renal damage. Furthermore, we investigated the effect of SPIRO on the transcription factors AP-1 and NF-κB. We treated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) from week 5 to 7 with SPIRO (20 mg/kg/d). Plasma aldosterone was significantly increased in dTGR vs. SPIRO treated and non-transgenic (SD) rats (p

Ro 46-2005 is a new synthetic non-peptide endothelin (ET) receptor antagonist. In binding experiments, Ro 46-2005 proved to be equipotent (IC50 200–500 nM) for inhibition of [125I]ET-1 binding on the two known ET receptor subtypes (ETA and ETB). Scatchard analysis was consistent with a competitive binding mode. Ro 46-2005 also inhibited the functional consequences of ET-1 stimulation: the ET-1-induced release of arachidonic acid from rat mesangial cells was inhibited with an IC50 of 1.8 μM.

We used Ro 46-8443, non-peptidic antagonist selective of endothelin ETB receptors, to study the role of ETB receptors in rat hypertension models. In normotensive rats, Ro 46-8443 decreased blood pressure, but in SHR and DOCA rats, it induced a pressor effect, due to blockade of ETB-mediated release of nitric oxide since L-NAME prevented it. In rats rendered hypertensive by chronic L-NAME, Ro 46-8443 did not induce a pressor but depressor effect. Thus, in DOCA rats and SHR, Ro 46-8443 reveals a predominant influence of endothelial ‘vasorelaxant’ ETB receptors, while in normotensive rats the prevailing role of ETB receptors seems to be in mediating a vasoconstrictor tone.

The present study aimed to characterize endothelin (ET) receptors mediating the effects of ET-1 in the rat fundic strip. Functional studies demonstrated that ET-1 and the ETB receptor-selective agonist sarafotoxin S6c elicit contraction of the rat fundic strip via high-affinity ETB receptors. Contraction was potentiated by the ETA receptor antagonist BQ 123 (10 microM) and the mixed antagonist bosentan (10 microM), suggesting that ET-1 also activates ETA and ETB receptors that inhibit contraction. This ET-1-mediated inhibition of contraction was blocked by an inhibitor of Ca(2+)-activated K+ channels, apamin (50 microM). Competitive binding studies revealed the presence of one BQ 123-sensitive and two BQ 123-insensitive binding sites that showed different affinity for ET-3 in the pM and nM range, respectively. These observations demonstrate the presence of multiple receptors in the rat fundus and suggest that smooth muscle contraction is counteracted by activation of Ca(2+)-activated K+ channels by ET-1.

Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT1 receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-κB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT1 receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT1 receptor blockade. Deletion of both AP-1 and NF-κB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT1 receptor blockade in this model are mediated via the inhibition of NF-κB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.

Abstract —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, AT 1 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. α L β 2 integrin and α 4 β 1 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), AT 1 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, AT 1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR.

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-β 1 , and the transcription factors AP-1 and nuclear factor (NF)-κB. 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-β 1 were little changed. VAL and SPIRO suppressed this upregulation. Both AP-1 and NF-κB 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-κB, and bFGF. Mineralocorticoid receptor blockade downregulates these effectors and reduces angiotensin II–induced cardiac damage.