Biomedical subjects
Publications and source records attributed to E H Ohlstein.
In the 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists, a compound containing an imidazopyridine arginine mimetic was discovered which had sufficient potency and i.v. pharmacokinetics for demonstration of efficacy in a rat restenosis model.
BACKGROUND: We have previously shown that elevated release of endothelin-1 is associated with increased pulmonary vascular resistance (PVR) immediately after reperfusion of the transplanted lung. In the present study, we investigated the effect of ET receptor blockage on pulmonary hemodynamics and function in an ex vivo lung reperfusion model after 6 hr of cold ischemia. METHODS: Eighteen rabbits were divided into three groups: no ischemia followed by 3 hr of reperfusion (group I) and 6 hr of cold ischemia followed by 3 hr of reperfusion with either blood (group II) or blood + SB209670 (mixed ETA/ETB receptor antagonist) (group III). RESULTS: Shortly after reperfusion, mean pulmonary artery pressure, PVR, and pulmonary edema were increased, and pulmonary compliance and PO2 were decreased in group II compared with group I. Treatment with SB209670 resulted in a significant decrease in mean pulmonary artery pressure, PVR, and pulmonary edema, and improvement in pulmonary compliance and PO2. CONCLUSION: The data suggest an important role for ET-1 in lung ischemia/reperfusion injury and that the use of ET receptor antagonist immediately after transplantation may provide a new therapeutic tool in the management of early graft dysfunction.
During the development of atherosclerotic lesions, lipoprotein(a) [Lp(a)], a highly atherogenic lipoprotein, accumulates within fibrin clots attached to blood vessel walls. As Lp(a) accumulates within the fibrin clot with time, fatty streaks are formed that develop into occlusive atherosclerotic plaques. It is not understood, however, which mechanisms are involved in the binding of Lp(a) to fibrin and, hence, the stable incorporation of Lp(a) into the fibrin clot. The results of the present study demonstrate that factor XIIIa, a transglutaminase that catalyzes the formation of amide bonds between endo-gamma-glutaminyl and endo-epsilon-lysyl residues of proteins, is capable of cross-linking Lp(a) to fibrinogen, the soluble precursor of fibrin. Biochemical assays were conducted to demonstrate that factor XIIIa cross-links Lp(a) with fibrinogen in a time- and concentration-dependent manner. Additionally, immunohistochemical studies revealed that factor XIII protein expression colocalizes with Lp(a) expression in human atherosclerotic plaques. It is proposed that factor XIIIa-mediated cross-linking of Lp(a) to fibrin effectively increases the local concentration of Lp(a) within a fibrin clot. The accumulation of Lp(a) within the blood vessel promotes an antifibrinolytic environment, foam cell formation, the generation of a fatty streak, and an increase in smooth muscle cell content, all of which may contribute to the pathogenesis of atherosclerosis.
BACKGROUND: Alleviating vascular restenosis after percutaneous transluminal angioplasty remains a formidable challenge. Although multiple factors have been implicated in the pathophysiology of this vascular remodeling disorder, only limited therapeutic success has been achieved. Endothelin (ET)-1 has recently been implicated in the pathogenesis of neointimal growth. We report the in vivo efficacy of SB 217242, a nonpeptide dual ET(A)/ET(B) receptor antagonist with high oral bioavailability, in the rat carotid artery balloon angioplasty model. METHODS AND RESULTS: The lumen volumes of carotid arteries were estimated serially with magnetic resonance imaging (MRI) at baseline and at day 7 and day 14 after balloon catheter-induced denudation of the carotid arterial wall in the rat. Histomorphometric analysis was performed at day 14 after surgery to quantitate intimal hyperplasia. Statistical analysis was performed with ANOVA followed by post hoc Newman-Keuls multiple comparison test. In comparison to vehicle-treated animals, a 20% protection (P<0.05) from reduction was shown in the estimated lumen volume with long-term administration of SB 217242 (15 mg/kg BID p.o.). Histologic analyses indicated a 42% decrease (P<0.05) in neointimal growth. The MRI lumen volumes had a significant correlation with the corresponding histologic indices. CONCLUSIONS: Serial MRI provides the opportunity to assess the progression of vascular lumen volume in vivo after balloon angioplasty. MRI measurements can, in conjunction with in vitro histologic measurements, contribute to the understanding of the actions of pharmacologic agents in experimental models of neointima formation. With the use of serial MRI and histologic measurements, it is demonstrated that protection from both lumen volume reduction and neointima formation is obtained in this model by use of a potent, nonpeptide dual ET(A)/ET(B) receptor antagonist, SB 217242. Furthermore, this study provides additional support to the implication of ET-1 in the pathophysiology of neointima formation.
Carvedilol, a multiple action neurohumoral antagonist, has been reported recently to significantly reduce mortality in congestive heart failure (CHF) patients. In addition to being a beta-adrenoceptor antagonist, one of the unique aspects of the biological effects of carvedilol is that it is also a potent antioxidant with antimitogenic properties. As there is a significant correlation between plasma immunoreactive endothelin-1 (ET-1) levels and the severity of CHF, the present study was designed to determine the effects of carvedilol on ET-1 biosynthesis in cultured human coronary artery endothelial cells (HCAECs). HCAECs were treated with carvedilol 15 min prior to the addition of serum and ET-1 levels were measured in the cell culture conditioned medium 24 h later. Carvedilol (10 microM) significantly inhibited basal ET-1 production in HCAECs by 62 +/- 8%. Carvedilol produced a concentration-dependent inhibition of serum-mediated stimulation of ET-1 biosynthesis with an IC50 = 1.2 microM. PreproET-1 mRNA expression was also inhibited by carvedilol. Other beta-adrenoceptor antagonists, such as propranolol (10 microM) or celiprolol (10 microM), did not effect ET-1 biosynthesis. Furthermore, the antioxidant probucol (10 microM) did not effect ET-1 production. Immunohistochemical analysis of HCAECs demonstrated that resting HCAECs have expression of ET-1 and can be inhibited by carvedilol. The results of the present study demonstrate that serum stimulation of HCAECs produced an increase in ET-1 expression, and carvedilol treatment caused a marked decrease in stimulated ET-1 expression as compared to serum-treated HCAECs. These data indicate that carvedilol directly inhibits the biosynthesis of ET-1 in HCAECs, and this effect may contribute to its vasodilating and antiproliferative actions. Furthermore, these effects may contribute to the ability of carvedilol to improve clinical outcome in CHF patients.
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Carvedilol is a third-generation vasodilating beta-blocker initially approved for the treatment of hypertension. It lowers systemic arterial blood pressure without causing reflex tachycardia and preserves renal function. More recently, carvedilol has been shown to reduce morbidity and mortality in patients with congestive heart failure. This reduction may occur in part via beta-blockade and alpha 1-adrenoceptor blockade, the latter resulting in vasodilation. Importantly, carvedilol and several of its metabolites are potent antioxidants that may inhibit the oxidation of norepinephrine and the subsequent formation of toxic intermediates, such as reactive free radicals in the myocardium. As a result, carvedilol inhibits the expression of certain genes involved in myocardial damage, such as intracellular adhesion molecule-1, free-radical-induced activation of transcription factors, and programmed cell death or apoptosis. In this respect, carvedilol represents a new therapy for the treatment of hypertension and congestive heart failure and combines, in one molecule, a number of potentially beneficial properties.
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SB 209670 reduced basal mean arterial pressure (16%) without affecting left-circumflex coronary artery (LCX) flow, cardiac output, heart rate, or global/regional myocardial contractility. In vehicle-treated animals, i.e. endothelin (ET)-1 produced an initial hyperemic response in the LCX, followed by a secondary reduction in flow. This response was accomplished by decreases in LCX regional wall fractional shortening, +dP/dt and -dP/dt, but an increase in left anterior wall fractional shortening. ET-1 also produced dose-related, fatal ventricular fibrillation. Whereas SB 209670 administration did not inhibit the initial increase in coronary flow produced by ET-1, the secondary constrictor responses were markedly antagonized. SB 209670 also attenuated the reduction in LCX regional wall fractional shortening and converted the increase in left anterior wall contractility to a reduction in contractility. Although SB 209670 produced only a modest inhibition of the ET-1-mediated reductions in dP/dt, the induction of fatal ventricular arrhythmias was completely abolished. Therefore, the data are consistent with the hypothesis that the coronary ischemic and proarrythmic actions of ET-1 are distinct. Therefore, ET receptor antagonists may be useful in treatment of disturbances in cardiac rhythm.
Some endothelin (ET) receptor antagonists have been reported to elevate plasma immunoreactive endothelin-1 (irET-1). However, there is no information regarding the effects of ET receptor antagonists on cerebrospinal fluid (CSF) levels. To better understand the regulation of circulating and CSF ET-1, the effects of several nonpeptide antagonists with high, intermediate, or low affinity at the ETB receptor, as well as the potent ETB selective agonist sarafotoxin 6c (S6c), were characterized and compared. The effects of SB209670 (Ki ETA = 0.2 nM; Ki ETB = 12 nM), SB217242 (Ki ETA = 1.1 nM; Ki ETB = 111 nM), SB234551 (Ki ETA = 0.1 nM; Ki ETB = 500 nM), SB247083 (Ki ETA = 0.4 nM; Ki ETB = 467 nM), and S6c (Ki ETA = 950 nM; Ki ETB = 1 nM) on plasma irET-1 were determined by ELISA in the anesthetized dog after i.v. administration. Systemic administration of equivalent doses of the nonpeptide ET receptor antagonists produced dose-related elevations in plasma irET-1 which were correlated (p = 0.019) with affinity at the ETB receptor. There was no significant correlation with affinity at the ETA receptor. In addition, the plasma irET-1 and ET antagonist concentrations were linearly correlated (r = 0.98) throughout the time course after antagonist administration. There was no evidence of densensitization after three bolus administrations performed at 2-h intervals (SB209670, 1 and 3 mg/kg i.v.). Elevations in plasma irET-1 (four- to fivefold) were also observed after systemic administration of S6c (1 nmol/kg i.v.). The administration of L-NAME (200 micrograms/kg/min for 30 min), an inhibitor of nitric oxide (NO) synthase, increased blood pressure (33%) but did not alter plasma irET-1. In contrast, systemic administration of the ET receptor antagonists had little or no effect on the on irET-1 in the CSF. However, intracerebroventricular (i.c.v.) administration of SB209670 produced a dose-related (3-100 micrograms) increase in cisternal CSF levels of irET-1 without altering plasma irET-1. Systemic administration of ETB receptor antagonists and agonists rapidly increased plasma irET-1. These ETB receptor antagonist effects correlate linearly with affinity at the cloned human ETB receptor, do not exhibit desensitization, and do not appear to reflect inhibition of ETB-mediated NO production. The endothelial ETB receptor may represent a high-capacity storage/clearance site for circulating ET-1. ET receptor antagonists may also act extravascularly/abluminally to increase irET-1 in the CNS.
Competition radioligand binding with [125I]ET-1 at human cloned ETA and ETB receptors demonstrated ET-A selective affinity by SB 247083 (Ki 0.41 and 467 nM, respectively). Accordingly, similar competitive, functional ETA receptor antagonism was observed. In vitro, SB 247083 exhibited a Kb of 3.5 +/- 0.3 nM (ET-1--induced rat aortic contraction). SB 247083 was significantly less potent as a functional ETB antagonist (Kb 0.34 +/- 0.01 microM; S6c-induced rabbit pulmonary artery contraction). In contrast to ETB-selective and mixed ETA/B antagonists, and consistent with its ETA-selective profile, in vivo administration of SB 247083 was not associated with an elevation in plasma ET-1 levels. Pharmacodynamic and pharmacokinetic studies revealed that SB 247083 was effectively absorbed from the gastrointestinal tract. A single bolus dose inhibited the hemodynamic actions of ET-1 for up to 8 h, consistent with a molecule shown to be 46% bioavailable. Therefore, the present study demonstrates that SB 247083, a unique chemical entity, represents a potent class of nonpeptide, orally active ETA-selective antagonists.
The effects of a nonpeptide, orally active mixed endothelin (ET) ETA/ETB receptor antagonist, SB 217242, and an angiotensin-converting enzyme (ACE) inhibitor, ramipril, were evaluated after inter-renal aortic banding in the rat. Separate sham, vehicle, and treatment groups were compared in each study. In vehicle-treated animals in the ramipril group, aortic banding for 4 weeks produced significant cardiac hypertrophy (247 +/- 5 mg/100 g bw vs. 305 +/- 11 mg/100 g bw; p < 0.001), right (upstream) renal hypertrophy (380 +/- 6 mg/100 g bw vs. 559 +/- 28 mg/100 g bw; p < 0.001), and significant left (downstream) renal atrophy (405 +/- 4 mg/100 g bw vs. 192 +/- 25 mg/100 g bw; p < 0.001). Continuous ramipril treatment (1 mg/kg p.o. once daily), begun 3 days before aortic banding, inhibited cardiac hypertrophy (305 +/- 11 mg/100 g bw vs. 266 +/- 7 mg/100 g bw; p < 0.05) but did not alter renal hypertrophy or atrophy. In a similarly designed study, SB 217242 (30 mg/kg p.o. b.i.d.) had no effect on the development of cardiac hypertrophy (298 +/- 7 mg/100 g bw vs. 310 +/- 12 mg/100 g bw) or renal hypertrophy (561 +/- 15 mg/100 g bw vs. 575 +/- 19 mg/100 g bw), but abolished the development of renal atrophy (158 +/- 16 mg/100 g bw vs. 395 +/- 19 mg/100 g bw; p < 0.001). [125I]ET-1 radioligand binding experiments indicated that the density of both ETA and ETB receptors was increased dramatically (three- to fourfold) in the atrophic kidney cortex compared to sham or hypertrophic kidneys. In situ hybridization studies indicate an upregulation of ETB receptor mRNA in the glomeruli of atrophic kidneys within 5 days of aortic banding. In conclusion, an angiotensin-dependent mechanism may mediate cardiac hypertrophy associated with aortic banding, whereas ET-dependent mechanisms may mediate an atrophic response in the hypoperfused kidney, perhaps through an interaction with upregulated ETA and/or ETB receptors.
Although several factors have been implicated in the pathophysiology of vascular neointimal formation and restenosis after balloon angioplasty, current therapies have failed to alleviate these conditions. Because endothelin-1 (ET-1) has been implicated in the pathogenesis of the neointimal growth, this study examined the in vivo efficacy of SB 217242, a potent nonpeptide mixed ETA/ETB receptor antagonist with high oral bioavailability, in the rat carotid balloon angioplasty model. A novel magnetic resonance imaging (MRI) approach was used for evaluation. MRI is a high-resolution, noninvasive spatial imaging modality that can serially monitor arterial luminal caliber in vivo, along with histologic evaluation. In vivo luminal volumes for the left carotid artery (LCA) were measured by MRI at baseline (before surgery) and again at 14 days after angioplasty. SB 217242 (30 mg/kg/day p.o.) was administered daily for 14 days after angioplasty. On day 14, SB 217242 provided 20% protection (p < 0.05) on neointimal-mediated decrease in luminal volume as measured by MRI. The non-ballooned right carotid arteries of both groups had significantly increased luminal volume on day 14 with respect to baseline values, indicating compensation for the decreased luminal size of the LCA. Histologic analysis of the mean intimal-to-medial ratio demonstrated that SB 217242 provided 42% (p < 0.05) protection. These results demonstrate that ET receptor antagonism can inhibit neointimal formation after balloon angioplasty and that MRI technology can provide valuable insight for noninvasive assessment of vascular lesion development.
Infusion of endothelin-1 (ET-1) into conscious, chronically instrumented dogs (10 ng/kg.min i.v.) resulted in a significant increase in mean arterial pressure and significant reductions in renal plasma flow, glomerular filtration rate, and sodium excretion. Intravenous infusion of SB 209670 (30 micrograms/kg.min, i.v.) abolished these responses, whereas infusion of SB 234551 (30 micrograms/kg.min, i.v.) resulted in significant increases in renal plasma flow and urinary sodium excretion. These data indicate that SB 234551 can unmask ETB receptor-induced renal vasodilatation and inhibition of sodium reabsorption.
The cardiopulmonary profile of three different rat strains was compared after exposure to hypoxia (9% O2) for 0, 7, or 14 days. In Sprague-Dawley (SD), Wistar (W), and high altitude-sensitive (HAS) rats, pulmonary arterial pressure (PAP) rose 30, 58, and 85% respectively, after 7 days of hypoxia, and by 108, 116, and 167%, respectively, at 14 days compared to strain- and age-matched normoxic controls. Right ventricular hypertrophy (RVH), expressed as the ratio of right free wall/left wall + septum weight, in SD, W, and HAS was increased by 24, 53, and 48%, respectively, at 7 days, and by 51, 93, and 55% at 14 days compared to normoxic littermates. Histologically, marked medial thickening and luminal stenosis of small and medium-sized arteries were observed in all hypoxic rats, being most pronounced in the HAS rats at 14 days. Treatment of HAS rats with the ET receptor antagonist SB 217242 (3.6 or 10.8 mg/day i.p. by osmotic pump) significantly inhibited the hypoxia-induced increases in PAP (70-75% decrease). RVH was inhibited by 40% at the dose of 10.8 mg/day. Histologically, the SB 217242-treated rats had almost "normal" small and medium-sized arteries, comparable to those of the normoxic HAS controls. This study demonstrates an exaggerated PAP response to chronic hypoxia in HAS compared to SD and W rats. The inhibitory influence of SB 217242 on the functional and morphologic changes induced by hypoxia provides further evidence for a role for ET and the potential utility of ET receptor antagonists in the treatment of pulmonary hypertension.
Many beta-adrenoceptor antagonists are weak partial agonists, possessing significant intrinsic sympathomimetic activity (ISA). Under certain conditions, ISA may be deleterious through stimulation of beta 1- and/or beta 2-adrenoceptors in the heart. Drugs with ISA are particularly problematic in the treatment of congestive heart failure since agents that activate cardiac beta-adrenoceptors, such as xamoterol, have been associated with increases in the incidence of arrhythmia and mortality. Carvedilol was recently approved for the treatment of congestive heart failure, and bucindolol is currently in large clinical trials for this indication. In the present study, the ISA of bucindolol and carvedilol was evaluated in a standard model used to investigate ISA, the pithed rat. Both compounds produced dose-dependent inhibition of the positive-chronotropic effects of the non-selective beta-adrenoceptor agonist, isoproterenol, confirming that these drugs are beta-adrenoceptor antagonists. However, cumulative administration of bucindolol (10-1,000 micrograms/kg i.v.) in the pithed rat produced a significant dose-related increase in heart rate. The maximal increase in heart rate produced by bucindolol was 44% of that obtained with isoproterenol (90 +/- 6vs. 205 +/- 11 bpm, respectively). In marked contrast, cumulative administration of carvedilol (10-1,000 micrograms/kg i.v.) had no significant effect on resting heart rate in the pithed rat. The maximal increase in heart rate elicited by bucindolol (1,000 micrograms/kg i.v.) was inhibited by treatment with the competitive beta-adrenoceptor antagonist, propranolol (99 +/- 8.7 vs. 26 +/- 2.6 bpm), confirming that the ISA observed with bucindolol was mediated through stimulation of myocardial beta-adrenoceptors. Carvedilol, which had no ISA, antagonized the ISA of bucindolol, and was as effective as propranolol in blocking the ISA of bucindolol (99 +/- 8.7 vs. 27 +/- 2.3 bpm). In summary, bucindolol and carvedilol are both potent beta-adrenoceptor antagonists in the pithed rat: however, only bucindolol possesses beta-adrenoceptor-mediated ISA.
BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS: Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS: MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS: These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.
The purpose of this study was to determine whether treatment with an endothelin-1 (ET-1)-receptor antagonist could prevent ET-1-mediated ischemia-reperfusion injury and early allograft dysfunction. Eleven dogs were subjected to left lung allotransplantation. Donor lungs were preserved with modified Eurocollins solution and stored at 4 degrees C for 18 to 20 h. Animals received an intravenous infusion of either the ET-receptor antagonist SB209670 (n = 6) (15 microg/kg/min) or saline (control, n = 5), in a blinded fashion. The infusion started 30 min before transplantation and continued for up to 6 h after transplantation. Hemodynamic measurements, blood gas tensions, and plasma samples were obtained with animals functioning solely on the transplanted lung. Open-lung biopsies were obtained for wet-to-dry-weight ratios and histologic and immunohistochemical analyses. Survival at 6 h after transplantation was 40% in the control group and 100% in the treatment group. Pulmonary vascular resistance and lung tissue wet-to-dry-weight ratio were significantly lower in treated animals at 3 and 6 h after transplantation. Histology of the transplanted lungs revealed more intense airway and interstitial inflammatory infiltration and edema in the control group. Arterial and venous plasma ET-1 concentrations increased after transplantation; however, they were significantly higher in the treatment group. Immunohistochemical analysis revealed more intense ET-1 immunostaining in the airways and parenchyma of the treatment group. We conclude that treatment of lung allografts with the mixed endothelin A/endothelin B (ETA/ETB) receptor antagonist SB209670 can ameliorate ischemia-reperfusion injury, resulting in improved graft function and survival after lung transplantation.