In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

Hypertensive end-organ damage and premature mortality are p38 mitogen-activated protein kinase-dependent in a rat model of cardiac hypertrophy and dysfunction.

BACKGROUND: Numerous pathological mediators of cardiac hypertrophy (eg, neurohormones, cytokines, and stretch) have been shown to activate p38 MAPK. The purpose of the present study was to examine p38 MAPK activation and the effects of its long-term inhibition in a model of hypertensive cardiac hypertrophy/dysfunction and end-organ damage. METHODS AND RESULTS: In spontaneously hypertensive stroke-prone (SP) rats receiving a high-salt/high-fat diet (SFD), myocardial p38 MAPK was activated persistently during the development of cardiac hypertrophy and inactivated during decompensation. Long-term oral treatment of SFD-SP rats with a selective p38 MAPK inhibitor (SB239063) significantly enhanced survival over an 18-week period compared with the untreated group (100% versus 50%). Periodic echocardiographic analysis revealed a significant reduction in LV hypertrophy and dysfunction in the SB239063-treatment groups. Little or no difference in blood pressure was noted in the treatment or vehicle groups. Basal and stimulated (lipopolysaccharide) plasma tumor necrosis factor-alpha concentrations were reduced in the SB239063-treatment groups. In vitro vasoreactivity studies demonstrated a significant preservation of endothelium-dependent relaxation in animals treated with the p38 MAPK inhibitor without effects on contraction or NO-mediated vasorelaxation. Proteinuria and the incidence of stroke (53% versus 7%) were also reduced significantly in the SB239063-treated groups. CONCLUSIONS: These results demonstrate a crucial role for p38 MAPK in hypertensive cardiac hypertrophy and end-organ damage. Interrupting its function with a specific p38 MAPK inhibitor halts clinical deterioration.

Effect of endothelin receptor antagonist on lung allograft apoptosis and NOSII expression.

BACKGROUND: It is postulated that apoptosis contributes to ischemia-reperfusion graft dysfunction after lung transplantation. The purpose of this study was to determine whether the improvement in lung function that we previously observed with the use of an endothelin-1 (ET-1) receptor antagonist after ischemia-reperfusion injury is associated with a reduction in inducible nitric oxide synthase (NOSII) expression and programmed cell death. METHODS: Left lung canine allotransplantation was performed. Harvested lung blocks were preserved with modified Eurocollins solution and stored at 4 degrees C for 18 to 20 hours. Lung allografts were tested for the expression of NOSII by immunohistochemistry, and extent of apoptosis by terminal dUTP nick end-labeling (TUNEL). Animals blindly received either an intravenous infusion of saline (control) or the ET-1 receptor antagonist (SB209670) (15 microg/kg/min). Infusion began 30 minutes pretransplantation and continued to 6 hours posttransplantation. RESULTS: Immunohistochemical analysis demonstrated significantly stronger NOSII immunostaining in the allografts of the saline control group (36.5%+/-3.6%) compared with native right lungs (6.9%+/-1.3%, p < 0.001) or the ET-receptor antagonist treatment group (9.6%+/-1.4%, p < 0.001). The TUNEL staining revealed a significantly stronger labeling in the allografts of the saline treatment control group (40.7%+/-6.2%) compared with native right lungs (5.0%+/-0.6%, p < 0.005) or the ET receptor antagonist treatment group (14.1%+/-2.8%, p < 0.01). CONCLUSIONS: We conclude that treatment of lung allografts with the ET-1 receptor antagonist SB209670 reduces the area of NOSII expression and the extent of apoptosis, factors known to contribute to the process of prolonged ischemia-reperfusion injury.

Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia.

Mitogen-activated protein kinases (MAPKs) are involved in many cellular processes. The stress-activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke-induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre- and post-stroke and intravenously post-stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28-41%, and neurological deficits by 25-35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke-induced expression of IL-1beta and TNFalpha (i.e., cytokines known to contribute to stroke-induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063-induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.

SB 239063, a second-generation p38 mitogen-activated protein kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia.

The stress-activated mitogen-activated protein kinase (MAPK) p38 has been linked to the production of inflammatory cytokines/mediators/inflammation and death/apoptosis following cell stress. In these studies, a second-generation p38 MAPK inhibitor, SB 239063 (IC(50) = 44 nM), was found to exhibit improved kinase selectivity and increased cellular (3-fold) and in vivo (3- to 10-fold) activity over first-generation inhibitors. Oral SB 239063 inhibited lipopolysaccharide-induced plasma tumor necrosis factor production (IC(50) = 2.6 mg/kg) and reduced adjuvant-induced arthritis (51% at 10 mg/kg) in rats. SB 239063 reduced infarct volume (48%) and neurological deficits (42%) when administered orally (15 mg/kg, b.i.d.) before moderate stroke. Intravenous SB 239063 exhibited a clearance of 34 ml/min/kg, a volume of distribution of 3 l/kg, and a plasma half-life of 75 min. An i.v. dosing regimen that provided effective plasma concentrations of 0.38, 0.75, or 1.5 microg/ml (i.e., begun 15 min poststroke and continuing over the initial 6-h p38 activation period) was used. Significant and dose-proportional brain penetration of SB 239063 was demonstrated during these infusion periods. In both moderate and severe stroke, intravenous SB 239063 produced a maximum reduction of infarct size by 41 and 27% and neurological deficits by 35 and 33%, respectively. No effects of the drug were observed on cerebral perfusion, hemodynamics, or body temperature. Direct neuroprotective effects from oxygen and glucose deprivation were also demonstrated in organotypic cultures of rat brain tissue. This robust in vitro and in vivo SB 239063-induced neuroprotection emphasizes the potential role of MAPK pathways in ischemic stroke and also suggests that p38 inhibition warrants further study, including protection in other models of nervous system injury and neurodegeneration.

Endothelial protective and antishock effects of a selective estrogen receptor modulator in rats.

This study investigated whether idoxifene, a selective estrogen receptor modulator (SERM), exerted protective effects against ischemia-reperfusion-induced shock. Ovariectomized rats were treated with vehicle, idoxifene, or 17beta-estradiol for 4 days. Rats were subjected to splanchnic artery occlusion (SAO) followed by reperfusion (SOA/R). In vehicle-treated rats, SAO/R resulted in hypotension, hemoconcentration, increased plasma tumor necrosis factor (TNF)-alpha levels, intestinal neutrophil accumulation, and endothelial dysfunction. 17beta-Estradiol treatment increased plasma estradiol concentration and reduced SAO/R-induced tissue injury. Idoxifene treatment had no effect on plasma estradiol concentration but reduced SAO/R-induced hemoconcentration (+8.8 +/- 1.3 vs. +14 +/- 1.3% in the vehicle group, P < 0.01), TNF-alpha production (98 +/- 3.2 vs. 214 +/- 13 pg/ml, P < 0.01), and neutrophil accumulation (0.025 +/- 0.005 vs. 0.047 +/- 0.005 U/g protein, P < 0.01). It also improved endothelial function, prolonged survival time (172 +/- 3.5 vs. 147 +/- 8 min, P < 0.01), and increased survival rate (69 vs. 23%, P < 0.01). Moreover, treatment with 17beta-estradiol or idoxifene in vivo reduced TNF-alpha-induced endothelial dysfunction in vitro. Taken together, these results demonstrated that idoxifene exerted estrogen-like, endothelial-protective, and antishock effects in ovariectomized rats, suggesting that SERMs have therapeutic potential in tissue injury resulting from ischemia-reperfusion.

Potent vasodilator responses to human urotensin-II in human pulmonary and abdominal resistance arteries.

The peptide human urotensin-II (hUT-II) and its receptor have recently been cloned. The vascular function of this peptide in humans, however, has yet to be determined. Vasoconstrictor and vasodilator responses to hUT-II were investigated in human small muscular pulmonary arteries [approximately 70 microm internal diameter (ID)] and human abdominal resistance arteries (approximately 200 microm ID). Vasodilator responses were investigated in endothelin-1 (3 nM) precontracted vessels and, in the small pulmonary vessels, compared with the known vasodilators adrenomedullin, sodium nitroprusside, and acetylcholine. In human small pulmonary arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [-log M concentration causing 50% of the maximum vasodilator effect (pIC(50)) 10.4 +/- 0.5; percentage of reduction in tone (E(max)) 81 +/- 8% (vs. 23 +/- 11% in time controls), n = 5]. The order of potency for vasodilation was human urotensin-II = adrenomedullin (pIC(50) 10.1 +/- 0.4, n = 6) > sodium nitroprusside (pIC(50) 7.4 +/- 0.2, n = 6) = acetylcholine (pIC(50) 6.8 +/- 0.3, n = 6). In human abdominal arteries, hUT-II did not induce vasoconstriction but was a potent vasodilator [pIC(50) 10.3 +/- 0.7; E(max) 96 +/- 8% (vs. 43 +/- 16% in time controls), n = 4]. This is the first report that hUT-II is a potent vasodilator but not a vasoconstrictor of human small pulmonary arteries and systemic resistance arteries.

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

Platelet agonist F11 receptor is a member of the immunoglobulin superfamily and identical with junctional adhesion molecule (JAM): regulation of expression in human endothelial cells and macrophages.

The stimulatory mAb F11 binds two platelet membrane proteins of 32 and 35 kDa and causes activation of platelets when cross-linked with the FcgammaRII receptor. We used bioinformatics to identify expressed sequence tags from libraries of cytokine-stimulated human endothelial cell (EC) cDNAs. The protein sequence deduced from full-length F11 cDNA was identical to partial sequences of peptides derived from affinity-purified platelet F11 antigen. F11 mRNA is expressed in human EC, macrophages, and a variety of non-hematopoietic vascular tissues. Expression of F11 mRNA is modulated by cytokines in EC and is up-regulated by oxidized low-density lipoprotein in human macrophages. The F11 receptor contains two immunoglobulin-like domains in its 236-amino-acid-long extracellular region, and has identity to the recently described junctional adhesion molecule. The data indicate that the F11 antigen is a novel receptor or cell adhesion molecule belonging to the immunoglobulin superfamily.

Nitric oxide stimulatory and endothelial protective effects of idoxifene, a selective estrogen receptor modulator, in the splanchnic artery of the ovariectomized rat.

Estrogen is known to stimulate endothelial nitric oxide production and attenuate endothelial dysfunction after ischemia and reperfusion. However, estrogen therapy increases the risk of breast and endometrial cancer. The present study was designed to determine whether idoxifene, a selective estrogen receptor modulator without adverse effects on reproductive organs, may stimulate nitric oxide release and protect endothelial function. In U-46619 precontracted superior mesenteric arterial (SMA) segments isolated from ovariectomized rats, idoxifene and 17 beta-estradiol resulted in a comparable dose-dependent vasorelaxation (maximal relaxation: 75.3 +/- 4.9 and 71 +/- 4.7%, respectively). Treatment of the rings with N(omega)-nitro-L-arginine methyl ester completely blocked idoxifene- and 17 beta-estradiol-induced vasorelaxation. In vitro incubation of SMA rings with TNF alpha significantly reduced vasorelaxation to an endothelium-dependent vasodilator, acetylcholine (maximal relaxation: 73 +/- 3.7 versus 95 +/- 2.9% pre-TNF alpha, P <.01). Idoxifene, but surprisingly not 17 beta-estradiol, prevented TNF alpha-induced endothelial dysfunction (maximal relaxation: 86 +/- 2.6% in idoxifene-treated rings and 77 +/- 5.1% in 17beta-estrogen-treated rings). In vivo ischemia and reperfusion resulted in significant endothelial dysfunction as evidenced by decreased vasorelaxation to acetylcholine (maximal relaxation: 48 +/- 5.5 versus 92 +/- 3.9% in normal SMA rings), but a normal relaxation response to an endothelium-independent vasodilator, acidified NaNO(2) (95 +/- 3.2%). Treatment with idoxifene at either 1 or 2 mg/kg/day, or 17beta-estrogen at 1 mg/kg/day for 4 days significantly preserved endothelial function (P <.01 versus vehicle). Taken together, these results demonstrate that idoxifene is an endothelium-dependent vasodilator and exerts significant endothelial protective effects against TNF alpha- and ischemia-reperfusion-induced endothelial injury. These results suggest that selective estrogen receptor modulators have therapeutic potential in diseases where endothelial dysfunction plays an important role.

Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy.

The extracellular signal-regulated kinase (ERK) pathway is activated by hypertrophic stimuli in cardiomyocytes. However, whether ERK plays an essential role or is implicated in all major components of cardiac hypertrophy remains controversial. Using a selective MEK inhibitor, U0126, and a selective Raf inhibitor, SB-386023, to block the ERK signaling pathway at two different levels and adenovirus-mediated transfection of dominant-negative Raf, we studied the role of ERK signaling in response of cultured rat cardiomyocytes to hypertrophic agonists, endothelin-1 (ET-1), and phenylephrine (PE). U0126 and SB-386023 blocked ET-1 and PE-induced ERK but not p38 and JNK activation in cardiomyocytes. Both compounds inhibited ET-1 and PE-induced protein synthesis and increased cell size, sarcomeric reorganization, and expression of beta-myosin heavy chain in myocytes with IC(50) values of 1-2 microm. Furthermore, both inhibitors significantly reduced ET-1- and PE-induced expression of atrial natriuretic factor. In cardiomyocytes transfected with a dominant-negative Raf, ET-1- and PE-induced increase in cell size, sarcomeric reorganization, and atrial natriuretic factor production were remarkably attenuated compared with the cells infected with an adenovirus-expressing green fluorescence protein. Taken together, our data strongly support the notion that the ERK signal pathway plays an essential role in ET-1- and PE-induced cardiomyocyte hypertrophy.

Application of in vivo and ex vivo magnetic resonance imaging for evaluation of tranilast on neointima formation following balloon angioplasty of the rat carotid artery.

OBJECTIVE: Recent studies suggest that tranilast inhibits a variety of agents implicated in neointimal growth and restenosis in experimental animal models and humans. We report here a study evaluating the efficacy of tranilast in the rat carotid artery balloon angioplasty model, a model that mimics many aspects of the percutaneous transluminal angioplasty procedure in humans. Efficacy was determined based on in vivo and ex vivo magnetic resonance imaging (MRI) as well as by histomorphometry. The utility of this study, using a reverse paradigm, is to investigate if agents successful in the clinic can demonstrate efficacy in this animal model primary screen as measured by MRI and histomorphometry. METHODS: Tranilast (300 mg/kg/day, p.o.) was administered to Sprague-Dawley rats 3 days prior to balloon injury and continued for 14 days after injury. Three methods of measuring the vascular injury that occurs in this model were employed: (1) in vivo MRI, used to measure in vivo lumen volumes for the carotid artery once at baseline (pre-surgery) and again at 14 days post angioplasty; (2) ex vivo MRI (and histomorphometry), used to evaluate the total arterial wall thickness and the intima-to-media ratio; and (3) analysis of collagen density, used to evaluate the efficacy of tranilast to abrogate collagen synthesis and deposition following vascular injury. RESULTS: Tranilast provided 33% protection (P<0.05) from angioplasty-induced lumen narrowing as measured by MRI in vivo. The results of the ex vivo MR analysis of total wall thickness showed a 14% protection of angioplasty-induced narrowing (P<0.05), and the mean intima-to-media ratio showed a 39% (P<0.006) protection for the tranilast-treated rats. Histological analysis of the mean intima-to-media ratio demonstrated that tranilast provided 36% (P<0. 01) protection in the intima-to-media ratio. Further, treatment with tranilast showed a 52% reduction in collagen density of the intimal layer and a 70% reduction in collagen density of the medial layer of the injured arteries. CONCLUSION: The data obtained by in vivo MRI, ex vivo MRI, histology and collagen analysis demonstrate that tranilast provided significant beneficial effects in inhibiting neointimal formation in the rat carotid artery model. Also this study, to the best of our knowledge, is the first to harness complimentary information from various technologies, including lumen patency by in vivo MRI, neointimal formation by ex vivo MRI and conventional histomorphometry, and histological analysis for collagen density, to provide a comprehensive understanding of the pathology in this disease model.

Contractile responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat.

Responses to human urotensin-II (hU-II) were investigated in human and rat pulmonary arteries. Rat pulmonary arteries: hU-II was a potent vasoconstrictor of main pulmonary arteries (2 - 3 mm i.d.) (pEC(50), 8.55+/-0.08, n=21) and was approximately 4 fold more potent than endothelin-1 [ET-1] (P<0.01), although its E(max) was considerably less (approximately 2.5 fold, P<0.001). The potency of hU-II increased 2.5 fold with endothelium removal (P<0.05) and after raising vascular tone with ET-1 (P<0.01). E(max) was enhanced approximately 1.5 fold in the presence of N(omega)-nitro-L-arginine methylester (L-NAME, 100 microM, P<0.01) and approximately 2 fold in vessels from pulmonary hypertensive rats exposed to 2 weeks chronic hypoxia (P<0.05). hU-II did not constrict smaller pulmonary arteries. Human pulmonary arteries ( approximately 250 microm i.d.): in the presence of L-NAME, 3 out of 10 vessels contracted to hU-II and this contraction was highly variable. hU-II is, therefore, a potent vasoconstrictor of rat main pulmonary arteries and this response is increased by endothelial factors, vascular tone and onset of pulmonary hypertension. Inhibition of nitric oxide synthase uncovers contractile responses to hU-II in human pulmonary arteries.

Expression of monocyte chemotactic protein-3 mRNA in rat vascular smooth muscle cells and in carotid artery after balloon angioplasty.

Monocyte chemotactic protein-3 (MCP-3) is a CC chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells and dendritic cells. The activation of the target cells by MCP-3 is via specific chemokine receptors CCR2 and CCR3, of which CCR2 is shared with MCP-1. MCP-1 and CCR2 have been implicated in vascular diseases including atherosclerosis and restenosis, that are known to be involved in inflammation (accumulation of T lymphocytes and monocytes) and smooth muscle cell (SMC) activation (proliferation, migration and matrix deposition). To investigate a potential role of MCP-3 in vascular injury, the present work examined its mRNA expression in rat aortic SMCs stimulated with various inflammatory stimuli including LPS, TNF-alpha, IL-1beta, IFN-gamma and TGF-beta. A time- and concentration-dependant induction of MCP-3 mRNA in SMCs was observed by means of Northern analysis. A strikingly similar expression profile was observed for MCP-3 and MCP-1 mRNA in SMCs. Furthermore, MCP-3 mRNA expression was induced in rat carotid artery after balloon angioplasty. A significant induction in MCP-3 mRNA was observed in the carotid artery at 6 h (41-fold increase over control, P<0.001), 1 day (13-fold increase, P<0.001) and 3 days (6-fold increase, P<0.01) after balloon angioplasty as quantitated by reverse transcription and polymerase chain reaction. These data provide evidence for the cytokine-induced expression of MCP-3 in SMCs and in carotid artery after balloon angioplasty, suggesting a potential role of MCP-3 in the pathogenesis of restenosis and atherosclerosis.

In vitro and in vivo characterization of intrinsic sympathomimetic activity in normal and heart failure rats.

Clinical studies conducted with carvedilol suggest that beta-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many beta-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective beta1-adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED50 = 5 and 40 microgram/kg, respectively) and SHHF rats (ED50 = 6 and 30 microgram/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (beta1-adrenoceptor antagonist) but not by ICI 118551 (beta2-adrenoceptor antagonist) in neonatal rat. When the beta-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting beta-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct beta1-adrenoceptor-mediated ISA in normal and heart failure rats.

Application of serial in vivo magnetic resonance imaging to evaluate the efficacy of endothelin receptor antagonist SB 217242 in the rat carotid artery model of neointima formation.

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.

Application of magnetic resonance imaging for evaluation of the efficacy of SB 217242 in neointimal formation.

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.

Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size.

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.

Signal transduction mechanisms mediating the vascular actions of endothelin.

Endothelin (ET)-1, an endothelium-derived vasoactive polypeptide encoded in the human genome, is the most potent vasoconstrictor identified to date. In addition to its acute role in modulating vascular smooth muscle tone, ET-1 also plays a critical role in the long-term control of cellular growth within the vasculature and thus, modulates the chronic remodeling of the vascular tree. In order to produce such a diverse range of biological responses, this peptide is able to activate numerous distinct effector systems including phospholipase C, phospholipase D, phospholipase A2, adenylate and guanylate cyclases and numerous cytosolic/nuclear protein kinases. These actions, mediated via an interaction with two major subtypes of cell surface seven-transmembrane receptors (ET(A) and ET(B)), are coupled to their effector systems by several distinct types of guanine nucleotide regulatory proteins (both inhibitory and stimulatory G proteins). This review describes such intercations and how distinct pharmacological agents have been used to identify the diverse signaling mechanisms utilized by the ET isopeptides.

Identification, characterization, and functional role of phosphodiesterase type IV in cerebral vessels: effects of selective phosphodiesterase inhibitors.

The role of the phosphodiesterase type IV isozyme (PDE IV) in the regulation of cerebrovascular tone was investigated in the canine basilar artery in vitro and in vivo. The PDE isozymes extracted from the canine basilar artery were isolated by diethylaminoethanol (DEAE)-Sepharose affinity chromatography and identified based on sensitivity to isozyme-selective PDE inhibitors. [3H]cAMP hydrolysis was observed in one major and one minor peak of activity. The predominant peak was inhibited by the addition of cGMP (25%), siguazodan (26%), rolipram (39%), and the combination of siguazodan and rolipram (95%). Selective PDE IV inhibitors BRL 61063, rolipram, and denbufylline were equieffective inhibitors of [3H]-ccAMP hydrolysis mediated by PDE IV isolated from the canine basilar artery [concentrations producing 50% inhibition (IC50S) = 0.21 +/- 0.05 microM, 0.67 +/- 0.23 microM, and 0.73 +/- 0.16 microM, respectively]. In precontracted isolated ring segments of the canine basilar artery, selective PDE IV inhibitors produced potent and complete relaxation (IC50S < 150 nM). In contrast, zaprinast (a selective PDE V inhibitor) and siguazodan (a selective PDE III inhibitor) produced only weak relaxation of the basilar artery (IC50S = 4.5 microM and > 10 microM, respectively). Vasorelaxation produced by PDE IV inhibitors was not altered by removing the endothelium, 1-NAME, or adenosine receptor antagonism. In a canine model of acute cerebral vasospasm, all three selective PDE IV inhibitors reversed basilar artery spasm produced by autologous blood without altering mean arterial blood pressure. In contrast, prolonged treatment with BRL 61063 failed to alter the development of basilar spasm in the two hemorrhage canine models of chronic cerebral vasospasm. Denbufylline-induced relaxation in vitro was also significantly impaired in basilar arteries obtained from the model of chronic vasospasm. In conclusion, PDE IV appears to be the predominant isozyme regulating vascular tone mediated by cAMP hydrolysis in cerebral vessels. In addition, vasorelaxation modulated by PDE IV is compromised in chronic cerebral vasospasm associated with subarachnoid hemorrhage.