P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.

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.

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.

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.

TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease.

TL1 is a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family. TL1 is abundantly expressed in endothelial cells, but its function is not known. The present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 72 ng/ml). The effect of TL1 was not inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas expression in BPAEC at 8 and 24 h after treatment, and significantly activated stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK). The peak activities of SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold, respectively. TL1-induced apoptosis in the BPAEC was reduced by expression of a dominant-interfering mutant of c-Jun (62.8%, p < 0.05) or by a specific p38 inhibitor, SB203580 (1-10 microM) dose-dependently. TL1 also activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was significantly attenuated by the caspase inhibitor, ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC was caspase-3, which was based on substrate specificity and immunocytochemical analysis. These findings suggest that TL1 may act as an autocrine factor to induce apoptosis in endothelial cells via activation of multiple signaling pathways, including stress protein kinases as well as certain caspases.

Prolonged expression of interferon-inducible protein-10 in ischemic cortex after permanent occlusion of the middle cerebral artery in rat.

Focal cerebral ischemia elicits local inflammatory reaction as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain. Interferon-inducible protein-10 (IP-10) is a member of the C-X-C chemokine family that possesses potent chemoattractant actions for monocytes, T cells, and smooth muscle cells. To investigate a potential role of IP-10 in focal stroke, we studied the temporal expression of IP-10 mRNA after occlusion of the middle cerebral artery in rat by means of northern analysis. IP-10 mRNA expression after focal stroke demonstrated a unique biphasic profile, with a marked increase early at 3 h (4.9-fold over control; p < 0.01), a peak level at 6 h (14.5-fold; p < 0.001) after occlusion of the middle cerebral artery, and a second wave induction 10-15 days after ischemic injury (7.2- and 9.3-fold increase for 10 and 15 days, respectively; p < 0.001). In situ hybridization confirmed the induced expression of IP-10 mRNA and revealed its spatial distribution after focal stroke. Immunohistochemical studies demonstrated the expression of IP-10 peptide in neurons (3-12 h) and astroglial cells (6 h to 15 days) of the ischemic zone. To explore further the potential role of IP-10 in focal stroke, we demonstrated a dose-dependent chemotactic action of IP-10 on C6 glial cells and enhanced attachment of rat cerebellar granule neurons. Taken together, the data suggest that ischemia induces IP-10, which may play a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke.

Staurosporine-induced apoptosis in cardiomyocytes: A potential role of caspase-3.

Cardiomyocyte apoptosis has been demonstrated in animal models of cardiac injury as well as in patients with congestive heart failure or acute myocardial infarction. Therefore, apoptosis has been proposed as an important process in cardiac remodeling and progression of myocardial dysfunction. However, the mechanisms underlying cardiac apoptosis are poorly understood. The present study was designed to determine whether the family of caspase proteases and stress-activated protein kinase (SAPK/JNK) are involved in cardiac apoptosis. Cultured rat neonatal cardiac myocytes were treated with staurosporine to induce apoptosis as evidenced by the morphological (including ultrastructural) characteristics of cell shrinkage, cytoplasmic and nuclear condensation, and fragmentation. Nucleosomal DNA fragmentation in myocytes was further identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end-labeling (TUNEL). Staurosporine-induced apoptosis in myocytes was a time- and concentration-(0.25-1 micro M)-dependent process. Staurosporine-induced apoptosis in myocytes was reduced by a cell-permeable, irreversible tripeptide inhibitor of caspases, ZVAD-fmk, but not by the ICE-specific inhibitor, Ac-YVAD-CHO. At 10, 50 and 100 muM of ZVAD-fmk, staurosporine-induced myocyte apoptosis was reduced by 5.8, 39.1 (P<0.01) and 53.8% (P<0.01), respectively. Staurosporine, at 0.25-1 micro M, increased caspase activity in cardiomyocytes by five- to eight-fold, peaking at 4-8 h after stimulation. Based on substrate specificity analysis, the major component of caspases activated in myocytes was consistent with caspase-3 (CPP32). Moreover, the appearance of the 17-kD subunit of active caspase-3 in staurosporine-treated myocytes was demonstrated by immunocytochemical analysis. In contrast, staurosporine induced a rapid and transient inhibition of SAPK/JNK in myocytes. The SAPK activity in myocytes was reduced by 68.3 and 58.3% (P<0.01 v basal) at 10 and 30 min after treatment with 1 micro M of staurosporine, respectively. Our results suggest that staurosporine-induced cardiac myocyte apoptosis involves activation of caspases, mainly caspase-3, but not activation of the SAPK signaling pathway.

Carvedilol inhibits activation of stress-activated protein kinase and reduces reperfusion injury in perfused rabbit heart.

Stress-activated protein kinase (SAPK/JNK) has been implicated in the signaling pathway that leads to cell death. Carvedilol, a new vasodilating beta-adrenoceptor antagonist with potent antioxidant activity, has been shown to convey a high degree of cardioprotection in a variety of experimental models of myocardial ischemia as well as in patients with congestive heart failure. The present study was designed to explore whether the cardioprotective effects of carvedilol involve inhibition of SAPK activation. Ex vivo ischemia (30 min)-reperfusion (60-120 min) of the rabbit heart resulted in 67% reduction of pressure-rate product, 45% necrosis of left ventricular tissue and 62% loss of myocardial creatine kinase (P < 0.01 vs. basal). SAPK levels in the perfused hearts increased markedly following reperfusion (5.6-fold increase, P < 0.01 vs. basal). Carvedilol, at 10 microM, administered at time of reperfusion, enhanced recovery of pressure-rate product by 61%, reduced necrotic size by 65% and decreased myocardial creatine kinase loss by 62% (P < 0.01 vs. vehicle). Carvedilol also inhibited reperfusion-induced activation of SAPK by 61% (P<0.01 vs. vehicle). Carvedilol, at 1 microM, displayed a trend of cardioprotection and inhibition of SAPK activation. Our results suggest that SAPK may play a role in ischemia/reperfusion-induced cardiac injury and inhibition of SAPK activation by carvedilol may contribute to its cardioprotective effects.

Possible involvement of stress-activated protein kinase signaling pathway and Fas receptor expression in prevention of ischemia/reperfusion-induced cardiomyocyte apoptosis by carvedilol.

Carvedilol, a new vasodilating beta-adrenoceptor antagonist and a potent antioxidant, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Recent clinical studies in patients with heart failure have demonstrated that carvedilol reduces morbidity and mortality and inhibits cardiac remodeling. The present study was designed to explore whether the protective effects of carvedilol on the ischemic myocardium include inhibition of apoptosis of cardiomyocytes and, if so, to determine its mechanism of action. Anesthetized rabbits were subjected to 30 minutes of coronary artery occlusion followed by 4 hours of reperfusion. Detection of apoptosis of cardiomyocytes was based on the presence of nucleosomal DNA fragments on agarose gels (DNA ladder) and in situ nick end labeling. Carvedilol (1 mg/kg IV), administered 5 minutes before reperfusion, reduced the number of apoptotic myocytes in the ischemic area from 14.7 +/- 0.4% to 3.4 +/- 1.8% (77% reduction, P<.001). Propranolol, administered at equipotent beta-blocking dosage, reduced the number of apoptotic myocytes to 8.9 +/- 2.1% (39% reduction, P<.05). DNA ladders were observed in the hearts of all six vehicle-treated rabbits but only one of six carvedilol-treated rabbits (P<.01). Immunocytochemical analysis of rabbit hearts demonstrated an upregulation of Fas protein in ischemic cardiomyocytes, and treatment with carvedilol reduced both the intensity of staining as well as the area stained. Myocardial ischemia/reperfusion led to a rapid activation of stress-activated protein kinase (SAPK) in the ischemic area but not in nonischemic regions. SAPK activity was increased from 2.1 +/- 0.3 mU/mg (basal) to 8.9 +/- 0.8 mU/mg after 30 minutes of ischemia followed by 20 minutes of reperfusion. Carvedilol inhibited the activation of SAPK by 53.4 +/- 6.5% (P<.05). Under the same conditions, propranolol (1 mg/kg) had no effect on SAPK activation. Taken together, these results suggest that carvedilol prevents myocardial ischemia/reperfusion-induced apoptosis in cardiomyocytes possibly by downregulation of the SAPK signaling pathway, by inhibition of Fas receptor expression, and by beta-adrenergic blockade. The former two actions represent novel and important mechanisms that may contribute to the cardioprotective effects of carvedilol.

2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic fibroblast growth factor, insulin-like growth factor, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

Hypercholesterolemia impairs a detoxification mechanism against peroxynitrite and renders the vascular tissue more susceptible to oxidative injury.

Previous studies have shown that glutathione (GSH) plays a central role in the protection against peroxynitrite (ONOO-) toxicity. The present study evaluated the changes of the GSH cytoprotective system against ONOO- in hypercholesterolemia and determined the effects of carvedilol, a beta-blocker with free radical-scavenging activity, on these hypercholesterol-induced changes. New Zealand White rabbits were fed either a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with either carvedilol or propranolol. Eight weeks later, the rabbits were killed, and the thoracic aortas were isolated. Total GSH content of aortic tissue, vasorelaxation response of aortic rings to exogenous ONOO-, No regeneration from ONOO- by aortic homogenate, and ONOO(-)-induced aortic tissue injury were examined. Hypercholesterolemia decreased tissue GSH content (0.52 +/- 0.08 versus 0.86 +/- 0.04 mumol/g in control, P < .01), attenuated the vasorelaxation response to ONOO- (40 +/- 4.1% versus 76 +/- 3.2%, P < .01), reduced NO regeneration from ONOO- (387 +/- 40 versus 662 +/- 51 pmol, P < .01), and potentiated ONOO(-)-induced vascular tissue injury (37 +/- 4.4% versus 14 +/- 2.6% of increase in lactate dehydrogenase release after 3-morpholinosydnonimine exposure, P < .01). Treatment of the hypercholesterolemic rabbits with carvedilol, but not propranolol, significantly preserved tissue GSH content (0.79 +/- 0.05 mumol/g, P < .01 versus nontreated hypercholesterolemic rabbits), restored the vasorelaxation to ONOO- (61 +/- 2%, P < .01), increased NO regeneration from ONOO- (583 +/- 39 pmol, P < .01), and attenuated ONOO(-)-induced tissue injury (19 +/- 1.8%, P < .01). These results suggest that hypercholesterolemia impairs the GSH-mediated detoxification mechanism against ONOO- and renders the vascular tissue more susceptible to oxidative injury. Carvedilol, a novel vasodilating beta-blocker with antioxidant activity, significantly preserved this self-defense system and protected tissue from oxidant injury.

Interferon-inducible protein-10 involves vascular smooth muscle cell migration, proliferation, and inflammatory response.

Interferon-inducible protein-10 (IP-10) is a member of the C-X-C chemokine family. Using mRNA differential display, we isolated a rat homologue to murine and human IP-10 from lipopolysaccharide-stimulated carotid arteries. Our studies demonstrated that IP-10 is a potent mitogenic and chemotactic factor for vascular smooth muscle cells, the critical features of smooth muscle cells for their contribution to the pathogenesis of atherosclerosis and restenosis. IP-10 induced a concentration-dependent stimulation of DNA synthesis, cell proliferation, and cell migration of rat aortic smooth muscle cells. A concentration- and time-dependent IP-10 mRNA induction was observed in lipopolysaccharide- or interferon-gamma-stimulated, but not interleukin-1beta- or tumor necrosis factor-alpha-stimulated smooth muscle cells. A marked synergistic effect on IP-10 mRNA expression was observed when smooth muscle cells were challenged with interferon-gamma together with interleukin-1beta or tumor necrosis factor-alpha. Furthermore, IP-10 mRNA expression was induced in the rat carotid artery after balloon angioplasty. The mitogenic and chemotactic features of IP-10 for smooth muscle cells, along with its discrete induction in cultured vascular smooth muscle cells and in carotid arteries after balloon angioplasty (neointima formation) suggest that IP-10 may play an active and distinct role in vascular remodeling processes.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Demonstration of increased endothelial-leukocyte adhesion molecule-1 mRNA expression in rat ischemic cortex.

BACKGROUND AND PURPOSE: Leukocyte infiltration from circulating blood into ischemic brain tissue contributes significantly to ischemic injury. The role of adhesion molecules in leukocyte attachment and infiltration in ischemic tissue has been emphasized. The aim of the present study was to evaluate whether endothelial-leukocyte adhesion molecule-1 (ELAM-1 or E-selectin) mRNA expression is upregulated in focal brain ischemia. METHODS: Northern blot analysis with the use of poly(A) RNA isolated from the ischemic and nonischemic rat cortex at 2 and 12 hours after permanent occlusion of the middle cerebral artery (PMCAO) was used to examine ELAM-1 mRNA expression. The temporal expression profile of ELAM-1 mRNA in the ischemic cortex was further evaluated with the use of a quantitative reverse transcription and polymerase chain reaction technique. RESULTS: A very low level of ELAM-1 mRNA was detected in the sham-operated cortex or in the nonischemic cortex. The expression of ELAM-1 mRNA in the focal ischemic cortex was significantly induced by PMCAO, reaching a peak level at 12 hours (6.9-fold increase compared with sham surgery cortical samples, P < .01) and remained elevated for up to 2 days (3.3-fold increase, P < .01) after PMCAO. CONCLUSIONS: The demonstration of upregulated ELAM-1 mRNA expression after focal stroke suggests that ELAM-1 may play an important role in leukocyte infiltration into the ischemic brain and that ELAM-1 may provide a potential therapeutic target in ischemic stroke. However, the demonstration of translated ELAM-1 and its cellular localization in the ischemic tissue is required when specific antibodies become available.

Expression of interleukin-6, c-fos, and zif268 mRNAs in rat ischemic cortex.

The expression of interleukin-6 (IL-6) mRNA in the focal ischemic rat cortex was studied by means of Northern hybridization. IL-6 mRNA was induced after permanent occlusion of the middle cerebral artery, reached a significant level at 3 h, and peaked at 12 h, i.e., approximately 10-fold increase in the ischemic zone compared with the nonischemic cortex or sham-operated controls. The increased IL-6 mRNA was elevated for at least 24 h. Low levels of IL-6 mRNA were detected in sham-operated rats or in the contralateral nonischemic cortex. The expression of c-fos and zif268 mRNAs, two early response genes, was rapid (increased by 1 h postischemia) and transient (returned to basal levels by 24 and 12 h, respectively), clearly having different kinetic patterns from that of IL-6 mRNA. The early response kinetic pattern of c-fos and zif268 mRNAs in focal ischemia suggests their transcriptional regulatory roles in response to ischemic insult, while the delayed induction pattern of IL-6 mRNA suggests a role for this pleiotropic cytokine in the inflammatory response to the focal ischemic damage of the brain.

Transforming growth factor-beta 1 exhibits delayed gene expression following focal cerebral ischemia.

Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotropic peptide growth factor. The expression of TGF-beta 1 mRNA in the focal ischemic cortex of rats was studied by means of Northern hybridization. A moderately low level of constitutively expressed TGF-beta 1 mRNA was detected following sham-surgery or in the contralateral (nonischemic) cortex. A significant increase of TGF-beta 1 mRNA level in the ischemic cortex was observed at 2 days (3.2-fold increase compared to sham-operated animals, p < 0.01, n = 4) following permanent occlusion of the middle cerebral artery (PMCAO). The elevated TGF-beta 1 mRNA expression was plateaued for up to 15 days (3.6-fold increase, p < 0.01) following PMCAO. This temporal profile for TGF-beta 1 mRNA expression in focal stroke was significantly delayed compared to that of TNF-alpha, IL-1 beta and IL-6 mRNA expressions as demonstrated previously which peaked at 12 h and decreased to almost basal levels by 5 days following PMCAO. Interestingly, the TGF-beta 1 mRNA expression profile was remarkably parallel with that of monocyte/macrophage accumulation in the ischemic cortex, as well as with the increased formation of extracellular matrix in the focal ischemic brain. These data suggest that TGF-beta 1 may play a role in anti-inflammatory process and in tissue remodeling following ischemic brain injury.

Neuroprotective effects of tetrodotoxin as a Na+ channel modulator and glutamate release inhibitor in cultured rat cerebellar neurons and in gerbil global brain ischemia.

BACKGROUND AND PURPOSE: Studies examining the role of tetrodotoxin-sensitive ion channels in hypoxic-ischemic neuronal damage have concluded that sodium influx is an important initiating event. We examined the neuroprotectant effect of tetrodotoxin on both cultured cerebellar neurons and on CA1 hippocampal neurons of gerbils exposed to brain ischemia. METHODS: We studied neuroprotective mechanisms using cultured rat cerebellar granule cells exposed to veratridine, which induced cytotoxicity, neurotransmitter release, and calcium influx. Survival of gerbil CA1 neurons was examined by direct neuron counts 7 days after 6 minutes of global ischemia with reperfusion. RESULTS: Tetrodotoxin protected cultured neurons in a dose-dependent manner from veratridine-induced toxicity (protective concentration [PC50] = 22 nmol/L). Veratridine induced [3H]aspartate efflux that was sodium dependent, only 25% calcium dependent, and was inhibited by tetrodotoxin (inhibitory concentration [IC50] = 60 nmol/L). Veratridine initiated increases in intracellular calcium that were also reversed by tetrodotoxin (IC50 = 63 nmol/L); reversal was dependent on the sodium-calcium exchanger and the sodium-potassium pump. Neuroprotection of 90% (n = 10; P = .001 versus vehicle) of gerbil CA1 hippocampal neurons was achieved by pretreatment with 2 ng of tetrodotoxin delivered three times intracerebroventricularly, without causing hypothermia. CONCLUSIONS: Sodium channel blockers like tetrodotoxin may have utility in treatment of ischemic neuronal injury by preventing excessive neuronal depolarizations, limiting excitotoxic glutamate release through reversal of the sodium-dependent glutamate transporter, preventing intracellular calcium overload, preserving cellular energy stores, and allowing recovery of ionic homeostasis through operation of the sodium-calcium exchanger.

Concomitant cortical expression of TNF-alpha and IL-1 beta mRNAs follows early response gene expression in transient focal ischemia.

The expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) mRNAs was significantly increased in the rat ischemic cortex following temporary occlusion of the middle cerebral artery (TMCAO) with reperfusion. Northern blot analysis demonstrated that the induction of TNF-alpha and IL-1 beta mRNAs occurred as early as 1 h after reperfusion, exhibiting a 4.6-fold increase (p < 0.05, n = 4) and 6.8-fold increase (p < 0.05, n = 4) in the ischemic cortex over control, respectively. TNF-alpha mRNA reached its peak at 3 h (8.0-fold, p < 0.05), whereas IL-1 beta mRNA reached its peak at 6 h (29.5-fold, p < 0.05). Both cytokine mRNA levels remained elevated for up to 2 d after reperfusion. In contrast to the time course of these cytokine mRNAs, c-fos and zif268 mRNAs, two early response genes, displayed a greater and earlier time-response profile. The early induction of c-fos and zif268 mRNAs in temporary brain ischemia with reperfusion suggests their roles in transcriptional regulation. The later concomitant expression of TNF-alpha and IL-1 beta suggests that these cytokines play an important role in the inflammatory response associated with focal ischemia.