Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat.

A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its "apical" position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype.

Carvedilol-liposome interaction: evidence for strong association with the hydrophobic region of the lipid bilayers.

Carvedilol (Kredex, Coreg) is a multiple action antihypertensive drug that has been shown to protect cell membranes from lipid peroxidative damages. In this study the physical and structural effects of carvedilol on lipid bilayers are investigated by fluorescence techniques, differential scanning calorimetry and other physical methods. Carvedilol binds to liposomal membranes (9:1 DMPC:DMPG) strongly with an apparent binding constant on the order of 10(4) M-1 in PBS (pH 7.4). The characteristic changes in its intrinsic fluorescence properties when bound to liposomes suggest that this compound is situated in a non-polar environment. The Stern-Volmer and bimolecular quenching constants, determined using nitrate as the fluorescence quencher, for the free and bound carvedilol indicate that the carbazole moiety is at a depth of > 11 A in the lipid bilayer. Fluorescence anisotropy measurements show that, unlike the membrane probes DPH and TMA-DPH, carvedilol is relatively mobile, and does not have a rigidly-defined molecular orientation in the bilayers. Differential scanning calorimetry results indicate that carvedilol is an effective membrane "fluidizer' as it dose-dependently lowers the gel to liquid crystalline transition temperature and broadens the endothermic transition. Comparative studies of interactions of carbazole, 4-OH carbazole and carvedilol with the model liposomal membranes reveal a possible role of membrane-partitioning in their antioxidant efficacy. These findings are discussed in perspective with the membrane biophysical properties of different classes of therapeutic significant lipid antioxidants in mind.

Monocyte chemoattractant protein-1 is upregulated in rats with volume-overload congestive heart failure.

BACKGROUND: Chemokines are potent proinflammatory and immune modulators. Increased expression of chemokines, eg, monocyte chemoattractant protein-1 (MCP-1), has recently been described in clinical and experimental heart failure. The present report is aimed at exploring the expression, localization, and binding site regulation of MCP-1, a member of the C-C chemokine family, in a rat model of volume-overload congestive heart failure (CHF). METHODS AND RESULTS: An aortocaval fistula was surgically created between the abdominal aorta and inferior vena cava. Rats with CHF were further subdivided into compensated and decompensated subgroups. Northern blot analysis and real-time quantitative polymerase chain reaction demonstrated upregulation of MCP-1 mRNA expression correlating with the severity of CHF (288+/-22, 502+/-62, and 826+/-138 copies/ng total RNA for sham, compensated, and decompensated animals, respectively; n=5, P:<0.05). MCP-1 protein was localized by immunohistochemistry in cardiomyocytes, vascular endothelium and smooth muscle cells, infiltrating leukocytes, and interstitial fibroblasts, and its intensity increased with severity of CHF. In addition, rats with CHF displayed a significant decrease of (125)I-labeled MCP-1 binding sites to myocardium-derived membranes (384.3+/-57.0, 181.3+/-8.8, and 123.3+/-14.1 fmol/mg protein for sham, compensated, and decompensated animals, respectively). CONCLUSIONS: Volume-overload CHF in rats is associated with alterations in the expression, immunohistochemical localization, and receptor binding of the MCP-1 chemokine in the myocardium. These changes were more pronounced in rats with decompensated CHF. The data suggest that activation of the MCP-1 system may contribute to the progressive cardiac decompensation and development of CHF in rats with aortocaval fistula.

Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice.

Factor XI (FXI) and factor IX (FIX) are zymogens of plasma serine proteases required for normal hemostasis. The purpose of this work was to evaluate FXI and FIX as potential therapeutic targets by means of a refined ferric chloride (FeCl(3))-induced arterial injury model in factor-deficient mice. Various concentrations of FeCl(3) were used to establish the arterial thrombosis model in C57BL/6 mice. Carotid artery blood flow was completely blocked within 10 min in C57BL/6 mice by application of 3.5% FeCl(3). In contrast, FXI- and FIX-deficient mice were fully protected from occlusion induced by 5% FeCl(3), and were partially protected against the effect of 7.5% FeCl(3). The protective effect was comparable to very high doses of heparin (1000 units kg(-1)) and substantially more effective than aspirin. While FXI and FIX deficiencies were indistinguishable in the carotid artery injury model, there was a marked difference in a tail-bleeding-time assay. FXI-deficient and wild-type mice have similar bleeding times, while FIX deficiency was associated with severely prolonged bleeding times (>5.8-fold increase, P < 0.01). Given the relatively mild bleeding diathesis associated with FXI deficiency, therapeutic inhibition of FXI may be a reasonable strategy for treating or preventing thrombus formation.

Apoptosis in cardiac diseases: stress- and mitogen-activated signaling pathways.

Apoptosis is a form of cell death that involves discrete genetic and molecular programs, de novo protein expression and a unique cellular phenotype. Evidence for the existence of apoptosis in the human heart has been reported in various cardiac diseases, including ischemic and non-ischemic heart failure, myocardial infarction and arrhythmias. Among the most potent stimuli that elicit cardiomyocyte apoptosis are: oxygen radicals (including NO), cytokines (FAS/TNF alpha-receptor signaling), stress conditions (chemical or physical, e.g., radiation), sphingolipid metabolites (ceramide) and autocoids, e.g., angiotensin II. Apoptosis of cardiac myocytes may contribute to progressive pump-failure, arrhythmias and cardiac remodeling. The recognition of numerous molecular targets associated with cardiomyocyte apoptosis may provide novel therapeutic strategies for diverse cardiac ailments, as recently suggested by pharmacologic studies in experimental animals. This review paper is aimed to highlight the role of protein kinase signaling pathways in apoptosis with special attention to the stress-activated protein kinases (SAPK) and mitogen-activated protein kinases (MAPK) systems.

The use of mRNA differential display for discovery of novel therapeutic targets in cardiovascular disease.

Recent advances in molecular biology techniques have provided powerful tools for discovery of novel genes relevant to both biological and pathological processes. mRNA differential display is an emerging technique for novel gene discovery and it has been successfully applied to many physiological and pathological conditions including normal development, cell differentiation, cancer, cardiovascular disease, inflammation and CNS disorders. In the present work, we briefly illustrate the critical procedure and highlight most recent technical improvements and modifications of this technology. Based upon the successful applications of this technique in cardiovascular research, it may provide a valuable and powerful investigational tool for the identification of novel therapeutic targets in cardiovascular diseases.

Enhanced leucocyte adhesion to interleukin-1 beta stimulated vascular smooth muscle cells is mainly through intercellular adhesion molecule-1.

OBJECTIVE: The aim was to investigate whether interleukin-1 beta (IL-1 beta) plays a role in modulating the adhesion of monocytes and neutrophils to vascular smooth muscle cells, and to identify what molecules on these cells may be involved in the adhesion. METHODS: Rat aortic smooth muscle cells were challenged with IL-1 beta and tested for adhesion of prelabelled monocytes and neutrophils. Northern analysis, reverse transcription/polymerase chain reaction (RT/PCR), and immunocytochemical staining were used to measure the changes of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in response to IL-1 beta stimulation. Neutralising antibody against ICAM-1 was used to demonstrate a role of ICAM-1 in this IL-1 beta induced adhesion. RESULTS: IL-1 beta induced the adhesion of monocytes and neutrophils to aortic smooth muscle cells in a concentration and time dependent manner. IL-1 beta-induced adhesion was inhibited by preincubation of the cells with an IL-1 receptor antagonist (IL-1ra). Northern analysis and RT/PCR showed that ICAM-1 mRNA represents a predominant adhesion molecule induced by IL-1 beta, and that the expression of ICAM-1 mRNA precedes and parallels the induced adhesion profiles of aortic smooth muscle cells for leucocytes. Immunocytochemical staining confirmed the IL-1 beta induced ICAM-1 expression on the smooth muscle cells. Moreover, a monoclonal anti-rat ICAM-1 antibody produced a concentration dependent inhibition of the IL-1 beta induced adhesion of monocytes and neutrophils to the smooth muscle cells. CONCLUSIONS: IL-1 beta actively regulates functional ICAM-1 expression in vascular smooth muscle cells. The IL-1 beta-induced expression of ICAM-1 on the smooth muscle cells may be an important contributor to the increased adhesion by monocytes and neutrophils to these cells and suggests that IL-1 beta might play a role in the proinflammatory and immune functions of the modified smooth muscle cells during atherosclerosis and restenosis.

Cardioprotective effects of carvedilol, a novel beta adrenoceptor antagonist with vasodilating properties, in anaesthetised minipigs: comparison with propranolol.

OBJECTIVE: The aim was to evaluate in a minipig model of acute myocardial infarction the cardioprotection provided by the beta adrenoceptor blocking and vasodilating activities present in carvedilol; comparison was made to the pure beta adrenoceptor antagonist, propranolol. METHODS: Experiments were performed in 25 Yucatan minipigs (9-12 kg), randomly assigned to receive vehicle (n = 7), carvedilol 0.3 mg.kg-1 (n = 6), carvedilol 1 mg.kg-1 (n = 6), or propranolol 1 mg.kg-1 (n = 6). Myocardial infarction was produced by occlusion of the left anterior descending coronary artery for 45 min followed by 4 h of reperfusion. Vehicle, carvedilol (0.3 and 1 mg.kg-1) or propranolol (1 mg.kg-1) were given intravenously 15 min before the coronary artery occlusion. At the end of the reperfusion period, infarct size was determined using Evans blue dye and triphenyltetrazolium chloride staining. Infarct volumes were visualised using computer assisted three dimensional image analysis of the stained myocardial tissue sections. Myeloperoxidase activity was measured in tissue samples removed from normal, infarcted, and at risk areas. RESULTS: Carvedilol (1 mg.kg-1) reduced infarct size by over 90% without producing pronounced changes in systemic haemodynamic variables. The ability of carvedilol to reduce infarct size was clearly dose dependent. Thus infarct size, which represented 27.5(SEM 2.3)% of the area at risk in the vehicle treated group, was only 13.1(4.0)% (p < 0.05) and 2.4(1.5)% (p < 0.01) in pigs treated with carvedilol at 0.3 and 1 mg.kg-1, respectively. In animals treated with propranolol (1 mg.kg-1), infarct size represented 10.9(2.4)% of the area at risk (p < 0.05). The 60% and 91% reductions in infarct size produced by propranolol (1 mg.kg-1) and carvedilol (1 mg.kg-1), respectively, were clearly evident upon three dimensional image analysis. The reduction in infarct size was significantly greater for carvedilol (1 mg.kg-1) compared to propranolol (1 mg.kg-1) at equivalent beta adrenoceptor blocking doses. Pretreatment with propranolol did not reduce the increases in myeloperoxidase activity observed in the area at risk or in the infarcted area. In contrast, carvedilol produced a dose dependent reduction in myeloperoxidase activity in these areas. CONCLUSIONS: Carvedilol limits myocardial necrosis resulting from coronary artery occlusion and reperfusion in a more pronounced manner than the pure beta adrenoceptor antagonist, propranolol. The cardioprotective effect of carvedilol, which reduced infarct size by 91%, may result from the combined effects of beta adrenoceptor blockade and vasodilatation, and possibly also from inhibition of intracellular calcium overload in cardiac cells resulting from antagonism of myocardial alpha 1 adrenoceptors and/or calcium channel blockade. The cardioprotection provided by carvedilol may ultimately be of benefit in hypertensive patients who are at risk for acute myocardial infarction.

Carvedilol, a new vasodilating beta adrenoceptor blocker antihypertensive drug, protects endothelial cells from damage initiated by xanthine-xanthine oxidase and neutrophils.

OBJECTIVE: Oxygen radical mediated endothelial injury plays an important role in cardiovascular disease. Carvedilol, a new beta blocker and antihypertensive agent, has been shown to have antioxidant activity. The aim of this study was to determine whether carvedilol protects oxygen radical induced endothelial injury. METHODS: Cultured bovine pulmonary artery (BPAEC) and human umbilical vein endothelial cells (HUVEC) were used and oxygen radicals were generated by xanthine-xanthine oxidase or phorbol myristate acetate (PMA) activated human neutrophils. Cell injury was assessed by lactate dehydrogenase (LDH) release and cell death, or 51 Cr release from prelabelled BPAEC. The electron paramagnetic resonance (EPR) spin trapping technique was used to detect the amount of radical spin adducts formed in cell lipids. RESULTS: Carvedilol dose dependently inhibited xanthine-xanthine oxidase induced LDH release from BPAEC and HUVEC, with IC50 values of 3.8 microM and 2.6 microM, respectively, and significantly reduced cell death by xanthine-xanthine oxidase. Other beta blockers tested (propranolol, labetalol, pindolol, and celiprolol) showed a mild effect or no effect at all. Increasing the time of pretreatment with carvedilol enhanced its cell protective effect against oxidative stress. Carvedilol also protected BPAEC dose dependently from PMA activated, neutrophil induced cell injury. Carvedilol had no effect on xanthine oxidase activity. EPR study confirmed that xanthine-xanthine oxidase induced the formation of lipid derived radicals in cell lipids and carvedilol scavenged free radicals, as indicated by the decreased EPR signal. CONCLUSIONS: Carvedilol protects endothelial cells against oxygen radical mediated cell injury and death by scavenging free radicals. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent.

Brain cooling during transient focal ischemia provides complete neuroprotection.

A review of the effects of reducing brain temperature on ischemic brain injury is presented together with original data describing the systematic evaluation of the effects of brain cooling on brain injury produced by transient focal ischemia. Male spontaneously hypertensive rate were subjected to transient middle cerebral artery occlusion (TMCAO; 80, 120 or 160 min) followed by 24 h of reperfusion. During TMCAO, the exposed skull was bathed with isotonic saline at various temperatures to control skull and deeper brain temperatures. Rectal temperature was always constant at 37 degrees C. Initial studies indicated that skull temperature was decreased significantly (i.e. to 32-33 degrees C) just as a consequence of surgical exposure of the artery. Subsequent studies indicated that maintaining skull temperature at 37 degrees C compared to 32 degrees C significantly (p < 0.05) increased the infarct size following 120 or 160 min TMCAO. In other studies, 80 min TMCAO was held constant, but deeper brain temperature could be varied by regulating skull temperature at different levels. At 36-38 degrees C brain temperature, infarct volumes of 102 +/- 10 to 91 +/- 9 mm3 occurred following TMCAO. However, at a brain temperature of 34 degrees C, a significantly (p < 0.05) reduced infarct volume of 37 +/- 10 mm3 was observed. Absolutely no brain infarction was observed if the brain was cooled to 29 degrees C during TMCAO. Middle cerebral artery exposure and maintaining brain temperature at 37 degrees C without artery occlusion did not produce any cerebral injury. These data indicated the importance of controlling brain temperature in cerebral ischemia and that reducing brain temperature during ischemia produces a brain temperature-related decrease in focal ischemic damage. Brain cooling of 3 degrees C and 8 degrees C can provide dramatic and complete, respectively, neuroprotection from transient focal ischemia. Multiple mechanisms for reduced brain temperature-induced neuroprotection have been identified and include reduced metabolic rate and energy depletion, decreased excitatory transmitter release, reduced alterations in ion flux, and reduced vascular permeability, edema, and blood-brain barrier disruption. Cerebral hypothermia is clearly the most potent therapeutic approach to reducing experimental ischemic brain injury identified to date, and this is emphasized by the present data which demonstrate complete neuroprotection in transient focal stroke. Certainly all available information warrants the evaluation of brain cooling for potential implementation in the treatment of human stroke.

The role of inflammation and cytokines in brain injury.

The original notion that the brain represented an "immune-privileged" organ lacking the capability to produce an inflammatory response to an injury, would appear no longer tenable. Indeed, accumulating evidence during the last decade has shown that the CNS can mount a well-defined inflammatory response to a variety of insults including trauma, ischemia, transplantation, viral infections, toxins as well as neurodegenerative processes. Many aspects of this centrally-derived inflammatory response parallel, to some extent, the nature of such a reaction in the periphery. Through the recent application of molecular biological techniques, new concepts are rapidly emerging as to the molecular mechanisms associated with the development of brain injury. In particular, the importance of cytokines, especially TNF alpha and IL-1 beta, as well as adhesion molecules, has been emphasized in the propagation and maintenance of a CNS inflammatory response. This review will summarize recent observations as to the involvement of these inflammatory mediators in CNS injury and lay claim to the possibility that inhibitors of peripheral inflammation may also be of benefit in treating CNS injuries such as stroke, head trauma, Alzheimer's disease and multiple sclerosis.

Genetic hypertension and increased susceptibility to cerebral ischemia.

A review of the sensitivity of genetically hypertensive rats to cerebral ischemia was presented together with original data describing the systematic comparison of the effects of focal ischemia (permanent and temporary with reperfusion) performed in hypertensive and normotensive rats (i.e., blood pressures verified in conscious instrumented rats). Microsurgical techniques were used to isolate and occlude the middle cerebral artery (MCAO) of spontaneously hypertensive (SHR), Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats at the level of the inferior cerebral vein. Following permanent (24 h) MCAO, persistent and similar decreases in local microvascular perfusion (i.e., to 15.6 +/- 1.7% of pre-MCAO levels) were verified in the primary ischemic zone of the cortex for all strains using Laser-Doppler flowmetry. A contralateral hemiplegia that occurred following MCAO, evidenced by forelimb flexion and muscle weakness, was greater in SHR (neurological grade = 2.0 +/- 0.1) than SD (1.0 +/- 0.4) or WKY (0.7 +/- 0.4) rats (N = 7-9, p less than 0.05). SHR also exhibited sensory motor deficits following MCAO compared to sham-operation, with decreased normal placement response of the hindlimb (% normal = 20 vs. 83, N = 23-30, p decreased rota-rod (41 +/- 7 vs. 126 +/- 19 on rod, N = 10-15, p less than 0.05) and balance beam (25 +/- 5 vs. 116 +/- 29 s on beam, N = 5-7, p less than 0.05) performance. However, an index of general motor activity was not affected by permanent MCAO. Triphenyltetrazolium-stained forebrain tissue analyzed by planimetry revealed a significantly larger and more consistent cortical infarction in SHR (hemispheric infarction = 27.9 +/- 1.5%) compared to SD (15.4 +/- 4.1%) and WKY (4.0 +/- 2.4%) rats (N = 7-9, p less than 0.05), occupying predominantly the frontal and parietal areas. Also, a significant degree of ipsilateral hemispheric swelling (4.6 +/- 0.9%, N = 7-9, p less than 0.05) and increased brain water content (78.4 +/- 0.3% to 80.4 +/- 0.2%, N = 8-9, p less than 0.05) was identified in SHR that was not observed in SD or WKY rats. A novel model of temporary MCAO also was evaluated in the hypertensive and normotensive rat strains. Initially, the effect of increasing MCAO-time followed by 24 h reperfusion in SHR was studied. During temporary MCAO (20 to 300 min), persistent and stable decreases in local microvascular perfusion (i.e., to 15-20% of pre-MCAO levels) were verified in the primary ischemic zones of the cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia.

Ischemic stroke is a leading cause of death and disability in developed countries. Yet, in spite of substantial research and development efforts, no specific therapy for stroke is available. Several mechanism for neuroprotection have been explored including ion channels, excitatory amino acids and oxygen radicals yet none has culminated in an effective therapeutic effect. The review article on "inflammation and stroke" summarizes key data in support for the possibility that inflammatory cells and mediators are important contributing and confounding factors in ischemic brain injury. In particular, the role of cytokines, endothelial cells and leukocyte adhesion molecules, nitric oxide and cyclooxygenase (COX-2) products are discussed. Furthermore, the potential role for certain cytokines in modulation of brain vulnerability to ischemia is also reviewed. The data suggest that novel therapeutic strategies may evolve from detailed research on some specific inflammatory factors that act in spatial and temporal relationships with traditionally recognized neurotoxic factors. The dual nature of some mediators in reformatting of brain cells for resistance or sensitivity to injury demonstrate the delicate balance needed in interventions based on anti-inflammatory strategies.

Use of suppression subtractive hybridization for differential gene expression in stroke: discovery of CD44 gene expression and localization in permanent focal stroke in rats.

BACKGROUND AND PURPOSE: CD44 is a transmembrane glycoprotein involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. The present report describes the discovery of upregulated CD44 gene expression and its spatial and temporal distribution in the brain after focal stroke. METHODS: Rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). Suppression subtractive hybridization (SSH) strategy was used to identify differentially expressed genes. Northern blotting and real-time polymerase chain reaction were used to evaluate the expression of CD44 and hyaluronan synthase 2 (HAS-2) mRNA. Western blotting and immunohistochemistry were used to examine CD44 expression and cellular distribution. RESULTS: CD44 upregulation after focal stroke was discovered by the SSH approach and confirmed by DNA sequencing. Northern blot using a pooled poly(A)+ RNA revealed 3 splice variants of CD44 mRNA, and their inducible expression started at 6 hours (5.3-fold increase over sham operation), peaked at 24 hours (28.6-fold increase), and persisted up to 72 hours (17.8-fold increase) after MCAO. A parallel induction profile of HAS-2 mRNA was observed in the ischemic brain tissue. The levels of CD44 were markedly elevated at 6 hours (1.8-fold increase over sham; n=3), 24 hours (2.9-fold, peak induction; P<0.01), and 72 hours (2.4-fold increase; P<0.05) after MCAO by means of Western analysis. Immunohistochemical and confocal microscopy confirmed that constitutive expression of CD44 is limited to microvessels in normal brain but is strongly induced after ischemia, where the immunoreactive signal mainly resided in endothelial cells and monocytes. Double-labeling immunohistochemistry demonstrated that a marked induction of CD44 in the ischemic lesion is dominantly located in microglia and a subset of macrophages. CONCLUSIONS: The discovery of concomitant induction of CD44 and HAS-2 mRNA expression and the localization of CD44 in the microglia, macrophages, and microvessels of the ischemic brain tissue suggest that an active interaction between CD44 and hyaluronan may occur and play a role in the known inflammatory response and tissue remodeling after stroke.

Effects of eprosartan on renal function and cardiac hypertrophy in rats with experimental heart failure.

Activation of the renin-angiotensin system may contribute to the derangement in renal and cardiac function in congestive heart failure. The present study evaluated the effects of eprosartan, a selective angiotensin II receptor antagonist, on renal hemodynamic and excretory parameters and on the development of cardiac hypertrophy in rats with aortocaval fistula, an experimental model of congestive heart failure. Infusion of eprosartan (1.0 mg/kg) in rats with aortocaval fistula produced a significant increase (+34%) in total renal blood flow and a sustained decrease (-33%) in the calculated renal vascular resistance. These effects on renal hemodynamics were more pronounced than those observed in sham-operated control rats and occurred despite a significant fall (-12%) in mean arterial blood pressure. Moreover, eprosartan caused a preferential increase in renal cortical blood perfusion and significantly increased glomerular filtration in rats with congestive heart failure. Chronic administration of eprosartan (5.0 mg/kg per day for 7 days through osmotic minipumps inserted intraperitoneally on the day of operation) resulted in a significant enhancement of urinary sodium excretion compared with nontreated rats with heart failure. Moreover, administration of eprosartan to salt-retaining rats with congestive heart failure resulted in a progressive increase and ultimate recovery in urinary sodium excretion. Finally, early treatment with eprosartan blocked the development of cardiac hypertrophy in rats with aortocaval fistula to a larger extent than the angiotensin-converting enzyme inhibitor enalapril. These findings emphasize the importance of angiotensin II in mediating the impairment in renal function and induction of cardiac hypertrophy in heart failure and further suggest that angiotensin II receptor blockade may be a useful treatment of these consequences in severe cardiac failure.

Antihypertensive actions of the novel nonpeptide endothelin receptor antagonist SB 209670.

Indirect evidence has implicated endothelin-1 in the pathogenesis of hypertension. In the present study we examined such a role directly with SB 209670, a novel nonpeptide endothelin receptor antagonist. The antihypertensive and hemodynamic effects of SB 209670 were examined in conscious, unrestrained spontaneously hypertensive (SHR), normotensive Wistar-Kyoto (WKY), and renin-hypertensive rats. Sustained intravenous infusion of SB 209670 (10 micrograms.kg-1.min-1 for 6 hours) produced a significant, reversible reduction in mean arterial pressure in SHR but not in WKY rats. The antihypertensive response to 10 micrograms.kg-1.min-1 SB 209670 (approximately 25 mm Hg reduction in blood pressure) was associated with bradycardia (16% decrease in heart rate) but only a minimal reduction (3%) in cardiac output, because stroke volume was evaluated (by 15%). Therefore, the antihypertensive effect of SB 209670 resulted from a decrease (13%) in total peripheral resistance. A sustained antihypertensive effect could also be observed after intraduodenal administration of SB 209670 (3 mg/kg) in conscious SHR (reduction of approximately 35 mm Hg 5 hours after administration). SB 209670 (3 mg/kg intravenous bolus) did not alter the pressor response or tachycardia observed in pithed SHR after stimulation of thoracolumbar sympathetic outflow. SB 209670 was also antihypertensive in renin-hypertensive rats, lowering blood pressure to an extent similar to that observed in SHR. Thus, the data presented provide evidence to support a role for endothelin-1 in the pathophysiology of two animal models of hypertension.

Cardiovascular and sympathetic responses to chronic arachidonate in SHR and WKY rats.

In rats between the ages of 4 and 12 or 14 weeks, repeated daily subcutaneous administration of arachidonate (AA) at a dose of 50 or 200 mg/kg significantly retarded the development of hypertension in spontaneously hypertensive rats (SHR) but did not alter the normal age-related increase in blood pressures (BP) of normotensive (WKY) rats. Heart rates (HR) and plasma levels of norepinephrine (NE), but not epinephrine, were lower in AA-treated SHR than in saline-treated animals. AA-treated SHR and WKY gained less weight than the saline-treated controls. In pithed AA-treated SHR, stimulation of the sympathetic outflow (50 V, for 1 minute at 0.3 or 3.0 Hz) and intravenous administration of NE (0.3 or 3.0 g/kg) evoked smaller pressor responses than in saline-treated controls, but the stimulation-evoked increases in plasma catecholamines were unchanged by AA treatment. These results indicated that, in SHR, chronic AA treatment reduces BP by mechanisms that do not directly affect NE release from sympathetic nerves. There appears to be both reduced central nervous system activation of the sympathetic outflow and diminished responses to peripheral sympathetic stimulation and exogeneous NE which may be secondary to the reduced vascular hypertrophy that usually accompanies the development of high BP in SHR.

Carvedilol, a new antihypertensive agent, prevents lipid peroxidation and oxidative injury to endothelial cells.

The protective effects of carvedilol, a new beta-adrenergic receptor blocker and vasodilating antihypertensive agent, against oxygen free radical-mediated injury were studied in cultured bovine endothelial cells and compared with five other beta-blockers. Carvedilol dose-dependently inhibited oxygen radical-induced lipid peroxidation (50% inhibition at 2.6 mumol/L) and glutathione depletion (50% inhibition at 1.8 mumol/L) in the cells. Under the same conditions, other beta-blockers--propranolol, labetalol, pindolol, atenolol, and celiprolol--had only mild or no effect. Moreover, carvedilol protected against oxygen radical--mediated cell damage, as assessed by cellular lactate dehydrogenase release, with a 50% inhibition at 4.1 mumol/L and increased the cell survival in a dose-dependent manner, whereas other beta-blockers had mild or no effects. Pretreatment of the cells with carvedilol for 7 days significantly enhanced the protective effects of carvedilol. Using 2-methyl-2-nitrosopropane as a trapping agent, the spin adduct in cell lipids was monitored by electron paramagnetic resonance. Carvedilol dose-dependently decreased the intensity of the free radical signals, indicating its free radical-scavenging ability. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent.

Inflammatory mediators and stroke: new opportunities for novel therapeutics.

Contrary to previous dogmas, it is now well established that brain cells can produce cytokines and chemokines, and can express adhesion molecules that enable an in situ inflammatory reaction. The accumulation of neutrophils early after brain injury is believed to contribute to the degree of brain tissue loss. Support for this hypothesis has been drawn from many studies where neutrophil-depletion blockade of endothelial-leukocyte interactions has been achieved by various techniques. The inflammation reaction is an attractive pharmacologic opportunity, considering its rapid initiation and progression over many hours after stroke and its contribution to evolution of tissue injury. While the expression of inflammatory cytokines that may contribute to ischemic injury has been repeatedly demonstrated, cytokines may also provide "neuroprotection" in certain conditions by promoting growth, repair, and ultimately, enhanced functional recovery. Significant additional basic work is required to understand the dynamic, complex, and time-dependent destructive and protective processes associated with inflammation mediators produced after brain injury. The realization that brain ischemia and trauma elicit robust inflammation in the brain provides fertile ground for discovery of novel therapeutic agents for stroke and neurotrauma. Inhibition of the mitogen-activated protein kinase (MAPK) cascade via cytokine suppressive anti-inflammatory drugs, which block p38 MAPK and hence the production of interleukin-1 and tumor necrosis factor-alpha, are most promising new opportunities. However, spatial and temporal considerations need to be exercised to elucidate the best opportunities for selective inhibitors for specific inflammatory mediators.

Detection of tumor necrosis factor-alpha mRNA induction in ischemic brain tolerance by means of real-time polymerase chain reaction.

A short duration of ischemia (i.e., ischemic preconditioning) results in significant brain protection to subsequent severe ischemic insult. Because previous studies suggest that tumor necrosis factor-alpha (TNF-alpha) plays a role in both promoting ischemic damage and neuroprotection, the present work aimed to evaluate the expression of TNF-alpha mRNA in an established model of ischemic preconditioning using a transient 10-minute occlusion of the middle cerebral artery. Because the level of TNF-alpha mRNA expression in the brain was too low to be consistently detected by Northern technique, a real-time polymerase chain reaction method was applied to quantitate the absolute copy number of TNF-alpha transcript in rat brain after the preconditioning procedure. TNF-alpha mRNA was induced in the ipsilateral cortex as early as 1 hour (27 +/- 1 copies of mRNA per microgram of tissue compared to 11 +/- 3 copies in sham-operated samples) after preconditioning, reached a peak level at 6 hours (49 +/- 10 copies of transcript, n = 4, P < 0.01), and persisted up to 2 days. These data not only demonstrate the utility of real-time polymerase chain reaction for sensitive and accurate measurement of mRNA expression in normal and injured tissues but also suggest a potential role of TNF-alpha in the phenomenon of ischemic preconditioning.