Prevention and intervention studies with telmisartan, ramipril and their combination in different rat stroke models.

OBJECTIVES: The effects of AT1 receptor blocker, telmisartan, and the ACE inhibitor, ramipril, were tested head-to head and in combination on stroke prevention in hypertensive rats and on potential neuroprotection in acute cerebral ischemia in normotensive rats. METHODS: Prevention study: Stroke-prone spontaneously hypertensive rats (SHR-SP) were subjected to high salt and randomly assigned to 4 groups: (1) untreated (NaCl, n = 24), (2) telmisartan (T; n = 27), (3) ramipril (R; n = 27) and (4) telmisartan + ramipril (T+R; n = 26). Drug doses were selected to keep blood pressure (BP) at 150 mmHg in all groups. Neurological signs and stroke incidence at 50% mortality of untreated SHR-SP were investigated. Intervention study: Normotensive Wistar rats were treated s.c. 5 days prior to middle cerebral artery occlusion (MCAO) for 90 min with reperfusion. Groups (n = 10 each): (1) sham, (2) vehicle (V; 0.9% NaCl), (3) T (0.5 mg/kg once daily), (4) R (0.01 mg/kg twice daily), (5) R (0.1 mg/kg twice daily) or (6) T (0.5 mg/kg once daily) plus R (0.01 mg/kg twice daily). Twenty-four and 48 h after MCAO, neurological outcome (NO) was determined. Forty-eight h after MCAO, infarct volume by MRI, neuronal survival, inflammation factors and neurotrophin receptor (TrkB) were analysed. RESULTS: Stroke incidence was reduced, survival was prolonged and neurological outcome was improved in all treated SHR-SP with no differences between treated groups. In the acute intervention study, T and T+R, but not R alone, improved NO, reduced infarct volume, inflammation (TNFα), and induced TrkB receptor and neuronal survival in comparison to V. CONCLUSIONS: T, R or T+R had similar beneficial effects on stroke incidence and NO in hypertensive rats, confirming BP reduction as determinant factor in stroke prevention. In contrast, T and T+R provided superior neuroprotection in comparison to R alone in normotensive rats with induced cerebral ischemia.

Novel therapy approach in primary stroke prevention: simultaneous inhibition of endothelin converting enzyme and neutral endopeptidase in spontaneously hypertensive, stroke-prone rats improves survival.

OBJECTIVES: Stroke, frequently a consequence of hypertension, is one of the leading causes of death and neurological disabilities worldwide. In the ischemic brain, levels of endothelin-1, one of the most potent vasoconstrictors, are raised. Anti-inflammatory and neuroprotective effects of endothelin antagonists after stroke have been described in literature. Based on these findings, we investigated the protective effect of the endothelin converting enzyme/neutral endopeptidase blocker, SLV 338, in salt-loaded, stroke-prone, spontaneously hypertensive rats. METHODS: Male, 8-week-old spontaneously hypertensive stroke-prone rats were put on a high salt diet and treated with either 30 mg/kg or 100 mg/kg SLV 338 or vehicle for 27 weeks. Blood pressure, neurological outcome, body weight, and mortality were investigated throughout treatment. In weeks 1 and 9, animals were housed in metabolic cages for collection of urinary and blood samples and assessment of salt water and food intake. In weeks 22 and 27, additional blood samples were taken. At the end of the study, all brains were analyzed using magnetic resonance imaging. RESULTS: SLV 338 was well tolerated in all animals. Neurological outcome and infarct size were similar in all groups. Albuminuria was considerably delayed and the incidence of stroke significantly lowered in treated animals. In spontaneously hypertensive stroke-prone rats, treatment with SLV 338 significantly (P = 0·01) improved survival in comparison to the vehicle treated group in a blood pressure-independent manner. DISCUSSION: Our data in spontaneously hypertensive stroke-prone rats demonstrate that combined endothelin converting enzyme/neutral endopeptidase inhibition could offer a new therapeutic approach for primary stroke prevention and improvement of mortality. The mechanism seems to be blood pressure-independent.

Telmisartan prevents aneurysm progression in the rat by inhibiting proteolysis, apoptosis and inflammation.

OBJECTIVES: We investigated the effects of treatment with the angiotensin II type 1 receptor antagonist, telmisartan, on abdominal aortic aneurysm formation in normotensive rats. METHODS: Abdominal aortic aneurysm was induced by perfusion of an isolated aortic segment with elastase. Treatment with telmisattan (0.5 mg/kg per day) or hydralazine (15 mg/kg per day) was started after surgery and continued for 14 days. Sham-operated animals and vehicle-treated animals after aneurysm induction served as controls. Aortic diameter was measured using ultrasound before aneurysm induction and on days 7 and 14 after aneurysm induction. RESULTS: On day 14, aortic diameter was increased two-fold in the vehicle-treated group compared to sham-operated animals (2.02 +/- 0.12 vs. 0.87 +/- 0.02 mm, P < 0.005, n = 8). Telmisartan treatment significantly reduced aneurysmal size (1.65 +/- 0.06 vs. 2.02 +/- 0.12 mm in vehicle, P < 0.05, n = 8), whereas treatment with hydralazine had no effect. Matrix metallopeptidase 3, cathepsin D, nuclear factor kappa B, tumour necrosis factor alpha, transforming growth factor-1 beta, as well as caspase 3, p53 and Fas ligand proteins, were significantly downregulated in aortic tissue under telmisartan compared to vehicle treatment. Serum monocyte chemoattractant protein 1 levels were also significantly decreased. Telmisartan and hydralazine reduced blood pressure to a similar extent within the observation period. CONCLUSION: The angiotensin II type 1 receptor antagonist, telmisartan, prevents abdominal aortic aneurysm progression independently of blood pressure reduction by inhibiting proteolysis, apoptosis and inflammation in aortic tissue.

The beta-lactam antibiotic, ceftriaxone, dramatically improves survival, increases glutamate uptake and induces neurotrophins in stroke.

OBJECTIVE: Ceftriaxone has been reported to reduce neuronal damage in amyotrophic lateral sclerosis and in an in-vitro model of neuronal ischaemia through increased expression and activity of the glutamate transporter, GLT1. We tested the effects of ceftriaxone on mortality, neurological outcome, and infarct size in experimental stroke in rats and looked for underlying mechanisms. METHODS: Male normotensive Wistar rats received ceftriaxone (200 mg/kg intraperitoneal) as a single injection 90 min after middle cerebral artery occlusion (90 min with reperfusion). Forty-eight hours after middle cerebral artery occlusion, infarct size (MRI) and neurological deficits were estimated. GLT1 expression was determined by real time RT-PCR, immunoblotting and promoter reporter assay, astrocyte GLT1 activity by measuring glutamate uptake. Bacterial load in various organs was measured by real time RT-PCR, neurotrophins and IL-6 by immunoblotting. RESULTS: Ceftriaxone dramatically reduced early (24-h) mortality from 34.5% (vehicle treatment, n = 29) to 0% (P < 0.01, n = 19). In a subgroup, followed up for 4 weeks, mortality persisted at 0%. Ceftriaxone strongly tended to reduce infarct size, it significantly improved neuronal survival within the penumbra, reduced neurological deficits (P < 0.001) and led to an upregulation of neurotrophins (P < 0.01) in the peri-infarct zone. Ceftriaxone did not increase GLT1 expression, but increased GLT1 activity (P < 0.05). CONCLUSION: Ceftriaxone causes a significant reduction in acute stroke mortality in a poststroke treatment regimen in animal studies. Improved neurological performance and survival may be due to neuroprotection by activation of GLT1 and a stimulation of neurotrophins resulting in an increased number of surviving neurons in the penumbra.

Metabolic actions of estrogen receptor beta (ERbeta) are mediated by a negative cross-talk with PPARgamma.

Estrogen receptors (ER) are important regulators of metabolic diseases such as obesity and insulin resistance (IR). While ERalpha seems to have a protective role in such diseases, the function of ERbeta is not clear. To characterize the metabolic function of ERbeta, we investigated its molecular interaction with a master regulator of insulin signaling/glucose metabolism, the PPARgamma, in vitro and in high-fat diet (HFD)-fed ERbeta -/- mice (betaERKO) mice. Our in vitro experiments showed that ERbeta inhibits ligand-mediated PPARgamma-transcriptional activity. That resulted in a blockade of PPARgamma-induced adipocytic gene expression and in decreased adipogenesis. Overexpression of nuclear coactivators such as SRC1 and TIF2 prevented the ERbeta-mediated inhibition of PPARgamma activity. Consistent with the in vitro data, we observed increased PPARgamma activity in gonadal fat from HFD-fed betaERKO mice. In consonance with enhanced PPARgamma activation, HFD-fed betaERKO mice showed increased body weight gain and fat mass in the presence of improved insulin sensitivity. To directly demonstrate the role of PPARgamma in HFD-fed betaERKO mice, PPARgamma signaling was disrupted by PPARgamma antisense oligonucleotide (ASO). Blockade of adipose PPARgamma by ASO reversed the phenotype of betaERKO mice with an impairment of insulin sensitization and glucose tolerance. Finally, binding of SRC1 and TIF2 to the PPARgamma-regulated adiponectin promoter was enhanced in gonadal fat from betaERKO mice indicating that the absence of ERbeta in adipose tissue results in exaggerated coactivator binding to a PPARgamma target promoter. Collectively, our data provide the first evidence that ERbeta-deficiency protects against diet-induced IR and glucose intolerance which involves an augmented PPARgamma signaling in adipose tissue. Moreover, our data suggest that the coactivators SRC1 and TIF2 are involved in this interaction. Impairment of insulin and glucose metabolism by ERbeta may have significant implications for our understanding of hormone receptor-dependent pathophysiology of metabolic diseases, and may be essential for the development of new ERbeta-selective agonists.

Comparison between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain.

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively (P<0.05-0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P<0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFalpha in vivo (P<0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.

Candesartan but not ramipril pretreatment improves outcome after stroke and stimulates neurotrophin BNDF/TrkB system in rats.

OBJECTIVES: Drugs interfering with the renin-angiotensin system (RAS) have been shown to reduce the incidence of stroke in patients at risk and to afford neuroprotection in experimental brain ischemia. This study aimed to compare potential neuroprotective effects of systemic pretreatment with the angiotensin receptor blocker, candesartan, and the angiotensin-converting enzyme (ACE)-inhibitor, ramipril, in normotensive Wistar rats after focal cerebral ischemia, with special emphasis on the regulation of neurotrophins. METHODS: Equipotent subcutaneous doses of candesartan and ramipril were determined via inhibition of pressor responses to intravenously injected angiotensin II (Ang II) or angiotensin I (Ang I), respectively. Accordingly, animals were treated with candesartan (0.1 mg/kg body weight, twice daily), ramipril (0.01 and 0.1 mg/kg body weight, twice daily) or vehicle (0.9% saline, twice daily), respectively, 5 days prior to middle cerebral artery occlusion (MCAO) with reperfusion. Severity of stroke was estimated via infarct size [magnetic resonance imaging (MRI) 48 h after MCAO] and neurological outcome (24 h, 48 h after MCAO). Measurements of neurotrophins/receptors in brain tissue were performed 48 h after MCAO. RESULTS: Pretreatment with candesartan and ramipril (low dose) did not reduce blood pressure during MCAO, whereas ramipril high dose did. Candesartan, but not ramipril at any dose, significantly reduced stroke volume and improved neurological outcome. Poststroke mRNA and protein of the neurotrophin receptor, TrkB, were significantly elevated in animals treated with candesartan, but not ramipril. CONCLUSIONS: Systemic pretreatment with a sub-hypotensive, RAS-blocking dose of candesartan affords neuroprotection after focal ischemia, associated with increased activity of the neurotrophin BDNF/TrkB system. Ramipril at sub-hypotensive and hypotensive, RAS-blocking doses showed no significant neuroprotective effects.

PPARgamma-activating angiotensin type-1 receptor blockers induce adiponectin.

The adipose-specific protein adiponectin has been recently discovered to improve insulin sensitivity. Angiotensin type-1 receptor (AT1R) blockers (ARBs) reduce the incidence of type 2 diabetes mellitus by mostly unknown molecular mechanisms. To identify new antidiabetic mechanisms of ARBs, we studied the regulation of adiponectin by angiotensin II (Ang II) and different ARBs in murine 3T3-L1 adipocytes and obese Zucker rats. Adiponectin protein expression was markedly stimulated by Ang II (5 nmol/L), which was inhibited by blockade of the AT2R, and further enhanced by the ARB irbesartan. Irbesartan-mediated adiponectin upregulation started beyond the concentrations needed for AT1R blockade and was also present in the absence of Ang II, implicating an AT1R-independent mechanism of action. Recently, certain ARBs (irbesartan, telmisartan) were identified as ligands of the peroxisome proliferator-activated receptor (PPAR)gamma. Telmisartan also stimulated adiponectin protein expression, whereas the non-PPARgamma-activating ARB eprosartan had no effect. Blockade of PPARgamma activation by the PPARgamma antagonist GW9662 markedly inhibited irbesartan-induced adiponectin expression. Cognate mRNA levels of adiponectin were not affected by ARBs. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that irbesartan prevented the cellular depletion of adiponectin protein. Finally, administration of irbesartan to obese Zucker rats improved insulin sensitivity and attenuated adiponectin serum depletion. The present study demonstrates that AT2R activation and certain ARBs induce adiponectin in adipocytes, which was associated with an improvement of parameters of insulin sensitivity in vivo. ARB-induced adiponectin stimulation is likely to be mediated via PPARgamma activation involving a post-transcriptional mechanism.

Are angiotensin receptor blockers neuroprotective?

Stroke is one of the leading causes of invalidism and death in the industrialized world. Among others, the renin- angiotensin system (RAS) has been implicated in the pathogenesis and outcome of ischemic events, including stroke. Angiotensin II (Ang II), the major effector peptide of the RAS, exerts most of its well-defined physiologic and pathophysiologic actions, including those on the central and peripheral nervous system, through its Ang II type 1 (AT1) receptor subtype. This receptor not only contributes to stroke-related pathologic mechanisms (eg, hypertension, atherothrombosis, and cardiac hypertrophy) but also may be involved in postischemic damage to the brain. However, it has also been demonstrated that Ang II, via its AT2 receptor subtype, accelerates neuronal tissue regeneration after injury. In this article, we review the experimental evidence supporting the notion that blockade of brain AT1 receptors can be beneficial with respect to stroke incidence and outcome. We further delineate how AT2 receptors could be involved in neuronal regeneration following brain injury, such as stroke. In doing so, we also attempt to shed some light on the mechanisms by which AT1 receptor blockers, which leave the AT2 receptor unopposed, might exert protective actions in brain ischemia.

Enhanced susceptibility to erythrocyte "apoptosis" following phosphate depletion.

Among the sequelae of phosphate depletion is anaemia, due in part to a decreased life span of mature erythrocytes. Recent studies have disclosed that cellular stress leads to an increase of cytosolic Ca(2+) activity in erythrocytes thereby triggering cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane, both typical features of apoptosis. In the present experiments, phosphatidylserine exposure and cell size were measured by fluorescence-activated cell sorting (FACS) analysis of annexin binding and forward scatter, respectively. Erythrocytes from intact mice were compared with erythrocytes from mice exposed to a low-phosphate diet for 4 days. Annexin binding of freshly drawn erythrocytes was slightly but significantly enhanced by the low-phosphate diet. Furthermore, intracellular phosphate and ATP concentrations were significantly decreased in those erythrocytes whereas intracellular Ca(2+) activity was unaltered. Osmotic shock (exposure to 700 mOsm by addition of sucrose for 12 h), removal of Cl(-) (replaced by gluconate for 15 h) or removal of glucose (12 h) decreased cell volume and increased the number of annexin-binding erythrocytes. Interestingly, these effects were significantly larger in erythrocytes from phosphate-depleted animals. The experiments reveal a novel mechanism triggered by phosphate depletion that presumably contributes to the enhanced vulnerability and accelerated sequestration of erythrocytes and, thus, to anaemia.