Acute administration of the olive constituent, oleuropein, combined with ischemic postconditioning increases myocardial protection by modulating oxidative defense.

Oleuropein, one of the main polyphenolic constituents of olive, is cardioprotective against ischemia reperfusion injury (IRI). We aimed to assess the cardioprotection afforded by acute administration of oleuropein and to evaluate the underlying mechanism. Importantly, since antioxidant therapies have yielded inconclusive results in attenuating IRI-induced damage on top of conditioning strategies, we investigated whether oleuropein could enhance or imbed the cardioprotective manifestation of ischemic postconditioning (PostC). Oleuropein, given during ischemia as a single intravenous bolus dose reduced the infarct size compared to the control group both in rabbits and mice subjected to myocardial IRI. None of the inhibitors of the cardioprotective pathways, l-NAME, wortmannin and AG490, influence its infarct size limiting effects. Combined oleuropein and PostC cause further limitation of infarct size in comparison with PostC alone in both animal models. Oleuropein did not inhibit the calcium induced mitochondrial permeability transition pore opening in isolated mitochondria and did not increase cGMP production. To provide further insights to the different cardioprotective mechanism of oleuropein, we sought to characterize its anti-inflammatory potential in vivo. Oleuropein, PostC and their combination reduce inflammatory monocytes infiltration into the heart and the circulating monocyte cell population. Oleuropein's mechanism of action involves a direct protective effect on cardiomyocytes since it significantly increased their viability following simulated IRI as compared to non-treated cells. Οleuropein confers additive cardioprotection on top of PostC, via increasing the expression of the transcription factor Nrf-2 and its downstream targets in vivo. In conclusion, acute oleuropein administration during ischemia in combination with PostC provides robust and synergistic cardioprotection in experimental models of IRI by inducing antioxidant defense genes through Nrf-2 axis and independently of the classic cardioprotective signaling pathways (RISK, cGMP/PKG, SAFE).

Investigating and re-evaluating the role of glycogen synthase kinase 3 beta kinase as a molecular target for cardioprotection by using novel pharmacological inhibitors.

AIMS: Glycogen synthase kinase 3 beta (GSK3ß) link with the mitochondrial Permeability Transition Pore (mPTP) in cardioprotection is debated. We investigated the role of GSK3ß in ischaemia (I)/reperfusion (R) injury using pharmacological tools. METHODS AND RESULTS: Infarct size using the GSK3ß inhibitor BIO (6-bromoindirubin-3'-oxime) and several novel analogues (MLS2776-MLS2779) was determined in anaesthetized rabbits and mice. In myocardial tissue GSK3ß inhibition and the specificity of the compounds was tested. The mechanism of protection focused on autophagy-related proteins. GSK3ß localization was determined in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from Langendorff-perfused murine hearts (30'I/10'R or normoxic conditions). Calcium retention capacity (CRC) was determined in mitochondria after administration of the inhibitors in mice and in vitro. The effects of the inhibitors on mitochondrial respiration, reactive oxygen species (ROS) formation, ATP production, or hydrolysis were measured in SSM at baseline. Cyclosporine A (CsA) was co-administered with the inhibitors to address putative additive cardioprotective effects. Rabbits and mice treated with MLS compounds had smaller infarct size compared with control. In rabbits, MLS2776 and MLS2778 possessed greater infarct-sparing effects than BIO. GSK3ß inhibition was confirmed at the 10th min and 2 h of reperfusion, while up-regulation of autophagy-related proteins was evident at late reperfusion. The mitochondrial amount of GSK3ß was similar in normoxic SSM and IFM and was not altered by I/R. The inhibitors did not affect CRC or respiration, ROS and ATP production/hydrolysis at baseline. The co-administration of CsA ensured that cardioprotection was CypD-independent. CONCLUSION: Pharmacological inhibition of GSK3ß attenuates infarct size beyond mPTP inhibition.

Ranolazine triggers pharmacological preconditioning and postconditioning in anesthetized rabbits through activation of RISK pathway.

We tested the hypothesis that ranolazine (Ran) is cardioprotective in a model of ischemia /reperfusion and we elucidated the intracellular mechanism. Anesthetized rabbits were subjected to is chemia and reperfusion and were divided into 5 groups: 1) Control, 2) Preconditioning (PreC), 3) Postconditioning (PostC), 4) RanA and 5) RanB, respectively treated with intravenous ranolazine, either 10min before or during index ischemia. Ranolazine was initially given over 60s and then from the beginning and throughout the whole reperfusion period. The infarcted to the risk ratio was calculated (%I/R). In a second series consisting of respective to the first series groups, the animals were subjected to the same interventions up to the 10th min of reperfusion where tissue samples were taken for immunoblotting of Akt, eNOS, ERK½ and GSK3ß (RISK pathway). In a third series, RanA+Wort, RanB+Wort and Wort groups were treated with ranolazine as RanA and RanB groups but with the addition of the PI3 inhibitor Wortmaninn (Wort) and %I/R calculated. Ranolazine reduced the % I/R in RanA and RanB compared to the Control (23.1±1.7%, 17.6±2.0% vs 47.6±1.0%, P<0.05). %I/R reduction achieved in the RanA and RanB groups was comparable to that observed in PreC and PostC (16.3±2.1%, 26.2±2.1%, respectively P<0.05 vs Control). Phosphorylation of Akt, ERK½, eNOS and GSK3ß were higher in PreC, PostC and in both ranolazine treated groups. Wortmannin abrogated ranolazine's %I/R reduction (RanA+Wort 31.4±1.7%, RanB+Wort 32.4±2.4%). Ranolazine reduces %I/R and triggers cardioprotection with a similar to conditioning mechanism which upregulates the RISK pathway.

Synthesis and Pharmacological Evaluation of Novel Adenine-Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents.

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H2S slow-releasing moiety, coupled via a stable ether bond. The H2S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives 4 and 11, both containing 4-hydroxythiobenzamide moiety as H2S donor, were selected for further in vivo evaluation. Their ability to release H2S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection.

Transient carotid ischemia as a remote conditioning stimulus for myocardial protection in anesthetized rabbits: Insights into intracellular signaling.

BACKGROUND: We investigated the effectiveness of perconditioning (Perc) applied at different time points along with the role of RISK, SAFE, STAT5 and eNOS pathways. METHODS AND RESULTS: Anesthetized rabbits were subjected to 30-min ischemia/3-hour reperfusion. Perc, consisted of 4 cycles of 1-min ischemia/reperfusion, was applied in the carotid artery at different time points. Perc was started and ended during ischemia, started during ischemia and ended at the beginning of reperfusion, started at the end of ischemia and ended at reperfusion and started and ended during reperfusion. The PI3K inhibitor wortmannin, or the JAK-2 inhibitor AG490, was also applied and the infarct size was assessed. In another series assigned to the previous groups, the phosphorylation of Akt, PI3K, ERKs1/2, GSK3ß, STAT3, and STAT5 was evaluated. All Perc groups had smaller infarction compared to those without Perc, independently of PI3K or JAK-2 inhibition. STAT5 was the only molecule that was phosphorylated in parallel with cardioprotection. Since Src and angiotensin II mediate the STAT5 pathway, we administered the Scr inhibitor PP1 and the angiotensin II receptor antagonist valsartan. PP1 and valsartan prevented STAT5 phosphorylation, but did not abrogate the effect of Perc. Furthermore, the NOS inhibitor L-NAME was administered and abrogated the infarct size limiting effect of Perc. In parallel, the expression of cleaved caspase-3 was elevated only in the control and Perc-A-L-NAME groups. CONCLUSION: Perc reduces infarction independently of RISK, SAFE and STAT5 pathways. Src kinase and angiotensin II play a predominant role in STAT5 activation. eNOS may protect the myocardium through inhibition of apoptosis.

The natural olive constituent oleuropein induces nutritional cardioprotection in normal and cholesterol-fed rabbits: comparison with preconditioning.

Ischemic preconditioning, which is mediated by cell signaling molecules, protects the heart from ischemia-reperfusion injury by limiting the infarct size. Oleuropein, the main polyphenolic constituent of olives, reduced the infarct size in normal and cholesterol-fed rabbits when it was administered at a nutritional dose. The aim of the present study was to compare the effects of oleuropein and preconditioning in terms of the cell signaling and metabolism pathways underlying myocardial protection. Rabbits were randomly divided into six groups: the control group received 5 % dextrose for six weeks, the preconditioning group was subjected to two cycles of preconditioning with 5 min ischemia/10 min reperfusion, the O6 group was treated with oleuropein for six weeks, the Chol group was fed a cholesterol-enriched diet and 5 % dextrose for six weeks, and the CholO6 and CholO3 groups were treated with cholesterol and oleuropein for six and three weeks, respectively; oleuropein was dissolved in 5 % dextrose solution and was administered orally at a dose of 20 mg × kg(-1) × day(-1). All animals were subsequently subjected to 30 min myocardial ischemia followed by 10 min of reperfusion. At that time, myocardial biopsies were taken from the ischemic areas for the assessment of oxidative and nitrosative stress biomarkers (malondialdehyde and nitrotyrosine), and determination of phosphorylation of signaling molecules involved in the mechanism of preconditioning (PI3K, Akt, eNOS, AMPK, STAT3). The tissue extracts NMR metabolic profile was recorded and further analyzed by multivariate statistics. Oxidative biomarkers were significantly reduced in the O6, CholO6, and CholO3 groups compared to the control, preconditioning, and Chol groups. Considering the underlying signaling cascade, the phosphorylation of PI3K, Akt, eNOS, AMPK, and STAT-3 was significantly higher in the preconditioning and all oleuropein-treated groups compared to the control and Chol groups. The NMR-based metabonomic study, performed through the analysis of spectroscopic data, depicted differences in the metabolome of the various groups with significant alterations in purine metabolism. In conclusion, the addition of oleuropein to a normal or hypercholesterolemic diet results in a preconditioning-like intracellular effect, eliminating the deleterious consequences of ischemia and hypercholesterolemia, followed by a decrease of oxidative stress biomarkers. This effect is exerted through inducing preconditioning-involved signaling transduction. Nutritional preconditioning may support the low cardiovascular morbidity and mortality associated with the consumption of olive products.

Pharmacological postconditioning of the rabbit heart with non-selective, A1 , A2A and A3 adenosine receptor agonists.

OBJECTIVES: We investigated the effects of novel selective and non-selective adenosine receptor agonists (ARs) on cardioprotection. METHODS: Male rabbits divided into six groups were subjected to 30-min heart ischaemia and 3-h reperfusion: (1) control group, (2) postconditioning (PostC) group, (3) group A: treated with the non-selective agonist (S)-PHPNECA, (4) group B: treated with the A1 agonist CCPA, (5) group C: treated with the A2A agonist VT 7 and (6) group D: treated with the A3 agonist AR 170. The infarcted (I) and the areas at risk (R) were estimated as %I/R. In additional rabbits of all groups, heart samples were taken for determination of Akt, eNOS and STAT 3 at the 10th reperfusion minute. KEY FINDINGS: (S)-PHPNECA and CCPA reduced the infarct size (17.2 ± 2.9% and 17.9 ± 2.0% vs 46.8 ± 1.9% in control, P < 0.05), conferring a benefit similar to PostC (26.4 ± 0.3%). Selective A2A and A3 receptor agonists did not reduce the infarct size (39.5 ± 0.8% and 38.7 ± 3.5%, P = NS vs control). Akt, eNOS and STAT 3 were significantly activated after non-selective A1 ARs and PostC. CONCLUSIONS: Non-selective and A1 but not A2A and A3 ARs agonists are essential for triggering cardioprotection. The molecular mechanism involves both RISK and the JAK/STAT pathways.

Myocardial protection provided by chronic skeletal muscle ischemia is not further enhanced by ischemic pre- or postconditioning: comparative effects on intracellular signaling.

Chronic skeletal muscle ischemia protects the ischemic heart by preserving coronary flow and inducing arterioangiogenesis. We sought to determine the effect and the underlying molecular mechanisms of preconditioning (PreC) and postconditioning (PostC), applied in a model of chronic skeletal muscle ischemia. Male rabbits were divided into 3 series. In each series, the animals were subjected either to severe hind limb (HL) ischemia, by excision of the femoral artery, or to sham operation (SHO). After 4 weeks, all the animals underwent 30 minutes of regional heart ischemia and 3 hours reperfusion. The animals of the first series received no further intervention (HL and SHO groups), those of the second series underwent PreC (HL + PreC and SHO + PreC), and of the third series PostC (HL + PostC and SHO + PostC). Infarct size (I) and risk zones (R) were determined, and their ratio was calculated in percentage. Three additional series of experiments were performed with respective interventions up to the 10th minute of reperfusion, where sample tissue was obtained for assessment of protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), glycogen synthase kinase 3ß (GSK3ß), p44/42, signal transducer and activator of transcription (STAT) 3, and STAT5. All groups demonstrated significantly smaller percentage of I/R compared with the SHO group (HL: 14.4% ± 3.7%, HL + PreC: 13.1% ± 1.0%, SHO + PreC: 21.3% ± 1.6%, HL + PostC: 18.0% ± 1.1%, and SHO + PostC: 24.3% ± 1.7%, P < .05 vs 35.7% ± 4.4% in SHO). The PreC and PostC did not further reduce the infarct size in HL groups. The Akt, eNOS, GSK3ß, p44/42, and STAT3 were activated in all PreC or PostC groups regardless of the infarct size reduction. The STAT5 was activated only in the HL groups compared with the SHO groups. In conclusion, chronic skeletal muscle ischemia results in effective cardioprotection, which is not further enhanced with application of PreC or PostC. The Akt, eNOS, GSK3ß, p44/42, and STAT3 may only be considered as indicators of the intracellular changes taking place during protection. Activation of STAT5 is possibly the end effector, which is responsible for infarct size reduction provided by chronic skeletal muscle ischemia.

Effects of pre- and postconditioning on arrhythmogenesis in the in vivo rat model.

The antiarrhythmic potential of postconditioning in in vivo models remains poorly defined. We compared the effects of pre- and postconditioning on ventricular arrhythmogenesis against controls with and without reperfusion. Wistar rats (n = 40, 269 ± 3 g) subjected to ischemia (30 minutes)--reperfusion (24 hours) were assigned to the following groups: (1) preconditioning (2 cycles), (2) postconditioning (6 cycles), or (3) no intervention and were compared with (4) nonreperfused infarcts and (5) sham-operated animals. Infarct size was measured, and arrhythmogenesis was evaluated with continuous telemetric electrocardiographic recording, heart rate variability indices, and monophasic action potentials (MAPs). During a 24-hour observation period, no differences in mortality were observed. Reperfusion decreased infarct size and ameliorated sympathetic activation during the late reperfusion phase. Preconditioning decreased infarct size by a further 35% (P = .0017), but only a marginal decrease (by 18%, P = .075) was noted after postconditioning. Preconditioning decreased arrhythmias during ischemia and early reperfusion, whereas postconditioning almost abolished them during the entire reperfusion period. No differences were noted in MAPs or in the magnitude of sympathetic activation between the 2 interventions. Compared to postconditioning, preconditioning affords more powerful cytoprotection, but both interventions exert antiarrhythmic actions. In the latter, these are mainly evident during the ischemic phase and continue during early reperfusion. Postconditioning markedly decreases reperfusion arrhythmias during a prolonged observation period. The mechanisms underlying the antiarrhythmic effects of pre- and postconditioning are likely different but remain elusive.

Various models of cardiac conditioning in single or sequential periods of ischemia: comparative effects on infarct size and intracellular signaling.

BACKGROUND: Preconditioning (PreC) and postconditioning (PostC) reduce infarct size. We sought to determine the effects of PreC and PostC, alone or in combination, on infarct size and expression of intracellular signals in different ischemia models. METHODS: Male rabbits were subjected to myocardial ischemia followed by 3-hour reperfusion. In a first series we applied 3 ischemia models [a 20-min period (20), a 40-min period (40), and two sequential 20-min periods (20-20)] and 3 types of interventions [no intervention (controls, C), 2 cycles of 5-min ischemia/10-min reperfusion before index ischemia (PreC) and 6 cycles of 10-s ischemia/10-s reperfusion after index ischemia and/or between the sequential ischemic periods (PostC)] (12 groups in total). Infarct size (I) and area at risk (R) were assessed (%I/R). In a second series, samples were taken for western blot analysis of Akt phosphorylation. RESULTS: Overall, %I/R differed significantly among groups (p<0.001). In control groups, C-40 had a greater %I/R than C-20 (p=0.006). In intervention groups, no differences were found in %I/R. All intervention groups had significantly lower %I/R compared to C-40 group (p<0.001), whereas, compared to C-20-20 group, PreC-20-20, 20-PostC-20, 20-PostC-20-PostC and PreC-20-20-PostC groups had lower %I/R (all p<0.05). Akt was increased in all groups in which a significant %I/R reduction was achieved (p<0.05 versus all other groups). CONCLUSIONS: PreC and PostC, alone or in combination, are effective when an ischemic insult of a given duration is applied either as a single or as sequential periods. Protection from either intervention is associated with an enhanced Akt activation.

Ovariectomy reinstates the infarct size-limiting effect of postconditioning in female rabbits.

Gender seems to interfere with the cardioprotective effect of ischemic preconditioning (PreC) and postconditioning (PostC); PreC-conferred protection is weaker or lost in female animals after ovariectomy (Ov), while the role of PostC is still in dispute. We sought to investigate the effect of PostC in female rabbits, its interaction with Ov, and the potential implicated intracellular pathways. Intact or Ov adult female rabbits (n = 46) were subjected to 30 min ischemia and reperfusion with PostC (PostC or OvPostC), which consisted of six cycles of 30-s ischemia/30-s reperfusion at the end of ischemia, or without PostC (Fem or OvFem). Infarct size (I) and area at risk (R) were determined by TTC staining and fluorescent particles, respectively, after 3-h reperfusion in 30 out of 46 animals. Plasma levels of estradiol and nitrite/nitrate (NO x ) were evaluated. ERKs, p38-MAPK, and Akt assessment was performed in excised hearts 1-min after starting the final reperfusion period in the remaining 16 animals. Infarct size was significantly reduced only in OvPostC group (I/R ratio, 25.3 ± 2.7, vs 48.1 ± 2.0, 43.6 ± 4.2 and 55.1 ± 5.6 % in Fem, OvFem, and PostC groups, p < 0.05). In ovariectomized rabbits, plasma estradiol and NO x levels were lower than in the normal ones. Akt phosphorylation in ischemic regions was significantly higher in OvPostC group, whereas ERK1/2 and p38-MAPK activation was observed in all ovariectomized animals irrespective of PostC. PostC is not effective in female rabbits, but the protection is reinstated after Ov potentially via the RISK pathway.

Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine, as pharmacological post-conditioning agent.

Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop compounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of 3.8 µmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs 26.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could be used as mimics or enhancers of PostC.

Short-term statin administration in hypercholesterolaemic rabbits resistant to postconditioning: effects on infarct size, endothelial nitric oxide synthase, and nitro-oxidative stress.

AIMS: The effectiveness of postconditioning (POC) in hypercholesterolaemia is in dispute. We investigated the effects of 3-day lipophilc (simvastatin) or hydrophilic (pravastatin) statin treatment, without or with POC in normocholesterolaemic (Norm) and hypercholesterolaemic (Chol) rabbits. METHODS AND RESULTS: Norm or Chol rabbits were subjected to 30 min ischaemia and randomized in two series of 12 groups each: control, simvastatin (Sim), pravastatin (Prav), POC, Sim-POC, Prav-POC, Chol, Sim-Chol, Prav-Chol, POC-Chol, Sim-POC-Chol, Prav-POC-Chol. After ischaemia, rabbits of the first series underwent 3 h reperfusion, followed by infarct size, total cholesterol, and low density lipoprotein plasma level evaluation; animals of the second series underwent 10 min reperfusion followed by tissue sampling for nitrotyrosine (NT), malondialdehyde, endothelial nitric oxide synthase (eNOS), and Akt analyses. N-nitro-l-arginine methylester (L-NAME) was given in two additional groups (POC-L-NAME and Prav-Chol-L-NAME) for infarct size assessment. All interventions reduced infarction in Norm (24.3 ± 1.3, 25.9 ± 2.8, 27.9 ± 3.1, 23.3 ± 2.3, and 33.4 ± 2.5%, in POC, Sim, Prav, Sim-POC, and Prav-POC groups, respectively, vs. 49.3 ± 1.9% in control, P < 0.05), but only Prav did so in Chol animals (25.7 ± 3.3 and 25.3 ± 3.9% in Prav-Chol and Prav-POC-Chol vs. 50.9 ± 1.7, 44.8 ± 4.3, 41.5 ± 3.5, and 49.3 ± 5.5% in Chol, Sim-Chol, POC-Chol, and Sim-POC-Chol, respectively, P < 0.05). L-NAME abolished the infarct size-limiting effect of POC and Prav-Chol. Prav induced the greatest reduction in NT, while it was the only intervention that increased myocardial eNOS and Akt in Chol rabbits (P < 0.05 vs. all others). CONCLUSION: Prav, in contrast to same-dose Sim or POC, reduces infarction in Chol rabbits independently of lipid lowering, potentially through eNOS activation and nitro-oxidative stress attenuation.

Investigating the effect of antioxidant treatment on the protective effect of preconditioning in anesthetized rabbits.

Reactive oxygen and nitrogen species are critical in preconditioning (PC). We sought to determine the effect of N-2-mercaptopropionyl glycine (MPG) on infarct size and on the oxidative status. Rabbits were exposed to 30-minute regional ischemia of the heart, which was followed by 3-hour reperfusion: (1) a control group without further intervention, (2) a PC1 group that was subjected to one cycle of PC, (3) a PC4 group that was subjected to 4 cycles of PC, (4) an MPG group that was treated with MPG for 60 minutes, starting 10 minutes before reperfusion, (5) MPG-PC1, and (6) the MPG-PC4 groups that were treated with the same dose of MPG and with 1 or 4 cycles of PC, respectively. Blood samples were drawn and collected for metabonomic analysis. In another series of experiments, 6 groups respective to the described ones were subjected to 30-minute regional ischemia of the heart and 20 minutes of reperfusion, after which pieces of heart tissue were quickly excised for malondialdehyde, nitrotyrosine, and glutathione content assessment. All PC and MPG groups developed smaller infarct size compared with control (16.5% ± 3.9%, 13.7% ± 3.1%, 18.6% ± 5.0%, 9.7% ± 2.0%, 15.0% ± 2.8% vs. 48.05% ± 7.2%; P < 0.05). MPG did not prevent lipid peroxidation and nitrotyrosine formation but enhanced the glutathione content. PC and MPG induced similar nuclear magnetic resonance changes. Long MPG infusion reduces the infarct size without abolishing the effect of PC, providing novel insights into the activity of MPG in PC.

Hypertension study in anaesthetized rabbits: protocol proposal for AT1 antagonists screening.

INTRODUCTION: The aim of this study was to establish an optimized fast and safe protocol for the pharmacological screening of AT(1) antagonists. MATERIALS AND METHODS: The pharmaceutical prototype AT(1) antagonist losartan, its active metabolite EXP3174 and the synthetic compound MMK1 were analysed in order to validate the protocol. Ang II was continuously infused while the animals received the drugs in two procedures. RESULTS: In the post-treatment procedure drugs were administered either in a single bolus dose or in a sequential manner. When losartan was administered in a single bolus dose, efficacy was evident until the 7th min (p=0.012) whilst EXP3174 infusion extended the efficiency up to the end of the study (p=0.006). In addition, the sequential injections of losartan prolonged the inhibitory time interval until the end of the study (p=0.045). In the pre-treatment procedure, results suggested a dose-dependent inhibitory effect for both antagonists. The pressor response to Ang II was unchanged after MMK1 administration either in the post- or in the pre-treatment mode. CONCLUSIONS: The proposed protocol appears to be safe, simple and fast for the pharmacological screening of AT(1) antagonists and enables the evaluation of new antagonists using lower doses than any other reported in the literature.

Chronic hind limb ischemia reduces myocardial ischemia-reperfusion injury in the rabbit heart by promoting coronary angiogenesis/arteriogenesis.

BACKGROUND/AIM: Application of ischemic injury in a remote organ may provide protection of other tissues against ischemia. We hypothesized that ischemia in the rabbit hind limb protects against myocardial ischemia by increasing angiogenesis/arteriogenesis. MATERIALS AND METHODS: In the first experiment, severe limb ischemia (LI) was induced in 26 New Zealand White rabbits by excision of the femoral artery while another 26 served as controls (no ischemia; sham operation [SHO]). Four weeks later, the blood vessels of the subendocardial and intramyocardial areas of the excised hearts were counted. In the second experiment, 14 LI rabbits and 14 SHO controls were subjected to 30 min of regional heart ischemia and 3 h reperfusion. Infarct size and the areas-at-risk were determined. RESULTS: Compared with controls, LI rabbits showed more subendocardial (103+/-14 vs. 113+/-13 capillaries/mm2, respectively; p=0.01) and intramyocardial blood vessels (102+/-12 vs. 114+/-16 capillaries/mm(2), respectively; p=0.009). LI rabbits had significantly smaller infarct size compared with the SHO animals (infarct areas/areas-at-risk: 14.37+/-11.23% vs. 31.31+/-13.73%, respectively; p=0.003). CONCLUSION: Chronic hind LI reduces myocardial infarct size by promoting coronary angiogenesis/arteriogenesis in an experimental model.

Simvastatin in contrast to postconditioning reduces infarct size in hyperlipidemic rabbits: possible role of oxidative/nitrosative stress attenuation.

Postconditioning (POC) reduces lethal reperfusion injury under normal conditions, but its effectiveness under certain pathological states is in dispute. In the present study, we sought to determine the effect of chronic simvastatin treatment in hyperlipidemic animals with or without POC. Anesthetized rabbits were randomized into eight groups, as follows, and were subjected to 30-min myocardial ischemia followed by 3-h reperfusion. Normally fed animals: a Control group with no additional intervention, a Sim group treated with simvastatin for 3 weeks at a dose of 3 mg kg(-1), a POC group subjected to POC with eight cycles of 30-s ischemia/reperfusion, a Sim-POC group treated with simvastatin, and POC. Cholesterol fed (6 weeks) animals: a Chol group with no additional interventions, a Chol-Sim group treated with simvastatin for 3 weeks, a Chol-POC group subjected to POC, and a Chol-Sim-POC group treated with simvastatin and POC. Infarct size and plasma levels of malondialdehyde (MDA), nitrotyrosine (NT), NOx, total cholesterol, and LDL were evaluated. In a second series of experiments, heart tissue samples were taken for MDA, NT, and NOx assessment. Infarct size, circulating MDA, NT, NOx and cardiac MDA, NT, and NOx levels declined in POC and all Sim groups compared with Control, Chol, and Chol-POC (p < 0.05). Simvastatin also reduced total cholesterol and LDL plasma levels. In conclusion, a 3-week simvastatin treatment limits the infarct size and attenuates the oxidative and nitrosative stress both in normo- and in hyper-cholesterolemic rabbits subjected to ischemia-reperfusion irrespective of the presence of POC, while POC is effective only in normocholesterolemic animals.

Design and synthesis of nitrate esters of aromatic heterocyclic compounds as pharmacological preconditioning agents.

Ischemic preconditioning (IPC) constitutes an endogenous protective mechanism in which one or more brief periods of myocardial ischemia and reperfusion render the myocardium resistant to a subsequent more-sustained ischemic insult. Pharmacological preconditioning represents an ideal alternative of IPC. We now describe the design and synthesis of indole, quinoline, and purine systems with an attached pharmacophoric nitrate ester group. The indole and quinoline derivatives 4 and 5 possess structural features of the nitrate containing K(ATP) channel openers. Purine analogues 11 and 12, substituted at the position 6 by a piperidine moiety and at position 9 by an alkyl nitrate, could combine the effects of the nitrate containing K(ATP) channel openers and those of adenosine. Compound 13 bears the nicotinamide moiety of nicorandil instead of nitrate ester. Compounds 4, 5, and 11 reduced infarction and the levels of malondialdehyde (MDA) at reperfusion in anesthetized rabbits. Compounds 12 and 13 did not significantly reduce the infarct size. Analogues 4 and 5 increased cGMP and MDA during ischemia, while combined analogue 4 and mitoK(ATP) blocker 5-hydroxydecanoic acid (5-HD) abrogated this benefit suggesting an action through mitoK(ATP) channel opening. Treatment with derivative 11 combined with 5-HD as well as treatment with 11 and adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) did not abrogate cardioprotection. Compound 11 is a lead molecule for the synthesis of novel analogues possessing a dual mode of action through cGMP-mitoK(ATP) channel opening-free radicals and through adenosine receptors.

Acute administration of vitamin C abrogates protection from ischemic preconditioning in rabbits.

Vitamin C is considered to be an antioxidant agent that is broadly used. Free radicals are involved in the protective mechanism of preconditioning (PC), but some antioxidant compounds abolish this benefit. The aim of the present study was to evaluate the effect of vitamin C on the protective effect of PC with respect to infarct size and oxidative stress in anesthetized rabbits. Male rabbits were randomly divided into six groups and subjected to 30 min of myocardial ischemia and 3h of reperfusion with the following interventions per group: (1) Control (no intervention), (2) Vit C 150 group (i.v. vitamin C at a total dose of 150 mg/kg for 75 min, starting 40 min before the onset of long ischemia and lasting up to the 5th min of reperfusion), (3) Vit C 300 group (i.v. vitamin C at a total dose of 300 mg/kg as previously described), (4) PC group (two cycles of 5 min ischemia and 10 min reperfusion), (5) combined PC-Vit C 150 group and (6) combined PC-Vit C 300 group. Blood samples were taken at different time points for malondialdehyde (MDA) assessment as a lipid peroxidation marker and for superoxide dismutase (SOD) activity. At the end of the experiment the infarct size was determined. Vitamin C, at both doses, did not reduce the infarct size (35.5+/-4.1%, 38.3+/-7.0% vs. 44.9+/-3.3% in the control group) and diminished the protection afforded by PC (32.0+/-2.7%, 43.8+/-3.3% vs. 15.7+/-2.9% in the PC group, P<0.05). At reperfusion there was an elevation of circulating MDA levels in the control and PC groups while in both vitamin C groups the levels were decreased. SOD activity was enhanced in the PC group compared to the controls; vitamin C did not change SOD activity during ischemia-reperfusion. Vitamin C abrogates the beneficial effect of ischemic PC on infarct size and elicits antioxidant properties during ischemia-reperfusion.

p38-MAPK is involved in restoration of the lost protection of preconditioning by nicorandil in vivo.

Nicorandil, a selective mitochondrial K(ATP) channel opener, reinstates the waned protection after multiple cycles of preconditioning. In this study, we determined the signal transduction activated in heart after 3 or 8 cycles of preconditioning and prolonged ischemia in rabbits treated with placebo or nicorandil. In a first series (eight groups) we evaluated the (%) infarct to risk ratio after 30 min ischemia/3 h reperfusion and in a second series (six groups), we assessed the intracellular levels of cyclic GMP (c-GMP), protein kinase C (PKC) activity and p38-mitogen activated protein kinase (p38-MAPK) phosphorylation from heart samples taken during the long ischemia. Cardioprotection by 3 cycles of preconditioning (11.7+/-3.8% vs 45.9+/-5.2% in the control, P<0.001) was lost after 8 cycles (43.9+/-5.1%, P=NS vs control). Nicorandil restored it to the levels of classic preconditioning (13.7+/-2.4% vs 40.8+/-3.5% in respective controls, P<0.001). This was reversed by the p38-MAPK inhibitor SB203580 (48.8+/-5.1%) which had no protective effect in the control group (44.6+/-5.8%). In the placebo-treated rabbits, intracellular c-GMP and PKC were increased only in the group subjected to 3 cycles of preconditioning. Despite that nicorandil equalizes the intracellular levels of c-GMP, PKC and activated p38-MAPK at the long ischemia, specific alterations of p38-MAPK phosphorylation differentiate the protected groups. Our data delineate the signal transduction mechanism mediating the beneficial effect of nicorandil and imply that the recapture of the lost protection is due to a dynamic process of the intracellular mediators accompanied by an increase in p38-MAPK phosphorylation and not to an instantaneous event.