The Australian and New Zealand Haemostasis Registry: ten years of data on off-licence use of recombinant activated factor VII.

BACKGROUND: Recombinant activated factor VII (rFVIIa) has been widely used as an off-licence pan-haemostatic agent in patients with critical bleeding. However, outside the trauma setting, there is relatively little high quality evidence on the risks and benefits of this agent. The Haemostasis Registry was established to investigate the extent of use, dosing, safety and outcomes of patients after off-licence rFVIIa treatment of critical bleeding. MATERIALS AND METHODS: The Registry recruited non-haemophiliac patients treated with rFVIIa from 2000-2009 (inclusive) in Australia and New Zealand. Detailed information was gathered on patients' demographics, context of bleeding, rFVIIa administration, laboratory results, blood component and other therapies, and outcomes. Outcome measures included subjectively assessed effect of rFVIIa on bleeding (response), adverse events (thromboembolic and other) and 28-day mortality. RESULTS: The registry included 3,446 cases in 3,322 patients (median [IQR] age 56 [33-70] years, 65% (n=2,147) male). Clinical indications included cardiac surgery (45%), other surgery (18%), trauma (13%), medical bleeding (6%), liver disease (6%), and obstetric haemorrhage (5%). The median [IQR] dose was 91 [72-103] µg/kg and 77% received a single dose. Reduction or cessation of bleeding was reported in 74% and 28-day survival was 71% but outcomes varied depending on clinical context. pH strongly correlated with outcome measures; 81% of patients with pH <7.1 died. Approximately 11% of patients had thromboembolic adverse events. In multivariate analysis, pH prior to administration and bleeding context were independently associated with reported response to rFVIIa and 28-day mortality. DISCUSSION: The Haemostasis Registry is the largest dataset of its kind and provides observational data on the off-licence use of rFVIIa over a 10-year period. It has been an invaluable resource for rigorously tracking adverse events and helping to inform clinical practice.

Effectiveness of massive transfusion protocols on mortality in trauma: a systematic review and meta-analysis.

INTRODUCTION: The effectiveness of massive transfusion protocols (MTPs) has been assumed from low quality studies with multiple biases. This review aimed to (i) evaluate the association between the institution of an MTP and mortality and (ii) determine the effect of MTPs on transfusion practice post trauma. METHODS: A systematic review of studies that examined patient outcomes before and after the institution of an MTP in the same centre was conducted. The design and results of each study were described. Heterogeneity was assessed using the Q test and the I(2) statistic. Odds ratios (ORs) for dichotomous outcomes from each study were pooled. RESULTS: There were eight studies that satisfied inclusion criteria with marked heterogeneity in study populations (I(2) = 72.1%, P = 0.001). Two studies showed significantly improved mortality following implementation of an MTP, and six studies showed no significant change. Pooled OR for the effect of an MTP on short-term mortality was 0.73 (95% confidence interval: 0.48-1.11). The effect of MTPs on transfusion practice was varied. CONCLUSION: Despite the popularity of MTPs and directives mandating their use in trauma centres, in before-after studies, MTPs have not always been associated with improved mortality. Evidence-based standardization of MTPs, improved compliance and analysis of broader endpoints were identified as areas for further research.

Use of recombinant activated factor VII in Jehovah's Witness patients with critical bleeding.

INTRODUCTION: The Australian and New Zealand Haemostasis Registry (ANZHR) included patients who received off-licence recombinant activated factor VII (rFVIIa) for critical bleeding from 2000 to 2009. Approximately 1.3% of the ANZHR patients were Jehovah's Witnesses (JWs). We compared them with the non-JW patients in the registry. METHODS: Patient characteristics (e.g. gender, context of bleeding), factors influencing rFVIIa use (e.g. body temperature and pH) and outcomes (e.g. bleeding response (stopped/attenuated or unchanged) to rFVIIa, mortality) were compared between JW and non-JW patients using Fisher's exact chi-square tests and Kruskal-Wallis tests. RESULTS: A total of 42 JW and 3134 non-JW patients were included in the analysis. Approximately 99% (n = 3098) of non-JWs received blood products compared with only 30% (n = 13) of JWs (P < 0.01). The distribution of gender and contexts of critical bleeding in the two groups was significantly different. Approximately 17% of the non-JW patients were hypothermic (T < 35°C) and about 19% were acidotic (pH < 7.2) at the time of initial rFVIIa administration. Conversely, none of the JWs were hypothermic and only one was acidotic. The proportion of positive responders to rFVIIa (stopped/attenuated bleeding following rFVIIa use) was similar in both groups (75% non-JWs, 74% JWs; P = 1.0). Approximately 28% of non-JW and 17% of JW patients were deceased by day 28 following rFVIIa use (P = 0.16). DISCUSSION: Several factors were observed to be significantly different between JW and non-JW patients, yet the proportions of responders to rFVIIa were similar in both groups. The actual factors influencing response to rFVIIa are yet to be determined.

Off-label use of recombinant factor VIIa in pediatric patients.

OBJECTIVE: To examine off-label recombinant factor VIIa (rFVIIa) use in pediatric patients including clinical indications, dose, adverse events, and outcomes. METHODS: All pediatric patients entered into the Haemostasis Registry from 75 participating hospitals were analyzed. RESULTS: Three hundred and eighty-eight pediatric patients received off-label rFVIIa from 2003 to 2009. Median age was 12 months (interquartile range 1 month to 11 years). Clinical context included cardiac surgery (52.1%), medical (11.6%), other surgery (10.8%), hematology/oncology (10.3%), trauma (9.3%), intracranial hemorrhage (3.1%), and liver disease (2.8%). Twenty-six patients received extracorporeal membrane oxygenation at the time of rFVIIa administration. Median first dose was 114 µg/kg (interquartile range 90-181; range 7-2250). Thirty-four percent received >1 dose. There was a reduction in usage of red blood cells, platelets, fresh-frozen plasma, and cryoprecipitate in the 24 hours after the first dose for all patients (all P values < .001). Thromboembolic adverse events (TEAs) were reported in 5.4%. No association between TEA and size of first dose was found. Where data were available, 82% of patients were subjectively classified as responding to rFVIIa. Overall 28-day mortality was 27%. In multivariate analysis, pH values before administration and clinical context were independently associated with response to first dose and 28-day mortality. CONCLUSIONS: There was a significant reduction in blood product administration after rFVIIa and a subjective response rate of 82%. Both pH and clinical context were associated with response to rFVIIa and mortality. Overall, 5.4% had a TEA reported.

Use of recombinant factor VIIa in patients with amniotic fluid embolism: a systematic review of case reports.

BACKGROUND: Patients with amniotic fluid embolism (AFE) (major cardiac and pulmonary symptoms plus consumptive coagulopathy) have high circulating tissue factor concentrations. Recombinant factor VIIa (rVIIa) has been used to treat hemorrhage in AFE patients even though rVIIa can combine with circulating tissue factor and form intravascular clots. A systematic review was done of case reports from 2003 to 2009 of AFE patients with massive hemorrhage who were and were not treated with rVIIa to assess the thrombotic complication risk. METHODS: MEDLINE was searched for case reports of AFE patients receiving rVIIa (rVIIa cases) and of AFE patients who received surgery to control bleeding but no rVIIa (cohorts who did not receive rVIIa). Additional AFE case reports were obtained from the Food and Drug Administration, the Australian and New Zealand Haemostasis Registry, and scientific meeting abstracts. The risk of a negative outcome (permanent disability or death) in rVIIa cases versus cohorts who did not receive rVIIa was calculated using risk ratio and 95% confidence interval. RESULTS: Sixteen rVIIa cases and 28 cohorts were identified who did not receive rVIIa. All patients had surgery to control bleeding. Death, permanent disability, and full recovery occurred in 8, 6, and 2 rVIIa cases and 7, 4, and 17 cohorts who did not receive rVIIa (risk ratio 2.2, 95% CI 1.4-3.7 for death or permanent disability vs. full recovery). CONCLUSION: Recombinant factor VIIa cases had significantly worse outcomes than cohorts who did not receive rVIIa. It is recommended that rVIIa be used in AFE patients only when the hemorrhage cannot be stopped by massive blood component replacement.

CXCR4 antagonism attenuates the cardiorenal consequences of mineralocorticoid excess.

BACKGROUND: Extensive evidence implicates aldosterone excess in the development and progression of cardiovascular disease states including hypertension, metabolic syndrome, cardiac hypertrophy, heart failure, and cardiorenal fibrosis. Recent studies show that activation of inflammatory cascade may play a specific role in the sequelae of mineralocorticoid activation, although the linking mechanism remains unclear. We tested the possibility that secondary stimulation of the stromal-derived factor 1/CXC chemokine receptor 4 (SDF-1/CXCR4) pathway plays a contributory role. METHODS AND RESULTS: We investigated the effect of the highly selective CXCR4 antagonist AMD3465 (6 mg/kg per day for 6 weeks through minipump) in dexoycorticosterone acetate (DOCA)-treated, uninephrectomized mice. CXCR4 antagonism significantly attenuated the induction of cardiac fibrosis, renal fibrosis, hypertension, and left ventricular hypertrophy by DOCA. Mineralocorticoid excess also stimulated the accumulation of T-lymphocytes in the heart and kidney and this was significantly blunted by CXCR4 inhibition. CONCLUSIONS: Taken together, these data strongly implicate the SDF-1/CXCR4 axis in the pathogenesis of mineralocorticoid excess induced hypertension, inflammation, and cardiorenal fibrosis. This insight provides a new potential therapeutic approach for the treatment of specific aspects of mineralocorticoid mediated cardiovascular disease.

Recirculating delivery improves myocardial cell engraftment.

INTRODUCTION: Despite pharmacological advances for heart failure, morbidity and mortality remain unacceptably high. As a result, alternative approaches such as cell therapy have been suggested to hold potential promise. However, a major obstacle is the optimization of cell delivery to the heart. Therefore, we investigated the efficacy of a percutaneous recirculation system for the delivery of cells to the heart. METHODS: Ovine fibroblasts were delivered to the ovine heart (3 x 10(7) cells) using the V-Focus system, a "closed" recirculatory system that draws blood from the coronary sinus and returns it to the coronary artery via an oxygenator, or intracoronary (IC) infusion, followed by a 2-hour recovery period. Animals were euthanized and cardiac tissue collected to determine presence of cells. RESULTS: There was a significant difference (P < 0.05) in the number of cells delivered to the heart by the V-Focus compared to direct coronary infusion for left ventricular freewall (V-Focus 1.39 +/- 0.63/mm(2), IC 0.11 +/- 0.06/mm(2)), septum (V-Focus 3.18 +/- 0.88/mm(2), IC 0.38 +/- 0.19/mm(2)), and right ventricle (V-Focus 0.46 +/- 0.23/mm(2), IC 0.05 +/- 0.04/mm(2)). CONCLUSIONS: These results suggest that potential therapeutic cells are optimally delivered to the large animal heart using the V-Focus cardiac delivery system in an ovine heart.

Reduced L-arginine transport contributes to the pathogenesis of myocardial ischemia-reperfusion injury.

Myocardial injury due to ischemia-reperfusion (I-R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I-R process induces a state of insufficient L-arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I-R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia-reoxygenation significantly decreased L-arginine uptake and NO production (42 +/- 2% and 71 +/- 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L-arginine transporter, CAT1, and NVCMs supplemented with L-arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia-reoxygenation. In contrast, L-arginine deprived NVCM had significantly worsened responses to hypoxia-reoxygenation. In isolated perfused mouse hearts, L-arginine infusion during reperfusion significantly improved left ventricular function after I-R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L-arginine availability as a key factor in the pathogenesis of I-R injury. Increasing L-arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I-R. Restoration of L-arginine availability may therefore be a valuable strategy to ameliorate I-R injury.

Persistent beneficial effect of postconditioning against infarct size: role of mitochondrial K(ATP) channels during reperfusion.

UNLABELLED: This study tested the hypothesis that inhibition of myocardial injury and modulation of mitochondrial dysfunction by postconditioning (Postcon) after 24 h of reperfusion is associated with activation of K(ATP) channels. Thirty dogs undergoing 60 min of ischemia and 24 h of reperfusion (R) were randomly divided into four groups: CONTROL: no intervention at R; Postcon: three cycles of 30 s R alternating with 30 s re-occlusion were applied at R; 5-hydroxydecanoate (5-HD): the mitochondrial K(ATP) channel blocker was infused 5 min before Postcon; HMR1098: the sarcolemmal K(ATP) channel blocker was administered 5 min before Postcon. After 24 h of R, infarct size was smaller in Postcon relative to CONTROL (27 +/- 4%* Vs. 39 +/- 2% of area at risk), consistent with a reduction in CK activity (66 +/- 7* Vs. 105 +/- 7 IU/g). The infarct-sparing effect of Postcon was blocked by 5-HD (48 +/- 5%(dagger)), but was not altered by HMR1098 (29 +/- 3%*), consistent with the change in CK activity (102 +/- 8(dagger) in 5-HD and 71 +/- 6* IU/g in HMR1098). In H9c2 cells exposed to 8 h hypoxia and 3 h of reoxygenation, Postcon up-regulated expression of mito-K(ATP) channel Kir6.1 protein, maintained mitochondrial membrane potential and inhibited mitochondrial permeability transition pore (mPTP) opening evidenced by preserved fluorescent TMRE and calcein staining. The protective effects were blocked by 5-HD, but not by HMR1098. These data suggest that in a clinically relevant model of ischemia-reperfusion (1) Postcon reduces infarct size and decreases CK activity after prolonged reperfusion; (2) protection by Postcon is achieved by opening mitochondrial K(ATP) channels and inhibiting mPTP opening. *P < 0.05 Vs. CONTROL; P < 0.05 Vs. Postcon.

Inhibition of myocardial apoptosis by postconditioning is associated with attenuation of oxidative stress-mediated nuclear factor-kappa B translocation and TNF alpha release.

Oxidative stress-stimulated nuclear factor-kappa B (NF-kappa B) activation has been associated with rapid transcription of TNF-alpha and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-kappa B and release of TNF-alpha secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 +/- 0.08 vs. 0.8 +/- 0.06* microM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-kappa B to nuclei (167% +/- 21% vs. 142% +/- 18%*), decreased the level of plasma TNF-alpha (1,994 +/- 447 vs. 667 +/- 130* pg/mL), and inhibited caspase-3 activity (0.57% +/- 0.1% vs. 0.21% +/- 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% +/- 1% vs. 11% +/- 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-kappa B expression (42% +/- 8%*) and reduction of release of TNF-alpha (231 +/- 72* pg/mL). Caspase-3 activity (0.33% +/- 0.1%*) and apoptotic cells (12% +/- 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-kappa B-TNF-alpha signaling pathway. *P < 0.05 Postcon and NAC vs. Control.

Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation.

Opioids introduced at reperfusion (R) following ischemia (I) reduce infarct size much like postconditioning, suggesting the hypothesis that postconditioning increases cardiac opioids and activates local opioid receptors. Anesthetized male rats subjected to 30 min regional I and 3 h R were postconditioned with three cycles of 10 s R and 10 s reocclusion at onset of R. Naloxone (NL), its peripherally restricted analog naloxone methiodide, delta-opioid receptor (DOR) antagonist naltrindole (NTI), kappa-opioid receptor antagonist norbinaltorphimine (NorBNI), and mu-opioid receptor (MOR) antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were administered intravenously 5 min before R. The area at risk (AAR) was comparable among groups, and postconditioning reduced infarct size from 57 +/- 2 to 42 +/- 2% (P < 0.05). None of the antagonists alone altered infarct size. All antagonists abrogated postconditioning protection at higher doses. However, blockade of infarct sparing by postconditioning was lost, since tested doses of NL, NTI, NorBNI, and CTAP were lowered. The efficacy of NorBNI declined first at 3.4 micromol/kg, followed sequentially by NTI (1.1), NL (0.37), and CTAP (0.09), suggesting likely MOR and perhaps DOR participation. Representative small, intermediate, and large enkephalins in the AAR were quantified (fmol/mg protein; mean +/- SE). I/R reduced proenkephalin (58 +/- 9 vs. 33 +/- 4; P < 0.05) and sum total of measured enkephalins, including proenkephalin, peptide B, methionine-enkephalin, and methionine-enkephalin-arginine-phenylalanine (139 +/- 17 vs. 104 +/- 7; P < 0.05) compared with shams. Postconditioning increased total enkephalins (89 +/- 8 vs. 135 +/- 5; P < 0.05) largely by increasing proenkephalin (33 +/- 4 vs. 96 +/- 7; P < 0.05). Thus the infarct-sparing effect of postconditioning appeared to involve endogenously activated MORs and possibly DORs, and preservation of enkephalin precursor synthesis in the AAR.

PAR-2 activation at the time of reperfusion salvages myocardium via an ERK1/2 pathway in in vivo rat hearts.

Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given 10 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERK1/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 +/- 2% vs. 53 +/- 1%, P < 0.05), and SP had no effect on infarct size (53 +/- 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 +/- 2% vs. PD + PAR-2 AP, 50 +/- 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 +/- 2%) but not by LY (PAR-2 AP, 36 +/- 2% vs. LY + PAR-2 AP, 38 +/- 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC.

Preconditioning and postconditioning: innate cardioprotection from ischemia-reperfusion injury.

Reperfusion is the definitive treatment to salvage ischemic myocardium from infarction. A primary determinant of infarct size is the duration of ischemia. In myocardium that has not been irreversibly injured by ischemia, reperfusion induces additional injury in the area at risk. The heart has potent innate cardioprotective mechanisms against ischemia-reperfusion that reduce infarct size and other presentations of postischemic injury. Ischemic preconditioning (IPC) applied before the prolonged ischemia exerts the most potent protection observed among known strategies. It has been assumed that IPC exerts protection during ischemia. However, recent data suggest that cardioprotection is also exerted during reperfusion. Postconditioning (PoC), defined as brief intermittent cycles of ischemia alternating with reperfusion applied after the ischemic event, has been shown to reduce infarct size, in some cases equivalent to that observed with IPC. Although there are similarities in mechanisms of cardioprotection by these two interventions, there are key differences that go beyond simply exerting these mechanisms before or after ischemia. A significant limitation of IPC has been the inability to apply this maneuver clinically except in situations where the ischemic event can be predicted. On the other hand, PoC is applied at the point of service in the hospital (cath-lab for percutaneous coronary intervention, coronary artery bypass grafting, and other cardiac surgery) where and when reperfusion is initiated. Initial clinical studies are in agreement with the success and extent to which PoC reduces infarct size and myocardial injury, even in the presence of multiple comorbidities.

Long-term inhibition of myocardial infarction by postconditioning during reperfusion.

Cardioprotection with postconditioning has been well demonstrated after a short period of reperfusion. This study tested the hypothesis that postconditioning reduces infarct size, vascular dysfunction, and neutrophil accumulation after a long-term reperfusion. Canines undergoing 60 min left anterior descending artery (LAD) occlusion were divided into two control groups of either 3 h or 24 h of full reperfusion and two postconditioning groups with three 30 s cycles of reperfusion and re-occlusion applied at the onset of either 3 h or 24 h of reperfusion. Size of the area at risk (AAR) and collateral blood flow during ischemia were similar among groups. In controls, infarct size as percentage of the AAR (30 +/- 3 vs. 39 +/- 2* %) by TTC staining, superoxide anion generation from the post-ischemic coronary arteries by lucigenin-enhanced chemiluminescence [(89 +/- 5 vs. 236 +/- 27* relative light units (RLU/mg)], and neutrophil (PMN) accumulation by immunohistochemical staining in the AAR (52 +/- 11 vs. 84 +/- 14* cells/mm(2) myocardium) significantly increased between 3 and 24 h of reperfusion. Postconditioning reduced infarct size (15 +/- 4 and 27 +/- 3.6 %), superoxide anion generation (24 +/- 4 and 43 +/- 11 RLU/mg), and PMN accumulation (19 +/- 6 and 45 +/- 8 cells/mm(2) myocardium) in the 3 and 24 h reperfusion groups relative to time-matched controls. These data suggest that myocardial injury increases with duration of reperfusion; reduction in infarct size and attenuation in inflammatory responses with postconditioning persist after a prolonged reperfusion. * p < 0.05 24 vs. 3 h control; p < 0.05 postconditioning vs. time-matched control.

Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling.

OBJECTIVE: Using non-selective and selective protein kinase C (PKC) epsilon and delta isoform inhibitors, we tested the hypothesis that the cardioprotective phenotype invoked by postconditioning (postcon) is dependent on PKC signalling. Furthermore, we determined whether postconditioning alters pPKCepsilon and/or pPKCdelta in cytosolic and mitochondrial fractions. METHODS: Male Sprague-Dawley rats underwent 30 min left coronary artery (LCA) occlusion followed by 3 h of reperfusion. Rats were randomised to the following groups: Untreated, no intervention either before or after LCA occlusion; Postcon, 3 cycles of 10-s full reperfusion and 10-s re-occlusion, initiated immediately at the onset of reperfusion; Chelerythrine (non-selective PKC inhibitor, 5 mg/kg)+/-postcon; Rottlerin (PKCdelta inhibitor, 0.3 mg/kg)+/-postcon; KIE1-1 (PKCepsilon inhibitor, 3.8 mg/kg)+/-postcon. A subset of rats was employed to assess pPKCepsilon and/or pPKCdelta in sham, Isch/RP (30-min LCA occlusion followed by 30-min reperfusion), and postcon-treated hearts. RESULTS: Infarct size, expressed as area of necrosis as a percentage of the area at risk, AN/AAR (%), was significantly reduced by postcon compared to control (untreated) rats (39+/-2% vs. 53+/-1% in control, P<0.001). Treatment with chelerythrine alone or the PKCepsilon antagonist KIE1-1 alone at reperfusion had no effect on infarct size compared to control. In contrast, the infarct-sparing effect of postcon was abrogated by non-selective PKC inhibition and PKCepsilon antagonism (50+/-2% and 50+/-1%, respectively, P<0.002). Inhibition of PKCdelta reduced infarct size to values comparable to that in postcon group (36+/-3% vs. 39+/-2%). However, postcon in the presence of PKCdelta inhibitor did not enhance the infarct-sparing effects (38+/-2%). In addition, pPKCepsilon in postcon hearts was significantly higher in the total cell homogenate (10338+/-1627 vs. 4165+/-608 in Isch/RP, arbitrary units), and pPKCdelta translocation to mitochondria was significantly less (>2-fold decrease) compared to Isch/RP. CONCLUSION: These data suggest that postcon modulates PKC during early reperfusion by increasing PKCepsilon expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCdelta to mitochondria and associated deleterious signalling.

Adenosine receptor-mediated coronary vascular protection in post-ischemic mouse heart.

This study evaluated the ability of A1 and A3 adenosine receptor (AR) agonism, and A1, A2A, A2B and A3AR antagonism (revealing "intrinsic" responses), to modify post-ischemic coronary dysfunction in mouse heart. Vascular function was assessed before and after 20 min global ischemia and 30-45 min reperfusion in Langendorff perfused C57/Bl6 mouse hearts. Ischemic insult impaired coronary sensitivity to the endothelial-dependent dilators ADP (pEC50=6.8+/-0.1 vs. 7.6+/-0.1, non-ischemic) and acetylcholine (pEC50=6.1+/-0.1 vs. 7.3+/-0.1 in non-ischemic), and for the mixed endothelial-dependent/independent dilator 2-chloroadenosine (pEC50=7.5+/-0.1 vs. 8.4+/-0.1, non-ischemic). Endothelium-independent dilation in response to nitroprusside was unaltered (pEC50=7.0+/-0.1 vs. 7.1+/-0.1 in non-ischemic). Pre-treatment with a selective A1AR agonist (50 nM CHA) failed to modify coronary dysfunction, whereas A1AR antagonism (200 nM DPCPX) worsened the effects of I/R (2-chloroadenosine pEC50=6.9+/-0.1). Conversely, A3AR agonism (100 nM Cl-IB-MECA) did reduce effects of I/R (pEC50s=8.0+/-0.1 and 7.3+/-0.1 for 2-chloroadenosine and ADP, respectively), whereas antagonism (100 nM MRS1220) was without effect. While A2AAR agonism could not be assessed (due to pronounced vasodilatation), A2AAR antagonism (100 nM SCH58261) was found to exert no effect, and antagonism of A2BARs (50 nM MRS1754) was also ineffective. The protective actions of A3AR agonism were also manifest as improved reactive hyperemic responses. Interestingly, post-ischemic coronary dysfunction was also limited by: Na+-H+ exchange (NHE) inhibition with 10 or 50 microM BIIB-513 (2-chloroadenosine pEC50s=7.8+/-0.1, either dose), an effect not additive with A3AR agonism; Ca2+ antagonism with 0.3 microM verapamil (2-chloroadenosine pEC50=7.9+/-0.1); and Ca2+ desensitization with 5 mM BDM (2-chloroadenosine pEC50=7.8+/-0.1). In contrast, endothelin antagonism (200 nM PD142893) and anti-oxidant therapy (300 microM MPG+150 U/ml SOD+600 U/ml catalase) were ineffective. Our data collectively confirm that ischemia selectively impairs endothelial function and reactive hyperemia independently of blood cells. Vascular injury is intrinsically limited by endogenous (but not exogenous) activation of A1ARs, whereas exogenous A3AR activation further limits dysfunction (improving post-ischemic vasoregulation). Finally, findings suggest this form of post-ischemic coronary injury is unrelated to endothelin or oxidant stress, but may involve modulation of Ca2+ overload and/or related ionic perturbations.

Myocardial protection in reperfusion with postconditioning.

Reperfusion is the definitive treatment for coronary occlusive disease. However, reperfusion carries the potential to exacerbate lethal injury, termed 'reperfusion injury'. Studies have suggested that reperfusion injury events are triggered during the early moments of reflow, and determine, in part, the severity of downstream manifestations of postischemic injury, including endothelial dysfunction, infarction and apoptosis. The application of brief iterative episodes of reflow (reoxygenation) and reocclusion (ischemia, hypoxia) at the immediate onset of reperfusion, which has been termed 'postconditioning' by the authors, reduces many manifestations of postischemic injury, notably infarct size, apoptosis, coronary vascular endothelial injury and reperfusion arrhythmias. Cardioprotection with postconditioning has been reported to be comparable with that observed using the gold standard maneuver ischemic preconditioning. In contrast to preconditioning, which exerts its effects primarily during the index ischemia, postconditioning appears to exert its effects during reperfusion alone. Postconditioning modifies the early phase of reperfusion in ways that are just beginning to be understood. It appears to first: reduce the oxidant burden and consequent oxidant-induced injury; secondly, attenuate the local inflammatory response to reperfusion; and thirdly, engage end effectors and signaling pathways implicated in other cardioprotective maneuvers, such as ischemic and pharmacologic preconditioning. Postconditioning seems to trigger the upregulation of survival kinases principally known to attenuate the pathogenesis of apoptosis and possibly necrosis. The postconditioning phenomenon has been reproduced by a number of independent laboratories and has been observed in both large and small animal in vivo models, as well as in ex vivo and cell culture models. In contrast to preconditioning, postconditioning may have widespread clinical application because it can be applied during reperfusion at the point of service for angioplasty, stenting, cardiac surgery and organ transplantation.

Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine.

OBJECTIVE: This study tested the hypothesis that brief cycles of iterative ischemia-reperfusion at onset of reperfusion (termed "postconditioning", post-con) delays washout of intravascular adenosine and thereby increases endogenous adenosine receptor (AR) activation during the early moments of reperfusion (R). METHODS: Isolated mouse hearts were subjected to 20 min global ischemia (I) and 30 min R with or without post-con (3 or 6 cycles of 10 s R&I). Intravascular purines in coronary effluent were analyzed by HPLC. To assess the functional role of endogenous AR activation in post-con, an open-chest rat model of myocardial infarction was employed. Rats were randomly divided into 11 groups: control, no intervention at R; post-con, three cycles of 10 s R followed by 10 s LCA re-occlusion immediately upon R. In the following interventions, drugs (or vehicle) were administered 5 min before R in the absence or presence (+/-) of post-con. Vehicle (DMSO < 300 microl/kg); 8-SPT (non-selective AR antagonist, 10 mg/kg) +/- post-con; DPCPX (A(1A)R antagonist, 0.1 mg/kg) +/- post-con; ZM241385 (A(2A)AR antagonist, 0.2 mg/kg) +/- post-con; MRS1523 (A(3)AR antagonist, 2 mg/kg) +/- post-con. RESULTS: In isolated mouse hearts, post-con reduced diastolic pressure during both early (26+/-3* vs. 37+/-3 mmHg at 5 min) and late (22+/-3* vs. 34+/-3 mmHg at 30 min) R. Post-con also hastened the early recovery of contractile function (developed pressure 39+/-6* vs. 16+/-2 mmHg at 5 min R), although differences did not persist at 30 min R. Importantly, post-con was associated with reduced adenosine washout (58+/-5* vs. 155+/-16 nM/min/g) at 2 min R suggesting greater retention time of intravascular adenosine. In rats, post-con significantly attenuated infarct size compared to control (40+/-3% vs. 53 +/- 2%* in control), an effect that was unaltered by DPCPX (42 +/- 2%) but was abrogated by 8-SPT (50 +/- 2%), ZM241385 (49 +/- 3%) or MRS1523 (52 +/- 1%) (P < 0.02). CONCLUSION: These data suggest that post-con involves endogenous activation of A(2A) and A3 but not A1AR subtypes. This activation may be linked to the delay in the washout of intravascular adenosine during the early minutes of R during which post-con is applied.

Age-related changes in ischemic tolerance in male and female mouse hearts.

Aging is associated with reduced tolerance to ischemic insult, and genesis of this intolerant phenotype is poorly understood. We characterized effects of aging and gender on cardiovascular function and cell damage during 20 min ischemia and 60 min reperfusion in isolated hearts from young adult (2-4 months), mature adult (8 months), middle-aged (12 months), aged (18 months), and senescent (24-28 months) C57/Bl6 mice. Aging substantially impaired recovery of ventricular contractility, with this change primarily evident within 12 months of age. In males ventricular developed pressure recovered to 72 +/- 8 mmHg in young hearts vs. only 44 +/- 7, 30 +/- 3, 24 +/- 5, and 27 +/- 4 mmHg in mature, middle-aged, aged and senescent hearts, respectively. This pattern was largely due to worsened diastolic dysfunction. Coronary flow recovered to below pre-ischemic levels in all ages, correlating with contractile recovery. However, coronary dysfunction (impaired responses to 2-chloroadenosine and ADP) was unaltered by senescence. Lactate dehydrogenase (LDH) loss, a marker for oncosis, increased to middle-age (approximately twofold), then fell with further aging to a value no longer different from that in young adult hearts. Similar patterns of change were observed in female hearts, although LDH efflux was significantly lower in mature adult and middle-aged female vs. male hearts, with functional tolerance also tending to be greater at these ages (though not achieving significance). Overall, our data reveal age-related ischemic intolerance develops well before senescence, being primarily evident by "middle-age". Phenotypic changes appear selective for myocardial vs. vascular injury, and functional vs. oncotic injury. Similar changes occur in males and females, though there is evidence of a protected phenotype in mature to middle-aged female vs. male hearts.

Mediators of coronary reactive hyperaemia in isolated mouse heart.

1. Mechanisms regulating coronary tone under basal conditions and during reactive hyperaemia following transient ischaemia were assessed in isolated mouse hearts. 2. Blockade of NO-synthase (50 muM L-NAME), K(ATP) channels (5 muM glibenclamide), A(2A) adenosine receptors (A(2A)ARs; 100 nM SCH58261), prostanoid synthesis (100 muM indomethacin), and EDHF (100 nM apamin+100 nM charybdotoxin) all reduced basal flow approximately 40%. Effects of L-NAME, glibenclamide, and apamin+charybdotoxin were additive, whereas coadministration of SCH58261 and indomethacin with these inhibitors failed to further limit flow. 3. Substantial hyperaemia was observed after 5-40 s occlusions, with flow increasing to a peak of 48+/-1 ml min(-1) g(-1). Glibenclamide most effectively inhibited peak flows (up to 50%) while L-NAME was ineffective. 4. With longer occlusions (20-40 s), glibenclamide alone was increasingly ineffective, reducing peak flows by approximately 15% after 20 s occlusion, and not altering peak flow after 40 s occlusion. However, cotreatment with L-NAME+glibenclamide inhibited peak hyperaemia by 70 and 25% following 20 and 40 s occlusions, respectively. 5. In contrast to peak flow changes, sustained dilation and flow repayment over 60 s was almost entirely K(ATP) channel and NO dependent (each contributing equally) with all occlusion durations. 6. Antagonism of A(2A)ARs with SCH58261 reduced hyperaemia 20-30% whereas inhibition of prostanoid synthesis was ineffective. Effects of A(2A)AR antagonism were absent in hearts treated with L-NAME and glibenclamide, supporting NO and K(ATP)-channel-dependent effects of A(2A)ARs. 7. EDHF inhibition alone exerted minor effects on hyperaemia and only with longer occlusions. However, residual hyperaemia after 40 s occlusion in hearts treated with L-NAME+glibenclamide+SCH58261+indomethacin was abrogated by cotreatment with apamin+charybdotoxin. 8. Data support a primary role for K(ATP) channels and NO in mediating sustained dilation after coronary occlusion. While K(ATP) channels (and not NO) are also important in mediating initial peak flow adjustments after brief 5-10 s occlusions, their contribution declines with longer 20-40 s occlusions. Intrinsic activation of A(2A)ARs is important in triggering K(ATP) channel/NO-dependent hyperaemia. Synergistic effects of combined inhibitors implicate interplay between mediators, with compensatory changes occurring in K(ATP) channel, NO, and/or EDHF responses when one is individually blocked.