TNF-α inhibitor protects against myocardial ischemia/reperfusion injury via Notch1-mediated suppression of oxidative/nitrative stress

TNF-α inhibitor reportedly protects against myocardial ischemia/reperfusion (MI/R) injury. It can also increase Notch1 expression in inflammatory bowel disease, revealing the regulation of Notch1 signaling by TNF-α inhibitor. However, the interaction between TNF-α inhibitor and Notch1 signaling in MI/R remains unclear. This study aimed to determine the involvement of TNF-α inhibitor with Notch1 in MI/R and delineate the related mechanism. Notch1-specific small interfering RNA (20 μg) or Jagged1 (a Notch ligand, 12 μg) was delivered through intramyocardial injection. Forty-eight hours after injection, mice received 30 min of myocardial ischemia followed by 3 h (for cell apoptosis and oxidative/nitrative stress) or 24 h (for infarct size and cardiac function) of reperfusion. Ten minutes before reperfusion, mice randomly received an intraperitoneal injection of vehicle, etanercept, diphenyleneiodonium, 1400W, or EUK134. Finally, downregulation of Notch1 significantly reversed the alleviation of MI/R injury induced by etanercept, as evidenced by enlarged myocardial infarct size, suppressed cardiac function, and increased myocardial apoptosis. Moreover, Notch1 blockade increased the expression of inducible NO synthase (iNOS) and gp^91phox, enhanced NO and superoxide production, and accelerated their cytotoxic reaction product, peroxynitrite. Furthermore, NADPH inhibition with diphenyleneiodonium or iNOS suppression with 1400W mitigated the aggravation of MI/R injury induced by Notch1 downregulation in mice treated with etanercept. Additionally, either Notch1 activation with Jagged1 or peroxynitrite decomposition with EUK134 reduced nitrotyrosine content and attenuated MI/R injury. These data indicate that MI/R injury can be attenuated by TNF-α inhibitor, partly via Notch1 signaling-mediated suppression of oxidative/nitrative stress.     

Diverse effects of L-arginine on cardiac function of rats subjected to myocardial ischemia and reperfusion in vivo

In vivo administration of L-arginine at different time points during the course of myocardialischemia and reperfusion(MI/R)has been shown to differentially regulate postischemic apoptosis.Cardiacfunction is one of the most important indexes used to judge the degree of myocardial injury.The presentstudy attempted to determine whether in vivo administration of L-arginine at different stages of MI/R has adiverse influence on cardiac function of ischemic reperfused hearts and,if so,to investigate the mechanismsinvolved.Male adult rats were subjected to 30 min myocardial ischemia followed by 5 h reperfusion.Anintravenous L-arginine bolus was given either 10 min before and 50 min after reperfusion(early treatment)or3 h and 4 h after reperfusion(late treatment).Early treatment with L-arginine markedly increased the leftventricular systolic pressure(LVSP)and dP/dt_(max),and decreased myocardial nitrotyrosine content.In strictcontrast,late treatment with L-arginine resulted in a significant decrease in LVSP and dP/dt_(max)from 4 h to 5h after reperfusion,and increase in toxic peroxynitrite formation as measured by nitrotyrosine.These resultssuggest that the administration of L-arginine at different time points during the course of MI/R leads todiverse effects on cardiac dysfunction.Early supplementation decreased the nitrative stress and improvedleft ventricular function.However,late treatment with L-arginine increased the formation of peroxynitriteand aggravated cardiac functional injury.     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

Tumor necrosis factor-alpha in mechanic trauma plasma mediates cardiomyocyte apoptosis

Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation (P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-alpha, IL-1beta, and IFN-gamma) or TNF-alpha alone, but not with IL-1beta or IFN-gamma alone, caused significant caspase-3 activation (P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-alpha antibody, and TP isolated from TNF-alpha(-/-) mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-alpha (anti-TNF-alpha antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) (P < 0.01). Taken together, our study demonstrated that there exists a TNF-alpha-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.     

Differential patterns of peroxynitrite mediated apoptosis in proximal tubular epithelial cells following ATP depletion recovery

Ischemia-reperfusion injury (IRI) is characterized by ATP depletion in the ischemic phase, followed by a rapid increase in reactive oxygen species, including peroxynitrite in the reperfusion phase. In this study, we examined the role of peroxynitrite on cytotoxicity and apoptosis in an in vitro model of ATP depletion-recovery. Porcine proximal tubular epithelial (LLC-PK₁) cells were ATP depleted for either 2 h (2/2) or 4 h (4/2) followed by recovery in serum free medium for 2 h. A subset of cells was treated with 100 μM of the peroxynitrite scavenger, iron (III) tetrakis (N-methyl-4′pyridyl) porphyrin pentachloride (FeTMPyP) 30 min prior to and during treatment/recovery. Treatment with FeTMPyP reduced cytotoxicity and superoxide levels at both the 2/2 and 4/2 time points, however FeTMPyP decreased nitric oxide only at the 2/2 time point. FeTMPyP also partially blocked caspase-3 and caspase-8 activation at both 2/2 and 4/2 time points. At the 4/2 time point, FeTMPyP also partially inhibited the ATP depletion mediated increase in tumor necrosis factor alpha (TNF-α) and decreased Bax and FasL gene expression. These data show that peroxynitrite induces apoptosis by activation of multiple pathways depending on length and severity of insult following ATP depletion-recovery.     

Nitrative thioredoxin inactivation as a cause of enhanced myocardial ischemia/reperfusion injury in the aging heart

Several recent studies have demonstrated that thioredoxin (Trx) is an important antiapoptotic/cytoprotective molecule. The present study was designed to determine whether Trx activity is altered in the aging heart in a way that may contribute to increased susceptibility to myocardial ischemia/reperfusion (MI/R). Compared to young animals, MI/R-induced cardiomyocyte apoptosis and infarct size were increased in aging animals (p<0.01). Trx activity was decreased in the aging heart before MI/R, and this difference was further amplified after MI/R. Trx expression was moderately increased and Trx nitration, a posttranslational modification that inhibits Trx activity, was increased in the aging heart. Moreover, Trx-aptosis-regulating kinase-1 (Trx-ASK1) complex formation was reduced and activity of p38 mitogen-activated protein kinase (MAPK) was increased. Treatment with FP15 (a peroxynitrite decomposition catalyst) reduced Trx nitration, increased Trx activity, restored Trx-ASK1 interaction, reduced P38 MAPK activity, attenuated caspase 3 activation, and reduced infarct size in aging animals (p<0.01). Our results demonstrated that Trx activity is decreased in the aging heart by posttranslational nitrative modification. Interventions that restore Trx activity in the aging heart may be novel therapies to attenuate MI/R injury in aging patients.     

Protective Effect of Metalloporphyrins against Cisplatin-Induced Kidney Injury in Mice

Oxidative and nitrative stress is a well-known phenomenon in cisplatin-induced nephrotoxicity. The purpose of this work is to study the role of two metalloporphyrins (FeTMPyP and MnTBAP), water soluble complexes, in cisplatin-induced renal damage and their ability to scavenge peroxynitrite. In cisplatin-induced nephropathy study in mice, renal nitrative stress was evident by the increase in protein nitration. Cisplatin-induced nephrotoxicity was also evident by the histological damage from the loss of the proximal tubular brush border, blebbing of apical membranes, tubular epithelial cell detachment from the basement membrane, or intra-luminal aggregation of cells and proteins and by the increase in blood urea nitrogen and serum creatinine. Cisplatin-induced apoptosis and cell death as shown by Caspase 3 assessments, TUNEL staining and DNA fragmentation Cisplatin-induced nitrative stress, apoptosis and nephrotoxicity were attenuated by both metalloporphyrins. Heme oxygenase (HO-1) also plays a critical role in metalloporphyrin-mediated protection of cisplatin-induced nephrotoxicity. It is evident that nitrative stress plays a critical role in cisplatin-induced nephrotoxicity in mice. Our data suggest that peroxynitrite is involved, at least in part, in cisplatin-induced nephrotoxicity and protein nitration and cisplatin-induced nephrotoxicity can be prevented with the use of metalloporphyrins.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia–reperfusion injury in hypertrophic heart

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91^phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.     

Endophilin A2-mediated alleviation of endoplasmic reticulum stress-induced cardiac injury involves the suppression of ERO1α/IP3R signaling pathway

Cardiac injury upon myocardial infarction (MI) is the leading cause of heart failure. The present study aims to investigate the role of EndoA2 in ischemia-induced cardiomyocyte apoptosis and cardiac injury. In vivo, we established an MI mouse model by ligating the left anterior descending (LAD) coronary artery, and intramyocardial injection of adenoviral EndoA2 (Ad-EndoA2) was used to overexpress EndoA2. In vitro, we used the siRNA and Ad-EndoA2 transfection strategies. Here, we reported that EndoA2 expression was remarkably elevated in the infarct border zone of MI mouse hearts and neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen and glucose deprivation (OGD) which mimicked ischemia. We showed that intramyocardial injection of Ad-EndoA2 attenuated cardiomyocyte apoptosis and reduced endoplasmic reticulum (ER) stress in response to MI injury. Using siRNA for knockdown and Ad-EndoA2 for overexpression, we validated that knockdown of EndoA2 in NRCMs exacerbated OGD-induced NRCM apoptosis, whereas overexpression of EndoA2 attenuates OGD-induced cardiomyocyte apoptosis. Mechanistically, knockdown of EndoA2 activated ER stress response, which increases ER oxidoreductase 1α (ERO1α) and inositol 1, 4, 5-trisphosphate receptor (IP₃R) activity, thus led to increased intracellular Ca²⁺ accumulation, followed by elevated calcineurin activity and nuclear factor of activated T-cells (NFAT) dephosphorylation. Pretreatment with the IP₃R inhibitor 2-Aminoethoxydiphenylborate (2-APB) attenuated intracellular Ca²⁺ accumulation, and pretreatment with the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the calcineurin inhibitor Cyclosporin A (CsA) inhibited EndoA2-knockdown-induced NRCM apoptosis. Overexpression of EndoA2 led to the opposite effects by suppressing ER-stress-mediated ERO1α/IP₃R signaling pathway. This study demonstrated that EndoA2 protected cardiac function in response to MI via attenuating ER-stress-mediated ERO1α/IP₃R signaling pathway. Targeting EndoA2 is a potential therapeutic strategy for the prevention of postinfarction-induced cardiac injury and heart failure.     

Inhibition of inducible nitric oxide synthase attenuates platelet adhesion in subpleural arterioles caused by lung ischemia-reperfusion in rabbits.

Oxidative stress, induced by lung ischemia-reperfusion, leads to platelet and leukocyte activation and may contribute to decreased alveolar perfusion by platelet adhesion to the arteriolar wall. We investigated the hypothesis that ischemia-reperfusion injury increases inducible nitric oxide synthase (iNOS) activity and subsequent generation of reactive nitrogen species with P-selectin-dependent platelet-endothelial interactions and vasoconstriction during lung reperfusion. Subpleural arterioles, labeled platelets, and leukocytes were examined in anesthetized, open-chest rabbits by intravital fluorescence microscopy. Ischemia was caused by reversible occlusion of the right pulmonary artery for 1 or 2 h (1IR and 2IR groups). During 2 h of reperfusion, postischemic platelet rolling and adhesion were independent from leukocyte-arteriolar wall interactions and correlated with pulmonary arteriolar constriction in proportion to the length of ischemia. In rabbits treated with an iNOS inhibitor (1400W) before occlusion (2IR + 1400W group), platelet-arteriolar wall interactions and vasoconstriction were prevented. iNOS expression and activity in ischemic lung tissue were markedly greater than control and also were proportional to ischemia duration. NOS activity, immunochemically detected P-selectin, and nitrotyrosine expression in ischemic lung tissue from animals subjected to ischemia-reperfusion, as well as the plasma level of soluble P-selectin, were significantly higher than in nonischemic lungs and were inhibited by pretreatment with 1400W. These results show that platelet adhesion and arteriolar constriction during early reperfusion in the ventilated lung can result from increased iNOS activity and is highly correlated with reactive nitrogen species and P-selectin expression.     

Nitrate tolerance aggravates postischemic myocardial apoptosis and impairs cardiac functional recovery after ischemia

Objectives: This study examined the effects of nitrate tolerance (NT) on myocardial ischemia reperfusion (MI/R) injury and elucidated the potential mechanisms involved. Furthermore, the effects of GSH on postischemic myocardial apoptosis in NT rats were investigated. Methods and results: Male Sprague–Dawley rats were randomized to receive nitroglycerin (60 μg/kg/h) or saline for 12 h followed by 40 min of MI and 4 h of reperfusion. Myocardial apoptosis, infarct size, nitrotyrosine formation, plasma CK and LDH activity, and cardiac function were determined. MI/R resulted in significant apoptotic cell death, which was further increased in animals with NT. In addition, NT further increased plasma CK and LDH activity, enlarged infarct size, and impaired cardiac functional recovery after ischemia. Myocardial nitrotyrosine, a footprint for cytotoxic reactive nitrogen species formation, was further enhanced in the NT heart after MI/R. Treatment of NT animals with exogenous GSH inhibited nitrotyrosine formation, reduced apoptosis, decreased infarct size, and improved cardiac functional recovery. Conclusion: Our results demonstrate that nitrate tolerance markedly enhances MI/R injury and that increased peroxynitrite formation likely plays a role in this pathologic process. In addition, our results suggest that GSH could decrease peroxynitrite formation and reduce MI/R injury in nitrate tolerant hearts.     

α-Lipoic Acid Reduces Infarct Size and Preserves Cardiac Function in Rat Myocardial Ischemia/Reperfusion Injury through Activation of PI3K/Akt/Nrf2 Pathway

Background

The present study investigates the effects and mechanisms of α-Lipoic acid (LA) on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in rat hearts subjected to in vivo myocardial ischemia/reperfusion (MI/R) injury.

Methodology/Principal Findings

Male adult rats underwent 30 minutes of ischemia followed by 3, 24, or 72 h of reperfusion. Animals were pretreated with LA or vehicle before coronary artery ligation. The level of MI/R- induced LDH and CK release, infarct size, cardiomyocyte apoptosis and cardiac functional impairment were examined and compared. Western blot analysis was performed to elucidate the mechanism of LA pretreatment. The level of inflammatory cytokine TNF-α released to serum and accumulated in injured myocardium as well as neutrophil accumulation in injured myocardium were also examined after MI/R injury. Our results reveal that LA administration significantly reduced LDH and CK release, attenuated myocardial infarct size, decreased cardiomyocytes apoptosis, and partially preserved heart function. Western blot analysis showed that LA pretreatment up-regulated Akt phosphorylation and Nrf2 nuclear translocation while producing no impact on p38MAPK activation or nitric oxide (NO) production. LA pretreatment also increased expression of HO-1, a major target of Nrf2. LA treatment inhibited neutrophil accumulation and release of TNF-α. Moreover, PI3K inhibition abolished the beneficial effects of LA.

Conclusions/Significance

This study indicates that LA attenuates cardiac dysfunction by reducing cardiomyoctyes necrosis, apoptosis and inflammation after MI/R. LA exerts its action by activating the PI3K/Akt pathway as well as subsequent Nrf2 nuclear translocation and induction of cytoprotective genes such as HO-1.     

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.     

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30?min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.     

Delineation of the effects of angiotensin type 1 and 2 receptors on HL-1 cardiomyocyte apoptosis

Angiotensin II (Ang II) exerts its effects by activating its receptors, primarily type 1 (AT1R) and type 2 (AT2R). While the role of AT1R activation in cardiomyocyte physiology is well known, the role of AT2R in cardiomyocyte apoptosis remains controversial. To define the precise role of AT1R and AT2R in this process, we transfected HL-1 cardiomyocytes with AT1R or AT2R cDNA, and examined markers of apoptosis. We found that AT1R overexpression was associated with upregulation of endogenous AT2R expression, but AT2R overexpression did not affect endogenous AT1R expression. Caspase-3 staining indicated that overexpression of AT1R as well as AT2R resulted in cardiomyocyte apoptosis with appropriate alterations in annexin V, Bax and Bcl2 expression. Overexpression of AT1R and AT2R markedly increased IL-1β (AT2R>AT1R), iNOS (AT2R>AT1R) and eNOS expression. AT2R-induced cell apoptosis could be blocked by the iNOS selective inhibitor 1,400?W, and did not require exogenous Ang II. These findings suggest that AT2R overexpression induces cardiomyocyte apoptosis, most likely via iNOS upregulation. AT1R-mediated cardiomyocyte apoptosis may be partially mediated by upregulation of endogenous AT2R.     

Intestinal ischemic preconditioning: Less xanthine accumulation relates with less apoptosis

Ischemic preconditioning has shown to reduce apoptosis in the intestinal mucosa during ischemia/reperfusion. This study evaluated if the decrease of apoptotic events found during preconditioning could be related with a reduction of the substrate (i.e., xanthine/hypoxanthine) available for xanthine oxidase (XO). Animals were randomly assigned to the following study groups: C, control; I/R, ischemia/reperfusion; P+I/R, ischemic preconditioning; P+I/R+H/X, ischemic preconditioning plus hypoxanthine/xanthine, and P+I/R+H/X+Allo, ischemic preconditioning plus hypoxanthine/xanthine plus allopurinol. Caspase-3 activity, DNA fragmentation and TUNEL staining increased in the I/R group compared to control. Ischemic preconditioning (P+I/R group) was able to reverse these apoptotic variables to a level similar to that of control rats. The addition of hypoxanthine/xanthine to rats subjected to ischemic preconditioning (P+I/R+H/X group) showed the highest apoptotic activity; however, further addition of allopurinol (P+I/R+H/X+Allo group) decreased significantly apoptotic activity and events. In conclusion, intestinal ischemic preconditioning is able to reduce apoptosis during the following sustained ischemia/reperfusion event because of a reduced accumulation of xanthine/hypoxanthine nucleotide.     

Differential response to myocardial reperfusion injury in eNOS-deficient mice

Two strains of endothelial nitric oxide synthase (eNOS)-deficient (-/-) mice have been developed that respond differently to myocardial ischemia-reperfusion (MI/R). We evaluated both strains of eNOS(-/-) mice in an in vivo model of MI/R. Harvard (Har) eNOS(-/-) mice (n = 12) experienced an 84% increase in myocardial necrosis compared with wild-type controls (P < 0.05). University of North Carolina (UNC) eNOS(-/-) (n = 10) exhibited a 52% reduction in myocardial injury versus wild-type controls (P < 0.05). PCR analysis of myocardial inducible NO synthase (iNOS) mRNA levels revealed a significant (P < 0.05) increase in the UNC eNOS(-/-) mice compared with wild-type mice, and there was no significant difference between the Har eNOS(-/-) and wild-type mice. UNC eNOS(-/-) mice treated with an iNOS inhibitor (1400W) exacerbated the extent of myocardial necrosis. When treated with 1400W, Har eNOS(-/-) did not exhibit a significant increase in myocardial necrosis. These data demonstrate that two distinct strains of eNOS(-/-) mice display opposite responses to MI/R. Although the protection seen in the UNC eNOS(-/-) mouse may result from compensatory increases in iNOS, other genes may be involved.     

Etanercept Attenuates Myocardial Ischemia/Reperfusion Injury by Decreasing Inflammation and Oxidative Stress

The protective role of etanercept in myocardial ischemia/reperfusion is not well understood. The aim of this study was to investigate whether etanercept modulates neutrophil accumulation, TNF-α induction and oxidative stress in an ischemia/reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia/reperfusion (MI/R) alone, MI/R+ etanercept. The results demonstrated that compared to MI/R, etanercept reduced myocardial infarction area, myocardial myeloperoxidase (MPO) levels, serum creatinine kinase (CK) and lactate dehydrogenase (LDH) levels, and both serum and myocardial TNF-α production. Etanercept also markedly enhanced the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and reduced the level of malondialdehyde (MDA) in MI/R rats. In summary, our data suggested that etanercept has protective effects against MI/R injury in rats, which may be attributed to attenuating inflammation and oxidative stress.