Sulforaphane Protects Rodent Retinas against Ischemia-Reperfusion Injury through the Activation of the Nrf2/HO-1 Antioxidant Pathway

Retinal ischemia-reperfusion (I/R) injury induces oxidative stress, leukocyte infiltration, and neuronal cell death. Sulforaphane (SF), which can be obtained in cruciferous vegetables such as broccoli, exerts protective effects in response to oxidative stress in various tissues. These effects can be initiated through nuclear factor E2-related factor 2 (Nrf2)-mediated induction of heme oxygenase-1 (HO-1). This investigation was designed to elucidate the neural protective mechanisms of SF in the retinal I/R rat model. Animals were intraperitoneally (i.p.) injected with SF (12.5 mg/kg) or vehicle (corn oil) once a day for 7 consecutive days. Then, retinal I/R was made by elevating the intraocular pressure (IOP) to 130 mmHg for 1 h. To determine if HO-1 was involved in the Nrf2 antioxidant pathway, rats were subjected to protoporphyrin IX zinc (II) (ZnPP, 30 mg/kg, i.p.) treatments at 24 h before retinal ischemia. The neuroprotective effects of SF were assessed by determining the morphology of the retina, counting the infiltrating inflammatory cells and the surviving retinal ganglion cells (RGCs) and amacrine cells, and measuring apoptosis in the retinal layers. The expression of Nrf2 and HO-1 was studied by immunofluorescence analysis and western blotting. I/R induced a marked increase of ROS generation, caused pronounced inflammation, increased the apoptosis of RGCs and amacrine cells and caused the thinning of the inner retinal layer (IRL), and these effects were diminished or abolished by SF pretreatment. Meanwhile, SF pretreatment significantly elevated the nuclear accumulation of Nrf2 and the level of HO-1 expression in the I/R retinas; however, ZnPP reversed the protective effects of SF on I/R retinas. Together, we offer direct evidence that SF had protective effects on I/R retinas, which could be attributed, at least in part, to the activation of the Nrf2/HO-1 antioxidant pathway.     

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways.     

Long Noncoding RNA-Sox2OT Knockdown Alleviates Diabetes Mellitus-Induced Retinal Ganglion Cell (RGC) injury

Retinal ganglion cell (RGC) injury is one of the important pathological features of diabetes-induced retinal neurodegeneration. Increasing attention has been paid to find strategies for protecting against RGC injury. Long noncoding RNAs (lncRNAs) have emerged as the key regulators of many cell functions. Here, we show that Sox2OT expression is significantly down-regulated in the retinas of STZ-induced diabetic mice and in the RGCs upon high glucose or oxidative stress. SOX2OT knockdown protects RGCs against high glucose-induced injury in vitro. Moreover, Sox2OT knockdown plays a neuroprotective role in diabetes-related retinal neurodegeneration in vivo. Sox2OT knockdown could regulate oxidative stress response in RGCs and diabetic mouse retinas. Sox2OT knockdown plays an anti-oxidative role via regulating NRF2/HO-1 signaling activity. Taken together, Sox2OT knockdown may be a therapeutic strategy for the prevention and treatment of diabetes-induced retinal neurodegeneration.     

Effects of antioxidant gene therapy on retinal neurons and oxidative stress in a model of retinal ischemia/reperfusion

Retinal ischemia/reperfusion (I/R) results in neuronal death and generation of reactive oxygen species. The aim of this study was to investigate the neuroprotective effect of manganese superoxide dismutase (SOD2) on retinal ganglion cells (RGCs) in an I/R-induced retinal injury model. One eye of each Wistar rat was pretreated with recombinant adeno-associated virus containing the SOD2 gene (AAV-SOD2) or recombinant AAV containing the GFP gene (AAV-GFP) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure for 1h, and reperfusion was established immediately afterward. The number of RGCs and the inner plexiform layer (IPL) thickness were measured by Fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h, and 5 days after injury. Superoxide anion, the number of RGCs, IPL thickness, malondialdehyde (MDA) level, 8-hydroxy-2-deoxyguanosine (8-OHdG) level, MnSOD (manganese superoxide dismutase) activity, and nitrotyrosine level were measured by fluorescence staining, immunohistochemistry, and enzyme-linked immunosorbent analysis at 5 days after I/R injury. Severe RGC loss, reduced IPL thickness, reduced MnSOD activity, and increased superoxide ion, MDA, 8-OHdG, and nitrotyrosine production were observed after I/R injury. Administration of AAV-SOD2 significantly reduced the levels of superoxide ion, MDA, 8-OHdG, and nitrotyrosine and prevented the damage to RGCs and IPL. Delivery of the antioxidant gene inhibited I/R-induced RGC and IPL damage by reducing oxidative stress and nitrative stress, suggesting that MnSOD may be relevant for the neuroprotection of the inner retina from I/R-related diseases.     

CircRNA expression profile and functional analysis in retinal ischemia-reperfusion injury

Retinal ischemia-reperfusion (I/R) is involved in the pathogenesis of many vision-threatening diseases. circRNAs act as key players in gene regulation and human diseases. However, the global circRNA expression profile in retinal I/R injury has not been fully uncovered. Herein, we established a murine model of retinal I/R injury and performed circRNA microarrays to identify I/R-related circRNAs. 1265 differentially expressed circRNAs were identified between I/R retinas and normal retinas. Notably, the detection of cWDR37 level in aqueous humor could discriminate glaucoma patients from cataract patients (AUC = 0.9367). cWdr37 silencing protected against hypoxic stress- or oxidative stress-induced retinal ganglion cell (RGC) injury. cWdr37 silencing alleviated IR-induced retinal neurodegeneration as shown by increased NeuN staining, reduced retinal reactive gliosis, and decreased retinal apoptosis. Collectively, this study provides a novel insight into the pathogenesis of retinal I/R injury. cWdr37 is a promising target for the diagnosis or treatment of I/R-related ocular diseases.     

Up-regulated Endogenous Erythropoietin/Erythropoietin Receptor System and Exogenous Erythropoietin Rescue Retinal Ganglion Cells after Chronic Ocular Hypertension

Aims Recent studies have showed that erythropoietin (EPO) is a neuroprotectant for central nerve system neurons in addition to being a hematopoietic cytokine in response to hypoxia. In this study, we investigate the role of the EPO/EPO receptor (EPOR) system in the rat retina after ocular hypertension injury that mimics glaucoma. Methods Elevated intraocular pressure was induced by laser coagulation of the episcleral and limbal veins. Expression of EPO and EPOR in the normal and glaucomous retinas was investigated by immunohistochemistry and Western blot. To examine the effects of endogenous EPO on the survival of retinal ganglion cells (RGCs) subjected to hypertensive injury, soluble EPOR was directly injected into the vitreous body. Recombinant human EPO was both intravitreally and systemically administrated to study the effect of exogenous EPO on the survival of RGCs after ocular hypertension injury. Results Immunohistochemistry studies identified Müller cells as the main source of EPO in the normal retina. Expression of EPO and EPOR proteins was increased significantly 2 weeks after ocular hypertension. RGCs, amacrine and bipolar cells all demonstrated an increased expression of EPOR after ocular hypertension. Neutralization of endogenous EPO with soluble EPOR exacerbated ocular hypertensive injury, suggesting a role of the EPO/EPOR system in the survival of RGCs after injury. Similarly, topical and systemic administration of recombinant human EPO rescues RGCs after chronic ocular hypertension. Conclusions These results indicate that an endogenous EPO/EPOR system participates in intrinsic recovery mechanisms after retina injury and RGCs might be rescued by exogenous administration of EPO.     

microRNA-193b protects against myocardial ischemia-reperfusion injury in mouse by targeting mastermind-like 1

The current study aimed to explore the functions and roles of microRNA-193b (miR-193b) in the myocardium with ischemia-reperfusion (I/R) injury and a potential therapeutic method for myocardial I/R injury. The mice were subjected to myocardial I/R with or without miR-193b pretreatment. The infarct size and myocardial enzymes were detected. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was conducted to investigate the effect of miR-193b on cardiomyocyte apoptosis. The expression levels of miR-193b and mastermind-like 1 (MAML1) were validated by quantitative real-time polymerase chain reaction and Western blot analysis. The results suggested that the miR-193b expression level was significantly downregulated in the myocardium with I/R injury compared with control group. miR-193b overexpression is able to reduce infarct size and myocardial enzymes after myocardial I/R injury. Furthermore, overexpression of miR-193b could alleviate the apoptosis level after myocardial I/R injury. Taken together, the present study demonstrated that upregulated miRNA-193b alleviated myocardial I/R injury via targeting MAML1.     

Deficiency for the Chemokine Monocyte Chemoattractant Protein-1 Aggravates Tubular Damage after Renal Ischemia/Reperfusion Injury

Temporal expression of chemokines is a crucial factor in the regulation of renal ischemia/reperfusion (I/R) injury and repair. Beside their role in the migration and activation of inflammatory cells to sites of injury, chemokines are also involved in other processes such as angiogenesis, development and migration of stem cells. In the present study we investigated the role of the chemokine MCP-1 (monocyte chemoattractant protein-1 or CCL2), the main chemoattractant for monocytes, during renal I/R injury. MCP-1 expression peaks several days after inducing renal I/R injury coinciding with macrophage accumulation. However, MCP-1 deficient mice had a significant decreased survival and increased renal damage within the first two days, i.e. the acute inflammatory response, after renal I/R injury with no evidence of altered macrophage accumulation. Kidneys and primary tubular epithelial cells from MCP-1 deficient mice showed increased apoptosis after ischemia. Taken together, MCP-1 protects the kidney during the acute inflammatory response following renal I/R injury.     

Autophagy in Retinal Ganglion Cells in a Rhesus Monkey Chronic Hypertensive Glaucoma Model

Primary open angle glaucoma (POAG) is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs). In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi) and late or degraded autophagic vacuoles (AVd) accumulated in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL) as determined by transmission electron microscopy (TEM) analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1) and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

Important role of apoptosis signal-regulating kinase 1 in ischemic acute kidney injury

We investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Blood urea nitrogen (BUN) and serum creatinine were significantly higher in ASK1+/+ mice than in ASK1−/− mice after I/R injury. Renal histology of ASK1+/+ mice showed significantly greater tubular necrosis and degradation. In ASK1−/− mice, phosphorylation of ASK1, JNK, and p38K, and the number of TUNEL-positive cells and infiltrated leukocytes decreased after I/R injury. Apoptotic changes were significantly decreased in cultured renal tubular epithelial cells (TECs) from ASK1−/− mice under hypoxic condition. Transfection with dominant-active ASK1 induced apoptosis in TECs. Protein expression of monocyte chemoattractant protein-1 (MCP-1) was significantly weaker in ASK1−/− mice after I/R injury. Transfection with dominant negative-ASK1 significantly decreased MCP-1 production in TECs. These results demonstrated that ASK1 is activated in I/R-induced AKI, and blockage of ASK1 attenuates renal tubular apoptosis, MCP-1 expression, and renal function.     

Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis,mitochondrial biogenesis and insulin sensitivity

Oncostatin M (OSM) exhibits many unique biological activities by activating Oβ receptor. However, its role in myocardial I/R injury in diabetic mice remains unknown. The involvement of OSM was assessed in diabetic mice which underwent myocardial I/R injury by OSM treatment or genetic deficiency of OSM receptor Oβ. Its mechanism on cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity were further studied. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through inhibition of inositol pyrophosphate 7 (IP7) production, thus activating PI3K/Akt/BAD pathway, decreasing Bax expression while up‐regulating Bcl‐2 expression and decreasing the ratio of Bax to Bcl‐2 in db/db mice. OSM enhanced mitochondrial biogenesis and mitochondrial function in db/db mice subjected to cardiac I/R injury. On the contrary, OSM receptor Oβ knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in cardiac I/R injured diabetic mice. Inhibition of IP7 production by TNP (IP6K inhibitor) exerted similar effects of OSM. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and adenine mononucleotide protein kinase/PGC‐1α pathway. OSM protects against cardiac I/R Injury by regulating apoptosis, insulin sensitivity and mitochondrial biogenesis in diabetic mice through inhibition of IP7 production.     

Apoptosis and activation of Erkl/2 and Akt in astrocytes postischemia

We have shown previously that in vitro ischemia could induce apoptosis in primary culture of astrocytes. In this paper we demonstrate that astrocytes in culture could undergo apoptosis during in vitro incubation postischemia. We also measured the changes of phosphorylated Erk1/2 (p-Erk1/2) and phosphorylated Akt (p-Akt) in order to determine whether these two pathways play a role in apoptosis. After 4 h in vitro ischemic incubation of cultured astrocytes, a limited amount of nuclear condensation was demonstrated by Hoechst 33342 staining. When ischemic incubation was halted and the cultures transferred to standard normoxic incubation (postischemia) conditions, DNA fragmentation and apoptosis were demonstrated by TUNEL and DNA laddering analysis. TUNEL-positive astrocytes began to appear at 6 h postischemia and increased in number from 12 h postischemia. Western blot analysis showed that both p-Erk1/2 and p-Akt were elevated in astrocytes subjected to 4 h of ischemia. Elevated p-Erk1/2 levels were sustained during the postischemia incubation for 12 h and decreased significantly afterward, but did not return to the levels in the control cultures that did not experience ischemic insult. In contrast, the p-Akt level continued to increase at 6 and 12 h postischemia before declining significantly. The changes in p-Erk1/2 and p-Akt correlated well with the appearance of apoptotic astrocytes in the culture.     

Dynamic alteration of adiponectin/adiponectin receptor expression and its impact on myocardial ischemia/reperfusion in type 1 diabetic mice

The present study determined the dynamic change of adiponectin (APN, a cardioprotective adipokine), its receptor expression, and their impact upon myocardial ischemia/reperfusion (MI/R) injury during type 1 diabetes mellitus (T1DM) progression, and involved underlying mechanisms. Diabetic state was induced in mice via multiple intraperitoneal injections of low-dose streptozotocin. The dynamic change of plasma APN concentration and cardiac APN receptor-1 and -2 (AdipoR1/2) expression were assessed immediately after diabetes onset (0 wk) and 1, 3, 5, and 7 wk thereafter. Indicators of MI/R injury (infarct size, apoptosis, and LDH release) were determined at 0, 1, and 7 wk of DM duration. The effect of APN on MI/R injury was determined in mice subjected to different diabetic durations. Plasma APN levels (total and HMW form) increased, whereas cardiac AdipoR1 expression decreased early after T1DM onset. With T1DM progression, APN levels were reduced and cardiac AdipoR1 expression increased. MI/R injury was exacerbated with T1DM progression in a time-dependent manner. Administration of globular APN (gAD) failed to attenuate MI/R injury in 1-wk T1DM mice, while an AMP-activated protein kinase (AMPK) activator (AICAR) reduced MI/R injury. However, administration of gAD (and AICAR) reduced infarct size and cardiomyocyte apoptosis in 7-wk T1DM mice. In conclusion, our results demonstrate a dynamic dysfunction of APN/AdipoR1 during T1DM progression. Reduced cardiac AdipoR1 expression and APN concentration may be responsible for increased I/R injury susceptibility at early and late T1DM stages, respectively. Interventions bolstering AdipoR1 expression during early T1DM stages and APN supplementation during advanced T1DM stages may potentially reduce the myocardial ischemic injury in diabetic patients.     

Myocardial Ablation of G Protein–Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein–coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.     

Dietary methionine restriction attenuates renal ischaemia/reperfusion‐induced myocardial injury by activating the CSE/H2S/ERS pathway in diabetic mice

Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)‐induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine‐restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R‐induced mediated myocardial injury and apoptosis were partially reversed by methionine‐restricted diet (MRD) via the down‐regulation of CSE expression and up‐regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose‐dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R‐induced myocardial injury.     

AGGF1 protects from myocardial ischemia/reperfusion injury by regulating myocardial apoptosis and angiogenesis

Angiogenic factor with G patch and FHA domains 1 (AGGF1) is a newly identified proangiogenic protein, which plays an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. This study investigated whether AGGF1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Wild-type (WT) C57BL/6 J mice were treated at 30 min prior to I/R injury with anti-AGGF1 neutralizing antibody (3 mg/kg) or recombinant human AGGF1 (rhAGGF1, 0.25 mg/kg). After I/R injury, the infarct size, the number of TUNEL-positive cardiomyocytes, Bax/Bcl2 ratio, inflammatory cytokine expression and angiogenesis were markedly increased as compared with sham control. Treatment of WT mice with anti-AGGF1 neutralizing antibody resulted in exaggeration of myocardial I/R injury but reducing angiogenesis. In contrast, administration of rhAGGF1 markedly reversed these effects. Furthermore, anti-AGGF1- or rhAGGF1-mediated effects on I/R-induced cardiac apoptosis, inflammation and angiogenesis were dose dependent. In addition, the protective effects of AGGF1 on cardiomyocyte apoptosis and inflammation were confirmed in cultured cardiomyocytes after I/R. Finally, these effects were associated with activation of ERK1/2, Stat3 and HIF-1α/VEGF pathways and inhibition of activation of NF-κB, p53 and JNK1/2 pathways. In conclusion, we report the first in vivo and in vitro evidence that AGGF1 reduces myocardial apoptosis and inflammation and enhances angiogenesis, leading to decreased infarct size after I/R injury. These results may provide a novel therapeutic approach for ischemic heart diseases.