Reduced Infarct Area Research Articles

Guanxin Danshen Formulation improved the effect of mesenchymal stem cells transplantation for the treatment of myocardial infarction probably via enhancing the engraftment

Although intravenous injection is the most convenient and feasible approach for mesenchymal stem cells (MSCs) delivery, the proportion of donor stem cells in the target myocardium after transplantation is small. It is believed that TCM enhances the effect of stem cell therapy by improving the hostile microenvironment and promoting the migration and survival of stem cells. Guanxin Danshen (GXDS) formulation is one of the main prescriptions for clinical treatment of ischemic heart diseases in China. The purpose of this study was to evaluate the effects of GXDS formulation administration combined with MSCs transplantation on cardiac function improvement, apoptosis, angiogenesis and survival of transplanted cells in an acute model of acute myocardial infarction (MI). After being labeled with GFP, MSCs were transplanted via intravenous injection. Meanwhile, GXDS dripping pills were given by intragastric administration for 4 weeks from 2 days before MI. Echocardiography showed moderate improvement in cardiac function after administration of GXDS formulation or intravenous transplantation of MSCs. However, GXDS formulation combined with MSCs transplantation significantly improved cardiac function after MI. The myocardial infarct size in rats treated with MSCs was similar to that in rats treated with GXDS formulation. However, GXDS formulation combined with MSCs transplantation significantly reduced infarction area. In addition, GXDS formulation combined with MSCs transplantation not only decreased cell apoptosis according to the TUNEL staining, but also enhanced angiogenesis in the peri-infarction and infarction area. Interestingly, the use of GXDS formulation increased the number of injected MSCs in the infarct area. Furthermore, GXDS formulation combined with MSCs transplantation increased SDF-1 levels in the infarcted area, but did not affect the expression of YAP. Our study provided a more feasible and accessible strategy to enhance the migration of stem cells after intravenous injection by oral administration of GXDS formulation. The combination of GXDS formulation and stem cell therapy has practical significance and application prospects in the treatment of ischemic cardiomyopathy such as MI.

Intrathecal lentivirus-mediated RNA interference targeting nerve growth factor attenuates myocardial ischaemia–reperfusion injury in rat

BackgroundNerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia–reperfusion injury and explored the underlying mechanisms. MethodsIn adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia–reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. ResultsNGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia–reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5′-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. ConclusionsSpinal NGF contributes to myocardial ischaemia–reperfusion injury by mediating nociceptive signal transmission.

Effect of miR-26a targeting GSK-3β/β-catenin signaling pathway on myocardial apoptosis in rats with myocardial ischemia-reperfusion.

The aim of this study was to evaluate the effect of micro ribonucleic acid (miR)-26a on myocardial ischemia-reperfusion (I/R) injury in rats and to explore its potential mechanism. Our findings might help to provide references for clinical prevention and treatment of myocardial I/R. A total of 60 male Sprague-Dawley (SD) rats were randomly divided into three groups using a random number table, including: Control group (n=20), I/R group (n=20) and I/R + miR-26a siRNA group (n=20). I/R model was established via recanalization after ligation of left anterior descending coronary artery (LAD). The model of miR-26a knockdown was established in rats of I/R + miR-26a siRNA group via tail intravenous injection of miR-26a siRNA. Ejection fraction (EF%) and fractional shortening (FS%) of rats in each group were detected via echocardiography. The infarction area of each group was detected via 2,3,5-triphenyltetrazolium chloride (TTC) assay. Subsequently, morphological changes in myocardial cells of each group were detected via hematoxylin-eosin (H&E) staining. Myocardial apoptosis level was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. At the same time, the expression levels of pro-apoptotic proteins Bcl-2 associated X protein (Bax) and cleaved (C)-caspase3 in myocardial tissues of the three groups were determined using Western blotting. Finally, the effects of miR-26a knockdown on the expressions of glycogen synthase kinase (GSK)-3β/β-catenin signaling pathway-related proteins were detected via Western blotting and immunohistochemistry. The expression of miR-26a in myocardial tissues of I/R group increased significantly when compared with that in Control group (p<0.05). Knockdown of miR-26a significantly improved cardiac insufficiency caused by I/R, which also obviously increased both EF% and FS% in rats (p<0.05). In addition, knockdown of miR-26a significantly inhibited myocardial infarction caused by I/R injury, and reduced infarction area from (43.08±2.43) to (21.54±1.82) (p<0.05). The results of H&E staining revealed that in I/R + miR-26a siRNA group, myofilaments were arranged more orderly, the degree of degradation and necrosis was significantly lower, and cellular edema was significantly alleviated when compared with I/R group. Subsequent TUNEL staining demonstrated that rats in I/R + miR-26a siRNA group showed a remarkably lower level of myocardial apoptosis than I/R group (p<0.05). Meanwhile, the protein expression levels of Bax and C-caspase3 were remarkably declined in I/R + miR-26a siRNA group (p<0.05). Furthermore, the results of Western blotting showed that miR-26a siRNA could significantly reverse the inhibition of GSK-3β/β-catenin signaling pathway induced by I/R injury (p<0.05). Knockdown of miR-26a could significantly improve I/R-induced myocardial injury and promote cardiac function in rats. The possible underlying mechanism might be related to targeted regulation of miR-26a on GSK-3β/β-catenin signaling pathway. Therefore, miR-26a was expected to be a new therapeutic target for myocardial I/R injury.

IL33 attenuates ventricular remodeling after myocardial infarction through inducing alternatively activated macrophages ethical standards statement

Interleukin 33 (IL33) has been found to be cardioprotective on various cardiovascular pathologies. However, it is not clear whether IL33 may inhibit myocardial infarction-related ventricular remodeling through inducing macrophage polarization. The objective of present study is to assess whether IL33 can improve ventricular remodeling after myocardial infarction by inducing macrophage polarization.In this study, the direct influence of IL33 on the polarization of macrophages and its mechanism in vitro were investigated. The potential protective effects of IL33 on acute and chronic myocardial infarction (MI) in vivo as well as its underlying mechanism through macrophage polarization were also determined.We found that IL33 significantly enhanced M2 macrophage and decreased the proportion of M1 macrophage. Importantly, IL33 induced M2 macrophage polarization by activating the JAK/STAT signaling pathway. In vivo, IL33 weaken the inflammatory level and myocardial apoptosis after MI and improved the systolic and diastolic function of the heart. Furthermore, IL33 significantly reduced infarct area and prevented the progression of fibrosis by inducing M2 macrophage polarization. The protective effects of IL33 were suppressed by JAK/STAT signaling pathway inhibitor.Our findings highlighted that IL33 not only reduced the early inflammatory response and inhibited myocardial apoptosis, but also increased the number of M2 macrophage in the infarcted area, significantly reduced infarct area and prevented the progression of fibrosis by activating JAK/STAT pathway. Therefore, IL33 may be a novel cardiac protective cytokine for myocardial infarction.

RETRACTED: Repression of long non-coding RNA MEG3 restores nerve growth and alleviates neurological impairment after cerebral ischemia-reperfusion injury in a rat model

This study was performed to investigate effect of long non-coding RNA (lncRNA) MEG3 on nerve growth and neurological impairment in a rat model after cerebral ischemia-reperfusion injury (IRI) through the Wnt/β-catenin signaling pathway. Rat models of middle cerebral artery occlusion (MCAO) were established to stimulate an environment of cerebral IRI. The modeled rats were subjected to negative control (NC), MEG3, si-MEG3, classical Wnt pathway inhibitor DKK1 or classical Wnt pathway activator LiCl to validate the effect of MEG3 on neurological impairment and nerve growth. Neurological deficit scoring, fault-foot test and balance beam test were performed to assess neurological impairment. TTC staining, dry-wet weight method and Evan's blue (EB) staining were employed to determine infarct area, water content of brain tissues and blood-brain barrier (BBB) permeability, respectively. Neuronal apoptosis and necrosis were observed by TUNEL staining and Fluoro-Jade C staining. ELISA was adopted to identify levels of nerve growth factors to identify neurogenesis conditions, including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF) and basic fibroblast growth factor (bFGF). Nissl staining was used to detect the survival of neurons in brain tissues of rats. Western blot analysis was used to detect the expression of key proteins in Wnt/β-catenin signaling pathway in brain tissues. High expression of MEG3 was identified in rat models of MACAO, the brain tissues of which manifested obvious neurological impairment, increased infarct area, water content, BBB permeability, accelerated neuronal apoptosis and necrosis, increased surviving neurons, upregulated expression of key proteins in Wnt/β-catenin signaling pathway and elevated levels of BDNF, NGF and bFGF. With the treatment of si-MEG3, the MEG3 expression was reduced; whereby, modeled rats showed ameliorated neurological impairment, reduced infarct area, water content, BBB permeability, neuronal apoptosis and necrosis and significantly enhanced neurogenesis. The treatment of MEG3 exhibited an opposite trend. After treatment with DKK1, the effect of si-MEG3 was reversed. After treatment with LiCl, the effect of MEG3 was reversed. Based on the findings of this study, down-regulation of lncRNA MEG3 expression enhanced nerve growth and alleviated neurological impairment of rats after cerebral IRI through the activation of the Wnt/β-catenin signaling pathway.

Sitagliptin improves cardiac function after myocardial infarction through activation of autophagy in streptozotocin-induced diabetic mice.

The aim of this study was to investigate the protective effect of sitagliptin on cardiac function in mice with diabetes and myocardial infarction (MI), and to explore its possible mechanism using the mouse models of diabetes and MI. The models of diabetes and MI were established using C57BL/6 mice. All mice were randomly divided into 5 groups, including the control sham (CS) group, the control MI (CMI) group, the diabetes sham (DS) group, diabetes + MI (DMI) group and the DMI + sitagliptin (DMI+SGL) group. After modeling, mice in the DMI+SGL group were intragastrically administrated with sitagliptin (10 mg/kg/day) for 21 d and the survival rate of mice was recorded. Before and 7, 14, 21 days after MI, the cardiac function of mice in each group was detected via ultrasound. 21 days after MI, the area of MI was measured via 2,3,5-triphenyl tetrazolium chloride (TTC). Meanwhile, the degree of fibrosis in the peripheral region of MI was determined via Masson staining. Moreover, myocardial autophagosomes of mice in each group were observed by transmission electron microscope (TEM). 7 days after MI, the expressions of autophagy-related proteins LC3II and P65 were detected via Western blotting. The expressions of myocardial inflammatory factors, interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were detected via enzyme-linked immunosorbent assay (ELISA). In addition, the expression of nuclear factor-κB (NF-κB) was also detected via Western blotting. Sitagliptin increased survival rate, improved cardiac function, reduced infarction area, alleviated myocardial fibrosis, enhanced autophagy in the peripheral region and inhibited inflammation in the peripheral region in mice with diabetes after MI. Sitagliptin can improve cardiac function and reduce the mortality rate in diabetic rats after MI. The possible underlying mechanism may be related to the fact that sitagliptin activates autophagy and inhibits inflammatory response in diabetes after MI.

RETRACTED: Therapeutic effects of melatonin on cerebral ischemia reperfusion injury: Role of Yap-OPA1 signaling pathway and mitochondrial fusion

The role of OPA1-related mitochondrial fusion in brain reperfusion stress has remained elusive. The aim of our study is to explore whether melatonin alleviates cerebral IR injury by modulating OPA1-related mitochondrial fusion. We found that melatonin reduced infarct area and suppressed neuron death during reperfusion stress. Biological studies have revealed that IR-inhibited mitochondrial fusion was largely reversed by melatonin via upregulated OPA1 expression. Knocking down OPA1 abrogated the protective effects of melatonin on mitochondrial energy metabolism and mitochondrial apoptosis. In addition, we also found that melatonin modified OPA1 expression via the Yap-Hippo pathway; blockade of the Yap-Hippo pathway induced neuron death and mitochondrial damage despite treatment with melatonin. Altogether, our data demonstrated that cerebral IR injury is closely associated with defective OPA1-related mitochondrial fusion. Melatonin supplementation enhances OPA1-related mitochondrial fusion by activating the Yap-Hippo pathway, ultimately reducing brain reperfusion stress.

Nur77 promotes cerebral ischemia-reperfusion injury via activating INF2-mediated mitochondrial fragmentation.

Mitochondrial fragmentation drastically regulates mitochondrial homeostasis in brain illness. However, the role of mitochondrial fragmentation in cerebral ischemia-reperfusion (IR) injury remains unclear. Nur77, a regulator of mitochondrial homeostasis, is associated with heart and liver IR injury, but its effects on mitochondrial function in cerebral IR injury has not been studied intensively. The aim of our study is to explore whether cerebral IR injury is modulated by Nur77 via modification of mitochondrial homeostasis. Our results indicated that Nur77 was upregulated in reperfused brain tissues. Genetic ablation of Nur77 reduced infarction area and promoted neuron survival under IR burden. Biochemical analysis demonstrated that Nur77 deletion protected mitochondrial function, attenuated mitochondrial oxidative stress, preserved mitochondrial potential, and blocked mitochondria-related cell apoptosis. In addition, we illustrated that Nur77 mediated mitochondrial damage via evoking mitochondrial fragmentation that occurred through increased mitochondrial fission and decreased fusion. Besides, our results also demonstrated that Nur77 controlled mitochondrial fragmentation via upregulating INF2 in a manner dependent on the Wnt/β-catenin pathway; inhibition of the Wnt pathway abrogated the protective effect of Nur77 deletion on reperfused-mediated neurons. Altogether, our study highlights that the pathogenesis of cerebral IR injury is associated with Nur77 activation followed by augmented mitochondrial fragmentation via an abnormal Wnt/β-catenin/INF2 pathway. Accordingly, Nur77-dependent mitochondrial fragmentation and the Wnt/β-catenin/INF2 axis may represent novel therapeutic targets to reduce cerebral IR injury.

Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke.

ObjectiveInhibitors of the voltage‐gated K+ channel Kv1.3 are currently in development as immunomodulators for the treatment of autoimmune diseases. As Kv1.3 is also expressed on microglia and has been shown to be specifically up‐regulated on “M1‐like” microglia, we here tested the therapeutic hypothesis that the brain‐penetrant small‐molecule Kv1.3‐inhibitor PAP‐1 reduces secondary inflammatory damage after ischemia/reperfusion.MethodsWe studied microglial Kv1.3 expression using electrophysiology and immunohistochemistry, and evaluated PAP‐1 in hypoxia‐exposed organotypic hippocampal slices and in middle cerebral artery occlusion (MCAO) with 8 days of reperfusion in both adult male C57BL/6J mice (60 min MCAO) and adult male Wistar rats (90 min MCAO). In both models, PAP‐1 administration was started 12 h after reperfusion.ResultsWe observed Kv1.3 staining on activated microglia in ischemic infarcts in mice, rats, and humans and found higher Kv1.3 current densities in acutely isolated microglia from the infarcted hemisphere than in microglia isolated from the contralateral hemisphere of MCAO mice. PAP‐1 reduced microglia activation and increased neuronal survival in hypoxia‐exposed hippocampal slices as effectively as minocycline. In mouse MCAO, PAP‐1 dose‐dependently reduced infarct area, improved neurological deficit score, and reduced brain levels of IL‐1β and IFN‐γ without affecting IL‐10 and brain‐derived nerve growth factor (BDNF) levels or inhibiting ongoing phagocytosis. The beneficial effects on infarct area and neurological deficit score were reproduced in rats providing confirmation in a second species.InterpretationOur findings suggest that Kv1.3 constitutes a promising therapeutic target for preferentially inhibiting “M1‐like” inflammatory microglia/macrophage functions in ischemic stroke.

The protective effect and its mechanism of 3-n-butylphthalide pretreatment on cerebral ischemia reperfusion injury in rats.

To investigate the potential effect of 3-n-butylphthalide (NBP) pretreatment on the cerebral ischemia/reperfusion injury in rats and the relevant mechanism. A total of 90 rats was divided into three groups: Sham operation group (Sham group), ischemia-reperfusion group (I-R group), and NBP pretreatment group (NBP group 75 mg·kg-1·d-1 gavage). Pre-treatment was given once a day within 1 week before establishing the rat model of cerebral ischemia-reperfusion injury. The middle cerebral artery occlusion (MACO) rat models were established with the improved Longa-Zea method on the 7th day after ischemia for 2 h and reperfusion for 24 h in all the rats. We detected the cerebral infarction, the pathologic change of brain, the apoptosis of nerve cell, the production levels of reactive oxygen species (ROS), the content of malonaldehyde (MDA) and the activity of superoxide dismutase (SOD), the water content and the permeability of blood-brain barriers (BBB). In addition, we also observed the expressions of mitogen-activated protein kinase (MAPK, p-38, JNK, ERK1/2) and cleaved caspase-3 in the hippocampus tissues. Compared with Sham group, we discovered that NBP significantly reduced infarction area, cell apoptosis, BBB damage and water content. Further, we found that NBP could also decrease ROS and MDA, and increase SOD activity in brain tissues of rats with a cerebral ischemia-reperfusion injury. Moreover, results showed that NBP also inhibited the levels p38 and JNK. NBP protected the cerebral from I/R injury, providing ideas for the expansion of clinical adaptability of NBP and possible approaches for its application.

Evaluation of the effects of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model

We reported previously that Stachybotrys microspora triprenyl phenol-7 (SMTP-7) showed potential thrombolytic, anti-inflammatory and anti-oxidant effects that account for its excellent pharmacological activity such as having a wider therapeutic time window than tissue plasminogen activator (t-PA) and a significant protection against hemorrhage. The aim of the present study was to evaluate and compare the effect of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model. Thrombotic occlusion was produced in mice by inducing the transfer of acetic acid-induced thrombi from the right common carotid artery into the brain. SMTPs were evaluated by their effect on reducing infarct area, neurological score and edema. Furthermore, plasmin formation, anti-inflammatory and anti-oxidant activities were assessed by fibrin zymography, measuring pro-inflammatory gene expression, and thiobarbituric acid reactive substances (TBARS) assay, respectively. Treatment with either SMTP-22 or SMTP-43 (10mg/kg), which have similar plasmin formation, anti-inflammatory and anti-oxidant activities to SMTP-7, resulted in reduced infarct area, neurological score and edema. Coexistence of all these three activities appears to be important for the treatment of embolic infarction because SMTP-6, SMTP-25, and SMTP-44D (10mg/kg), which are each missing at least one of the three functions, were not as effective. Therefore, these results indicate that SMTP-22 and SMTP-43 have potential as medicinal compounds for the treatment of embolic cerebral infarction.

Genetic deletion of mGlu2 metabotropic glutamate receptors improves the short-term outcome of cerebral transient focal ischemia

We have recently shown that pharmacological blockade of mGlu2 metabotropic glutamate receptors protects vulnerable neurons in the 4-vessel occlusion model of transient global ischemia, whereas receptor activation amplifies neuronal death. This raised the possibility that endogenous activation of mGlu2 receptors contributes to the pathophysiology of ischemic neuronal damage. Here, we examined this possibility using two models of transient focal ischemia: (i) the monofilament model of middle cerebral artery occlusion (MCAO) in mice, and (ii) the model based on intracerebral infusion of endothelin-1 (Et-1) in rats. Following transient MCAO, mGlu2 receptor knockout mice showed a significant reduction in infarct volume and an improved short-term behavioural outcome, as assessed by a neurological disability scale and the “grip test”. Following Et-1 infusion, Grm2 gene mutated Hannover Wistar rats lacking mGlu2 receptors did not show changes in the overall infarct volume as compared to their wild-type counterparts, although they showed a reduced infarct area in the agranular insular cortex. Interestingly, however, mGlu2 receptor-deficient rats performed better than wild-type rats in the adhesive tape test, in which these rats did not show the laterality preference typically observed after focal ischemia. These findings support the hypothesis that activation of mGlu2 receptors is detrimental in the post-ischemic phase, and support the use of mGlu2 receptor antagonists in the experimental treatment of brain ischemia.

Cardioprotective effects of PKG activation by soluble GC activator, BAY 60-2770, in ischemia-reperfusion-injured rat hearts.

Soluble guanylate cyclase (sGC) has been suggested as a therapeutic target for cardiac ischemia-reperfusion (IR) injury. Until now, the molecular mechanism of BAY 60–2770, a sGC activator, in cardiac IR injury has not been assessed. To identify the cardioprotective effects of BAY 60–2770 in IR-injured rat hearts, IR injury was established by occlusion of LAD for 40 min and reperfusion for 7 days, and the effects of BAY 60–2770 on myocardial protection were assessed by echocardiography and TTC staining. 5 nM and 5 μM of BAY 60–2770 were perfused into isolated rat hearts in a Langendorff system. After 10- or 30-min reperfusion with BAY 60–2770, cGMP and cAMP concentrations and PKG activation status were examined. Hearts were also perfused with 1 μM KT5823 or 100 μM 5-HD in conjunction with 5 nM Bay 60–2770 to evaluate the protective role of PKG. Mitochondrial oxidative stress was investigated under hypoxia-reoxygenation in H9c2 cells. In IR-injured rat hearts, BAY 60–2770 oral administration reduced infarct size by TTC staining and improved left ventricular function by echocardiography. Tissue samples from BAY 60-2770-perfused hearts had approximately two-fold higher cGMP levels. BAY 60–2770 increased PKG activity in the myocardium, and the reduced infarct area by BAY 60–2770 was abrogated by KT-5823 in isolated myocardium. In H9c2 cardiac myoblasts, hypoxia-reoxygenation-mediated mitochondrial ROS generation was diminished with BAY 60–2770 treatment, but was recovered by pretreatment with KT-5823. BAY 60–2770 demonstrated a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. BAY 60–2770 has a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. These results demonstrated that BAY 60–2770 may be used as a therapeutic agent for cardiac IR injury.

Chronically elevated bilirubin protects from cardiac reperfusion injury in the male Gunn rat.

Bilirubin is associated with reduced risk of cardiovascular disease, as evidenced in conditions of mild hyperbilirubinaemia (Gilbert's Syndrome). Little is known regarding myocardial stress resistance in hyperbilirubinaemic conditions or whether life-long exposure modifies cardiac function, which might contribute to protection from cardiovascular disease. Hyperbilirubinaemic rats and littermate controls underwent echocardiography at 3, 6 and 12 months of age, with hearts subsequently assessed for resistance to 30 min of ischaemia. Heart tissue was then collected for assessment of bilirubin content. No difference in baseline cardiac function was evident until 6 months onwards, where Gunn rats demonstrated aortic dilatation and reduced peak ejection velocities. Additionally, duration of ventricular ejection increased progressively, indicating a negative inotropic effect of bilirubin in vivo. Ex vivo analysis of baseline function revealed reduced left ventricular pressure development (LVDP) and contractility in hyperbilirubinaemic rats. Furthermore, stress resistance was improved in Gunn hearts: post-ischaemic recoveries of LVDP (76 ± 22% vs. 29 ± 17% Control, P < 0.01) and coronary flow (96 ± 9% vs. 86 ± 16% Control, P < 0.01) were improved in Gunn hearts, accompanied by reduced infarct area (21 ± 5% vs. 47 ± 15% Control, P < 0.01), and ventricular malondialdehyde and protein carbonyl content. Expression of myocardial nitric oxide-regulating genes including Nos1 and Noa1 were not significantly different. These data reveal life-long hyperbilirubinaemia induces age-dependent hypocontractility in male Gunn rats, and improved stress resistance. In addition, bilirubin exerts sex-independent effects on vascular structure, myocardial function and ischaemic tolerance, the latter likely mediated via bilirubin's antioxidant properties.

The IL-2/Anti-IL-2 Complex Attenuates Cardiac Ischaemia-Reperfusion Injury Through Expansion of Regulatory T Cells

Background/Aims: Regulatory T cells (Tregs) can suppress immunologic damage in myocardial ischaemia/reperfusion injury (MIRI), however, the isolation and ex vivo expansion of these cells for clinical application remains challenging. Here, we investigated whether the IL-2/anti-IL-2 complex (IL-2C), a mediator of Treg expansion, can attenuate MIRI in mice. Methods: Myocardial I/R was surgically induced in male C57BL/6 mice, aged 8-10 weeks, that were randomly assigned to 1) sham group (Sham), 2) Phosphate Buffered Saline (PBS), 3) IL-2-anti-IL-2 Ab complex (IL-2C), or 4) sham group, 5) PBS, 6) IL-2C after MIRI, or 7) IL-2C, 8) IL-2C+anti-CD25 mAbs, or 9) IL-2C; 10) IL-2C+anti-TGF-β1 mAbs, 11) IL-2C+anti-IL-10 mAbs. The following parameters were measured at different time points: infarct area, myocardial apoptosis, splenocytes, the inhibitory function of Tregs, and presence of inflammatory factors. In addition, immunohistochemistry analysis was performed. Results: We observed that Tregs were activated in response to MIRI. IL-2C administered before MIRI induced Treg expansion in both spleen and heart, attenuated Th1 and Th17 cell numbers, improved myocardial function, and attenuated both infiltration of inflammatory cells and apoptosis after MIRI. Furthermore, IL-2C administration reduced expression of inflammatory cytokines in the heart and attenuated proliferation of splenic cells. Depletion of Tregs with anti-CD25 mAb abrogated the beneficial effects of IL-2C. However, IL-2C–mediated myocardial protection was not dependent on either IL-10 or TGF-β. In addition, IL-2C administration after MIRI did not reduce infarct area, but did improve myocardial function slightly and reduced myocardial fibrosis. Conclusion: Our results demonstrate that IL-2C–induced Treg expansion attenuates MIRI and improves myocardial recovery in vivo, suggesting that IL-2C is a promising therapeutic target for myocardial IRI.

Assessment of the MRI and Behavioral Test Results in a Focal Cerebral Ischemia-Reperfusion Model in the Rat after Separate and Combined Use of Mouse-Derived Neural Progenitor Cells, Human-Derived Neural Progenitor Cells and Atorvastatin.

To assess the efficacy of Neural progenitor cell (NPC) transplantation in ischemic stroke, and to investigate whether atorvastatin enhances therapeutic potency of NPC after stroke. The focal cerebral ischemia-reperfusion model was performed by transient occlusion of middle cerebral artery. Rats were assigned randomly to receive intracerebral transplantation of mouse NPC alone (mNPC), human NPC alone (hNPC), mouse NPC plus oral atorvastatin (mNPC+A), human NPC plus oral atorvastatin (hNPC+A), oral atorvastatin alone, or intracerebral Dulbecco"s Modified Eagle"s medium injection (control group). Adhesive removal, rotarod, cylinder tests, and magnetic resonance imaging (MRI) were used for assessment of rats during 4 weeks. After sacrification on 28th day, rats were investigated by immunofluorescent staining. The hNPC and mNPC groups showed significantly improved functional outcome and reduced infarct area ratio compared with the control group. The hNPC group had significantly better performance and lower infarct area ratio than the mNPC group. Addition of atorvastatin to stem cell therapy significantly improved functional outcome, although it did not affect the infarct area ratio on MRI. Anti-inflammatory response in the infarct area was higher in the mNPC group. NPC transplantation significantly reduced the amount of microglia and a significant increase in the amount of astrocytes. CD8a+ T lymphocyte and granzyme B activities were not detected in any of the subjects. Both hNPC and mNPC treatments significantly improved functional outcome, and reduced infarct area ratio after stroke. Atorvastatin enhanced the therapeutic potency of NPCs, including neurological improvement.

Abstract 11804: GsMTx4-D is a Cardioprotectant Against Myocardial Infarction During Ischemia and Reperfusion

Introduction: GsMTx4 is a selective inhibitor of cationic mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. Inhomogeneous local mechanical stresses due to hypercontracture and swelling, likely induce elevated MSC activity that can contribute to the cation imbalance observed during ischemic reperfusion injury. This study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant during ischemia and reperfusion and to evaluate its effects on cation influx and inflammatory kinases that are regulated by this influx. Hypothesis: GsMTx4 protects heart from ischemic insults through inhibiting the cationic mechanosensitive ion channels. Methods: Ischemia and reperfusion in the mouse heart involved ligating a coronary artery followed by release of the ligature. Results: GsMTx4-D was administered by acute intravenous injection during the ischemic event. Based on pharmacokinetic studies, GsMTx4-D dosage was set to achieve expected plasma concentrations between 50-5000 nM and heart tissue concentrations between 1-200 nM. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by ~40% for acute and pretreated animals, and for both 20 and 45 min ischemic challenges. Many indicators of cardiac output were indistinguishable from sham-treated control hearts after GsMTx4-D treatment and most showed improvement at both 4 and 48 hrs post ischemia. Premature ventricular beats immediately following reperfusion were also significantly reduced by intravenous injections of GsMTx4-D. Myocytes cultured under hypoxic conditions treated with 10 μM GsMTx4-D showed improved contractility and near normal contraction-related Ca 2+ influx. GsMTx4-D inhibited indicators of ischemic damage such as the apoptotic signaling system JNK/c-Jun, but also inhibited the energy response signaling system Akt kinase. Conclusions: GsMTx4-D is a potent cardioprotectant that decreases infarct area, increases cardiac output and decreases arrhythmias that are caused by ischemia and reperfusion. GsMTx4-D improved cardiomyocyte function and suppresses apoptosis, making it potentially useful in multidrug strategies to treat ischemic heart disease.

Comparsion between Intravenous Delivered Human Fetal Bone Marrow Mesenchymal Stromal Cells and Mononuclear Cells in the Treatment of Rat Cerebral Infarct.

Objective To compare the effecacy of human mesenchymal stromal cell (hMSC) with human mononuclear cell (hMNC) in treating rat cerebral infarct.Methods The SD rat models of cerebral infarct were established by distal middle cerebral artery occlusion (dMCAO). Rats were divided into four groups: sham,ischemia vehicle,MSC,and MNC transplantation groups. For the transplantation group,1×106 hMSCs or hMNCs were intravascularly transplanted into the tail vein 1 hour after the ischemia onset. The ischemia vehicle group received dMCAO surgery and intravascular saline injection 1,3,5,and 7 days after the ischemia onset,and then behavioral tests were performed. At 48 h after the ischemia onset,the abundance of Iba- 1,the symbol of activated microglia,was evaluated in the peri-ischemia striatum area; meanwhile,the neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in ipsilateral peri-ischemia striatum area were also measured. Results The relative infarct volume in ischemia vehicle group,hMSC group,and hMNC transplantation group were (37.85±4.40)%,(33.41±3.82)%,and (30.23±3.63)%,respectively. The infarct volumes of MSC group (t=2.100,P=0.034) and MNC group (t=2.109,P=0.0009) were significantly smaller than that of ischemia vehicle group,and that of MNC group was significantly smaller than that of MSC group (t=1.743,P=0.043). One day after transplantation,the score of ischemia vehicle group in limb placing test was (4.32±0.71)%,which was significantly lower than that in sham group (9.73±0.36)% (t=2.178,P=8.61×10-11). The scores of MSC and MNC group,which were (5.09±0.62)% (t=2.1009,P=0.024) and (5.90±0.68)% (t=2.1008,P=0.0001),respectively,were significantly higher than that of ischemia vehicle group; also,the score of MNC group was significantly higher than that of MSC group(t=2.1009,P=0.0165). The contralateral forelimb scores of MSC and MNC groups in beam walking test were (5.56±0.86)% (t=2.120,P=0.020) and (5.13±0.95)% (t=2.131,P=0.003),were both significantly lower than that of ischemia vehicle group [(6.47±0.61)%]. Three days after the transplantation,the limb placing test score of MNC group [(6.91±1.10)%] was significantly higher than that of ischemia vehicle group (5.80±0.82)% (t=2.110,P=0.027). The score of MSC group [(6.30±0.77)%] showed no statistic difference with that of ischemia vehicle group(t=2.101,P=0.199).The contralateral forelimb scores of MNC group in beam walking test [(4.34±0.58)%] was significantly lower than that of ischemia vehicle group [(5.31±0.65)%] (t=2.100,P=0.006) and MSC group [(4.92±0.53)%] (t=2.100,P=0.041); there was no statistic difference between MSC group and ischemia vehicle group (t=2.109,P=0.139). The relative abundance of Iba- 1 in sham,ischemia vehicle,MSC,and MNC groups was 1.00+0.00,1.72±0.21,1.23±0.08,and 1.48±0.06,respectively. The Iba-1 relative abundance of ischemia vehicle group was significantly higher than that of sham group (t=2.262,P=2.9×10-6). The Iba-1 relative abundances of both MSC (t=2.178,P=3.91×10-5)and MNC (t=2.200,P=0.007)groups were significantly lower than that of ischemia vehicle group. It was also significantly lower in MNC group than in MSC group also (t=2.120,P=7.09×10-6). Three days after transplantation,the BDNF and GDNF levels of MSC group,which were (531.127±73.176)pg/mg (t=2.109,P=0.003)and(127.780±16.733)pg/mg(t=2.100,P=2.76×10-5),respectively,were significantly higher than those of ischemia vehicle group,which were (401.988±89.006)pg/mg and (86.278±14.832) pg/mg,respectively. The BDNF and GDNF levels of MNC group,which were (627.429±65.646)pg/mg (t=2.144,P=0.017) and (153.117±20.443)pg/mg (t=2.109,P=0.010),respectively,were all significantly higher than that of MSC group. At day 7,the BDNF and GDNF levels of MSC group,which were (504.776±83.282)pg/mg (t=2.101,P=0.005) and (81.641±11.019)pg/mg (t=2.100,P=0.002),respectively,were significantly higher than those of ischemia vehicle group,which were (389.257±70.440)pg/mg and (64.322±9.855) pg/mg,respectively. The BDNF and GDNF levels of MNC group,which were (589.068±63.323)pg/mg (t=2.100,P=0.027) and (102.161±19.932)pg/mg (t=2.144,P=0.017),respectively,were all significantly higher than that of MSC group. Conclusions Both hMSC and hMNC are beneficial to the ischemia-damaged brain when they are intravascularly transplanted within 1 h after the onset of ischemia. The anti-inflammation ability and secretion of neurotrophic factors are the underlying mechanisms of the therapeutic effects. MNC is more effective than MSC in reducing infarct area and improving behaviors,which might be explained by the fact that MNC induces more GDNF and BDNF in brain than MSC.

Abstract 114: Downregulation of Small GTPase Rnd3 in Cardiomyocytes Leads to Hyperinflammatory Response in Post-myocardial Infarction Heart

Background: Hyperinflammatory response leads to maladaptive healing and remodeling after acute myocardial infarction (MI), while a balanced inflammatory response is essential for post-MI repair and remodeling. Most of the studies have focused on inflammatory cells, extracellular matrices and myofibroblasts in the regulation of acute MI-induced inflammatory response, the investigation of crucial role of cardiomyocytes in the initiation and regulation of the responsive inflammation in heart has been overlooked. Subjects and Methods: To demonstrate that small G protein Rnd3 functions as a negative regulator to refine the inflammatory response by preventing excessive inflammation in cardiomyocytes after acute MI. Genetic mouse models with cardiomyocyte-specific knockout and overexpression of Rnd3 were studied along with multiple cell culture approaches. An acute MI was introduced by ligation of the left anterior descending artery. Results: Mice with cardiomyocyte-specific deletion of Rnd3 (heterozygous, Rnd3 +/- ) showed a significantly higher mortality, worse cardiac function, and increased infarct size compared to wild-type control mice after MI. Massive inflammatory cell infiltration along with a profound increase in the expression levels of cytokines, MMP2 and MMP9 were observed in the infarcted and remote areas in Rnd3 +/- heart. Mechanistic studies revealed that Rnd3 bound to NFκB complex and attenuated NFκB activity by 1) physically interacting with NFκB component p65; 2) competing with NFκB component p50 for p65 binding; 3) blocking p65 nuclear import. The mechanisms were demonstrated in vitro and in vivo . Cardiomyocyte-specific overexpression of Rnd3 in mice inhibited hyperactivation of NFκB signaling, attenuated excessive inflammatory response, reduced infarct areas and ameliorated post-MI cardiac function. Conclusions: 1) This study demonstrates cardiomyocytes as an intrinsic and crucial component in the initiation of the inflammatory cascade in acute MI; 2) discover Rnd3 as a new “fine-tuning” factor situated at the nexus of the inflammatory response; 3) reveal a novel molecular mechanism through Rnd3-mediated NFκB signaling axes responsible for a balanced inflammatory response after acute MI.

Sestrin2, as a negative feedback regulator of mTOR, provides neuroprotection by activation AMPK phosphorylation in neonatal hypoxic-ischemic encephalopathy in rat pups

Hypoxic-ischemic encephalopathy is a condition caused by reduced oxygen and cerebral blood flow to the brain resulting in neurological impairments. Effective therapeutic treatments to ameliorate these disabilities are still lacking. We sought to investigate the role of sestrin2, a highly conserved stress-inducible protein, in a neonatal rat hypoxic-ischemic encephalopathy model. Ten-day-old rat pups underwent right common carotid artery ligation followed by 2.5 h hypoxia. At 1 h post hypoxic-ischemic encephalopathy, rats were intranasally administered with recombinant human sestrin2 and sacrificed for brain infarct area measurement, Fluoro-Jade C, immunofluorescence staining, Western blot, and neurological function testing. rh-sestrin2 reduced brain infarct area, brain atrophy, apoptosis, ventricular area enlargement, and improved neurological function. Western blot showed that sestrin2 expression levels were increased after treatment with rh-sestrin2, and sestrin2 exerts neuroprotective effects via activation of the adenosine monophosphate-activated protein kinase pathway which in turn inhibits mammalian target of rapamycin signaling resulting in the attenuation of apoptosis. In conclusions: Sestrin2 plays an important neuroprotective role after hypoxic-ischemic encephalopathy via adenosine monophosphate-activated protein kinase signaling pathway and serves as a negative feedback regulator of mammalian target of rapamycin. Administration of rh-sestrin2 not only reduced infarct area and brain atrophy, but also significantly improved neurological function.