Exosomes derived from SDF1-overexpressing mesenchymal stem cells inhibit ischemic myocardial cell apoptosis and promote cardiac endothelial microvascular regeneration in mice with myocardial infarction

Exosomes extracted from mesenchymal stem cells (MSCs) was reported to reduce myocardial ischemia/reperfusion damage. Besides, stromal-derived factor 1 (SDF1a) functions as cardiac repair after myocardial infarction (MI). Therefore, the present study aims to identify whether exosomes (Exo) released from SDF1-overexpressing MSCs display a beneficial effect on ischemic myocardial infarction. Initially, a gain-of-function study was performed to investigate the function of SDF1 in ischemic myocardial cells and cardiac endothelial cells. Coculture experiments were performed to measure potential exosomic transfer of SDF1 from MSCs to ischemic myocardial cells and cardiac endothelial cells. During the coculture experiments, exosome secretion was disrupted by neutral sphingomyelinase inhibitor GW4869 and upregulated exosomal SDF1 using SDF1 plasmid. Effects of Exo-SDF1 on cardiac function in MI mice were investigated in vivo. MSCs suppressed myocardial cell apoptosis and promoted microvascular regeneration of endothelial cells through secretion of exosomes. The addition of GW4869 led to increased apoptotic capacity of myocardial cells, decreased microvascular formation ability of endothelial cells, enhanced autophagy ability, and elevated Beclin-1 level as well as ratio of LC3II/LC3I. Overexpression of SDF1 and Exo-SDF1 inhibited apoptosis and autophagy of myocardial cells, but promoted tube formation of endothelial cells. The interference of PI3K signaling pathway promoted apoptosis and autophagy of myocardial cells, but inhibited tube formation of endothelial cells. SDF1 activated the PI3K signaling pathway. Exo-SDF1 protected cardiac function of MI mice and inhibited myocardial tissue damage. This study provided evidence that SDF1 overexpression in MSCs-derived exosomes inhibited autophagy of ischemic myocardial cells and promoted microvascular production of endothelial cells.     

缓激肽在骨骼肌缺血预适应对猪心肌坏死和凋亡中的作用

目的：研究缓激肽在骨骼肌缺血预适应对心肌坏死和心肌凋亡保护中可能的作用：方法：采用非开胸法建立猪心脏缺血／再灌注（I／R）模型,通过球囊堵塞左股动脉造成骨骼肌短暂缺血,使用缓激肽（BK）的B2受体拮抗剂烟酸已可碱（HOE-140）以及外源BK进行干预。分别观察各组对心肌坏死和凋亡的影响。结果：远端预处理后心肌坏死范围明显缩小,凋亡率明显降低：预处理前使用HOE-140可使对坏死范围的保护作用明显减弱;心肌I／R前使用外源BK注射,可缩小心肌梗死范围。但HOE-140及外源BK对以上凋亡指标无影响．结论：骨骼肌远端预适应可减少心肌坏死和心肌凋亡、BK可能参与对坏死面积的保护．但不参与对凋亡的保护作用。     

The effects of parathyroid hormone-related peptide on cardiac angiogenesis,apoptosis, and function in mice with myocardial infarction

It is known that parathyroid hormone-related peptide (PTHrP) contains a nuclear localization sequence (NLS, 87-107), which, together with its C-terminus (107-139), has been shown to positively regulate vascular smooth muscle cell (VSMCs) proliferation and vascular neointima formation, and inhibit cellular apoptosis. The role of PTHrP in ischemic cardiac diseases remains unclear. In this study, we attempted to determine whether PTHrP 87 to 139 can play a role in promoting cardiac function via enhancing angiogenesis after myocardial infarction (MI) occurred. MI was reproduced in C57BL/6 mice using a coronary artery ligation method. In total, three groups (n = 11 per group) of animals were used, and they were received either PTHrP 87 to 139 (80 µg/kg, treatment group) or saline (MI and Sham group) subcutaneously once a day for 4 weeks after MI. To measure cardiac function, an echocardiography was generated and cardiac tissue was harvested for immunohistological studies 4 weeks after operation. Our results show that, after MI, the cardiac function of the experimental mice was significantly impaired. PTHrP 87 to 139 treatment attenuated cardiac dysfunction in MI mice. Besides, as indicated by decreased heart weight/body weight and lung weight/body weight ratio, PTHrP 87 to 139 attenuated pulmonary congestion and cardiac hypertrophy. Masson staining revealed that PTHrP 87 to 139 attenuated myocardial fibrosis after MI. Also, terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling staining and the expression of cleaved caspase 3 suggested that MI-induced myocytes apotosis was inhibited by PTHrP 87 to 139. In addition to the significantly increased capillary density, PTHrP 87 to 139 treatment also induced p-Akt and several angiogenic factors. In conclusion, PTHrP 87 to 139 treatment preserved cardiac function after MI, and stimulated angiogenesis via upregulating vascular endothelial growth factor and basic fibroblast growth factor (bFGF) in infarct border zone of ischemic myocardium,. These results suggest that PTHrP 87 to 139 is of therapeutic potential for MI.     

Berbamine ameliorates isoproterenol-induced myocardial infarction by inhibiting mitochondrial dysfunction and apoptosis in rats

Berbamine (BBM), a bisbenzylisoquinoline alkaloid from roots, bark, and stem of Berberis plant such as Berberis aristata has a wide range of pharmacological activities. However, the evidence for the cardioprotective effect of BBM is inadequate and the molecular mechanism of BBM remains unclear. This study investigated the underlying molecular mechanism of BBM-mediated cardioprotection on isoproterenol (ISO)-induced mitochondrial dysfunction and apoptosis in rats. The assays of mitochondria antioxidant status, mitochondrial marker enzymes, and electron microscopic analysis of mitochondria revealed BBM significantly prevented the mitochondrial dysfunction induced by ISO. The ISO-induced elevation of mitochondrial oxidative stress was also curbed by BBM. Furthermore, pretreatment with BBM protected the heart tissue from ISO-induced apoptosis as evident from decreased terminal dUTP nickend-labeling positive cells and decreased expression of Bax, cytochrome c, cleaved caspase-9, and caspase-3, and poly (ADP-ribose) polymerase and increased expression of Bcl-2 in ISO-induced rats. These current findings suggest that BBM exerts a significant cardioprotective effect on ISO-induced myocardial infarction in rats.     

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.     

Mangiferin prevents myocardial infarction-induced apoptosis and heart failure in mice by activating the Sirt1/FoxO3a pathway

Myocardial infarction (MI) commonly leads to cardiomyocyte apoptosis and heart failure. Mangiferin is a natural glucosylxanthone extracted from mango fruits and leaves, which has anti-apoptotic and anti-inflammatory properties in experimental cardiovascular diseases. In the present study, we investigated the role and detailed mechanism of mangiferin in MI. We used ligation of the left anterior descending coronary artery to establish an MI model in vivo, and cardiomyocyte-specific Sirt1 knockout mice were used to identify the mechanism of mangiferin. For in vitro studies, oxygen and glucose deprivation (OGD) was used to mimic ischaemia in H9c2 cardiomyocytes. In mice, mangiferin treatment increased Sirt1 expression after MI, significantly reduced the infarct area, and prevented MI-induced apoptosis and heart failure. Mangiferin reduced OGD-induced cellular apoptosis in H9c2 cells. Meanwhile, Sirt1 knockout/silencing abolished the protective effects of mangiferin. Further studies revealed that mangiferin increased FoxO3a deacetylation by up-regulating Sirt1, thus preventing apoptosis, and adenovirus-mediated constitutive acetylation of FoxO3a restricted the anti-apoptotic effects of mangiferin in vivo and in vitro. Our results indicate that mangiferin prevents cardiomyocyte apoptosis and the subsequent heart failure by activating the Sirt1/FoxO3a pathway in MI, and suggest that mangiferin may have an interesting potential in following studies towards clinical evaluation.     

MFGE8 protects against CCl4-induced liver injury by reducing apoptosis and promoting proliferation of hepatocytes

Milk fat globule-EGF factor 8 (MFGE8) has been reported to play various roles in acute injury and inflammation response. However, the role of MFGE8 in liver injury is poorly investigated. The present research was designed to clarify the expression and function of MFGE8 in carbon tetrachloride (CCl₄)-induced liver injury. Using serum cytokine arrays, we selected a promising cytokine MFGE8 as the candidate in the process of hepatitis-fibrosis-hepatocellular carcinoma (HCC) progression, based on the elevated expression in both hepatic fibrosis and HCC models. We validated the increased expression of MFGE8 in liver tissues and serum samples of acute and chronic CCl₄-induced mice. Immunohistochemistry staining of mouse liver tissues indicated that elevated MFGE8 expression was mainly derived from the injured hepatocytes. In addition, MFGE8 expression in the supernatant of primary hepatocytes was accumulated with prolongation of culture time, and CCl₄ treatment further increased the expression of MFGE8. Moreover, a strong correlation between serum MFGE8 expression and liver transaminase activities suggested that MFGE8 may be a novel candidate in liver injury. Intriguingly, mice pretreated with MFGE8 were protected from CCl₄-induced liver injury through antiapoptosis role in the early stage and proproliferation role in the late stage. MFGE8 reduced apoptosis by inhibiting the activation of IRE1α/ASK1/JNK pathway and promoted proliferation by phosphorylation of ERK and AKT. Moreover, serum MFGE8 expression was increased in hepatitis patients while decreased in liver cirrhosis patients. All the results suggest MFGE8 as a novel marker and promising therapeutic agent of liver injury.     

Mitophagy‐regulated mitochondrial health strongly protects the heart against cardiac dysfunction after acute myocardial infarction

Autophagy including mitophagy serves as an important regulatory mechanism in the heart to maintain the cellular homeostasis and to protect against heart damages caused by myocardial infarction (MI). The current study aims to dissect roles of general autophagy and specific mitophagy in regulating cardiac function after MI. By using Beclin1^+/−, Fundc1 knockout (KO) and Fundc1 transgenic (TG) mouse models, combined with starvation and MI models, we found that Fundc1 KO caused more severe mitochondrial and cardiac dysfunction damages than Beclin1^+/− after MI. Interestingly, Beclin1^+/− caused notable decrease of total autophagy without detectable change to mitophagy, and Fundc1 KO markedly suppressed mitophagy but did not change the total autophagy activity. In contrast, starvation increased total autophagy without changing mitophagy while Fundc1 TG elevated total autophagy and mitophagy in mouse hearts. As a result, Fundc1 TG provided much stronger protective effects than starvation after MI. Moreover, Beclin1^+/−/Fundc1 TG showed increased total autophagy and mitophagy to a level comparable to Fundc1 TG per se, and completely reversed Beclin1^+/−‐caused aggravation of mitochondrial and cardiac injury after MI. Our results reveal that mitophagy but not general autophagy contributes predominantly to the cardiac protective effect through regulating mitochondrial function.     

一氧化氮对心肌缺血/再灌注损伤细胞凋亡和心功能的影响

目的:采取促进或抑制NO的方法,了解在重复可逆性心肌缺血/再灌注所致的心肌顿抑时,血液中一氧化氮(NO)的动态变化与细胞顿抑及心功能的影响.方法:新西兰兔15只,随机分为3组(n=5):对照组、在静脉内注射NO合成底物L-精氨酸为L-Arg组、静脉注射一氧化氮合酶抑制剂L-硝基-精氨酸为L-NNA组.用戊巴比妥钠静脉注射麻醉后,结扎前降支制成心肌缺血/再灌注模型,用电子自旋共振法测定血液中NO含量,同时记录左心室最大上升速率dp/dtmax.将兔心肌缺血10 min,共3次,第1、2次缺血后再灌注10 min,第3次缺血后再灌注120 min.结果:第1次缺血/再灌注5 min时NO升高的顺序依次为L-Arg组最大、对照组次之,而L-NNA组较缺血前降低.而dp/dtmax明显下降的是L-Arg组最大、对照组次之、L-NNA组最小.细胞凋亡指数:L-Arg组最大,对照组次之、L-NNA组最小.结论:再灌注早期NO的大量生成及细胞凋亡参与加重心肌顿抑的过程.     

Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice

The role of the proapototic Bax gene in ischemia-reperfusion (I/R) injury was studied in three groups of mice: homozygotic knockout mice lacking the Bax gene (Bax(-/-)), heterozygotic mice (Bax(+/-)), and wild-type mice (Bax(+/+)). Isolated hearts were subjected to ischemia (30 min, 37 degrees C) and then to 120 min of reperfusion. The left ventricular developed force of Bax-deficient vs. Bax(+/+) hearts at stabilization and at 120 min of reperfusion was 1,411 +/- 177 vs. 1,161 +/- 137 mg and 485 +/- 69 vs. 306 +/- 68 mg, respectively. Superior cardiac function of Bax(-/-) hearts after I/R was accompanied by a decrease in creatine kinase release, caspase 3 activity, irreversible ischemic injury, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes. Electron microscopic evaluation revealed reduced damage to mitochondria and the nuclear chromatin structure in Bax-deficient mice. In the Bax(+/-) hearts, the damage markers were moderate. The superior tolerance of Bax knockout hearts to I/R injury recommends this gene as a potential target for therapeutic intervention in patients with severe and intractable myocardial ischemia.     

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.     

Paracrine effect of CXCR4‐overexpressing mesenchymal stem cells on ischemic heart injury

It has been reported that CXCR4‐overexpressing mesenchymal stem cells (MSC^CX4) can repair heart tissue post myocardial infarction. This study aims to investigate the MSCCX4‐derived paracrine cardio‐protective signaling in the presence of myocardial infarction. Mesenchymal stem cells (MSCs) were divided into 3 groups: MSC only, MSC^CX4, and CXCR4 gene‐specific siRNA‐transduced MSC. Mesenchymal stem cells were exposed to hypoxia, and then MSCs‐conditioned culture medium was incubated with neonatal and adult cardiomyocytes, respectively. Cell proliferation–regulating genes were assessed by real‐time polymerase chain reaction (RT‐PCR). In vitro: The number of cardiomyocytes undergoing DNA synthesis, cytokinesis, and mitosis was increased to a greater extent in MSC^CX4 medium‐treated group than control group, while this proproliferative effect was reduced in CXCR4 gene‐specific siRNA‐transduced MSC–treated cells. Accordingly, the maximal enhancement of vascular endothelial growth factor, cyclin 2, and transforming growth factor‐β2 was observed in hypoxia‐exposed MSC^CX4. In vivo: MSCs were labeled with enhanced green fluorescent protein (EGFP) and engrafted into injured myocardium in rats. The number of EGFP and CD31 positive cells in the MSC^CX4 group was significantly increased than other 2 groups, associated with the reduced left ventricular (LV) fibrosis, the increased LV free wall thickness, the enhanced angiogenesis, and the improved contractile function. CXCR4 overexpression can mobilize MSCs into ischemic area, whereby these cells can promoted angiogenesis and alleviate LV remodeling via paracrine signaling mechanism.     

心肌缺血预适应循环血中微囊泡对大鼠心肌I/R损伤的作用

目的：研究心肌缺血预适应（IPC）大鼠循环血中微囊泡（MVs）对大鼠在体心肌缺血/再灌注（I/R）损伤的作用及相关机制。方法：反复短暂结扎/松开大鼠冠状动脉左前降支建立大鼠IPC模型,自腹主动脉取血,超速离心法分离循环血中的IPC-MVs,并对其进行流式鉴定。建立在体大鼠心肌I/R模型,股静脉注射IPC-MVs 7 mg/kg。HE染色观察心肌形态学变化,TTC染色检测心肌梗死范围,TUNEL染色检测心肌细胞凋亡率。比色法测定血清乳酸脱氢酶（LDH）活力,分光光度法测定心肌组织caspase 3活力,Western blot法检测心肌组织Bcl-2、Bax蛋白表达水平。结果：流式细胞术检测IPC-MVs浓度为4380±745个/μl。与I/R组比较,IPC-MVs能够减轻I/R大鼠心肌组织损伤,缩小心肌梗死范围（P<0.01）,减少心肌细胞凋亡数量（P<0.01）,明显降低血清LDH活力（P<0.01）,降低心肌组织caspase 3活力（P<0.01）,升高Bcl-2蛋白表达（P<0.01）,降低Bax蛋白表达（P<0.01）,升高Bcl-2/Bax比值（P<0.01）。结论：IPC-MVs显著减轻大鼠在体心肌I/R损伤,通过上调心肌组织中Bcl-2的蛋白表达,下调Bax的蛋白表达,升高Bcl-2/Bax比值,降低caspase 3活力而发挥心肌保护作用。     

Angiotensin IV protects cardiac reperfusion injury by inhibiting apoptosis and inflammation via AT4R in rats

Angiotensin IV (Ang IV) is formed by aminopeptidase N from Ang III by removing the first N-terminal amino acid. Previously, we reported that Ang III has some cardioprotective effects against global ischemia in Langendorff heart. However, it is not clear whether Ang IV has cardioprotective effects. The aim of the present study was to evaluate the effect of Ang IV on myocardial ischemia-reperfusion (I/R) injury in rats. Before ischemia, male Sprague-Dawley rats received Ang IV (1 mg/kg/day) for 3 days. Anesthetized rats were subjected to 45 min of ischemia by ligation of left anterior descending coronary artery followed by reperfusion and then, sacrificed 1 day or 1 week after reperfusion. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, and infarct size were measured. Quantitative analysis of apoptotic and inflammatory proteins in ventricles were performed using Western blotting. Pretreatment with Ang IV attenuated I/R-induced increases in plasma CK and LDH levels, and infarct size, which were blunted by Ang IV receptor (AT₄R) antagonist and but not by antagonist for AT₁R, AT₂R, or Mas receptor. I/R increased Bax, caspase-3 and caspase-9 protein levels, and decreased Bcl-2 protein level in ventricles, which were blunted by Ang IV. I/R-induced increases in TNF-α, MMP-9, and VCAM-1 protein levels in ventricles were also blunted by Ang IV. Ang IV increased the phosphorylation of Akt and mTOR. These effects were attenuated by co-treatment with AT₄R antagonist or inhibitors of downstream signaling pathway. Myocardial dysfunction after reperfusion was improved by Ang IV. These results suggest that Ang IV has cardioprotective effect against I/R injury by inhibiting apoptosis via AT₄R and PI3K-Akt-mTOR pathway.     

Overexpression of lncRNA Gm15621 alleviates apoptosis and inflammation response resulting from sevoflurane treatment through inhibiting miR-133a/Sox4

Sevoflurane is the most widely used anesthetic administered by inhalation. Exposure to sevoflurane can elicit learning deficits and abnormal cognitive disorder. In this study, we investigated the function of long noncoding RNA (lncRNA) Gm15621. Primary hippocampal neuron cells were used to analyze the function of lncRNA Gm15621 in vitro. The tunel, inflammation markers, and cell survival rates were detected to evaluate the function of lncRNA Gm15621. Dual-luciferase reporter assay was used to identify the interaction between microRNA 133a and Gm15621. We found that lncRNA Gm15621 located in the cytoplasm. The expression of lncRNA Gm15621 was decreased with the development of sevoflurane exposure. Overexpression of lncRNA Gm15621 significantly reduced the apoptosis and cell survival rates. The inflammation response was also attenuated in lncRNA Gm15621 overexpressed group. The dual-luciferase assay revealed that miR-133a was the direct target of lncRNA Gm15621. In addition, we also found that Sox4 was a downstream target of miR-133a and lncRNA Gm15621 exerted its biological functions by regulating the expression of Sox4. In summary, our findings revealed that lncRNA Gm15621 ameliorated the sevoflurane-induced neurotoxicity and the important role of Gm15621/miR-133a/Sox4 axis in cognitive disorder.