Angiopoietin‐like protein 4 treated bone marrow‐derived mesenchymal stem cells alleviate myocardial injury of patients with myocardial infarction

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  FIGURE 1. ANGPTL4 rescues BMSCs apoptosis induced by hypoxia/SD in a dose‐dependent manner. BMSCs, which were inoculated into six‐well plates at 1 × 104/well were randomly divided into six groups. Group 1, BMSCs were cultured by normoxia/serum supplement medium for 24 h. Group 2, BMSCs were cultured by hypoxia/SD medium for 24 h. Group 3, BMSCs treated with ANGPTL4 (1 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 4, BMSCs treated with ANGPTL4 (10 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 5, BMSCs treated with ANGPTL4 (100 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 6, BMSCs treated with ANGPTL4 (1000 ng/ml) were cultured by hypoxia/SD medium for 24 h. (a) Fluorescence‐activated cell sorting (FACs) were performed to detect BMSCs apoptosis. (b) Western blot was conducted to measure the levels of apoptosis‐related proteins, including Bax, Bcl‐2, cleaved caspase‐3, and cleaved caspase‐9. Student's t‐test or one‐way analysis of variance is used for comparison between two groups, and Tukey posttest is used for comparison between multiple groups. ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus sham group. Error bars represent SD. Data…

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  FIGURE 2. ANGPTL4 activates paracrine of BMSCs inhibited by hypoxia/SD in a dose‐dependent manner. BMSCs, which were inoculated into six‐well plates at 1 × 104/well were randomly divided into six groups. Group 1, BMSCs were cultured by normoxia/serum supplement medium for 24 h. Group 2, BMSCs were cultured by hypoxia/SD medium for 24 h. Group 3, BMSCs treated with ANGPTL4 (1 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 4, BMSCs treated with ANGPTL4 (10 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 5, BMSCs treated with ANGPTL4 (100 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 6, BMSCs treated with ANGPTL4 (1000 ng/ml) were cultured by hypoxia/SD medium for 24 h. (a) reverse transcription polymerase chain reaction was performed to assess the levels of VEGF, bFGF, HGF, and IGF in BMSCs. (b) enzyme‐linked immunosorbent assay was conducted to detect the levels of VEGF, bFGF, HGF, and IGF in BMSCs. Student's t‐test or one‐way analysis of variance is used for comparison between two groups, and Tukey posttest is used for comparison between multiple groups. ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus sham group. Error bars represent SD. Data…

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  FIGURE 3. ANGPTL4 alleviates oxidative stress of BMSCs induced by hypoxia/SD in a dose‐dependent manner. BMSCs, which were inoculated into six‐well plates at 1 × 104/well were randomly divided into six groups. Group 1, BMSCs were cultured by normoxia/serum supplement medium for 24 h. Group 2, BMSCs were cultured by hypoxia/SD medium for 24 h. Group 3, BMSCs treated with ANGPTL4 (1 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 4, BMSCs treated with ANGPTL4 (10 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 5, BMSCs treated with ANGPTL4 (100 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 6, BMSCs treated with ANGPTL4 (1000 ng/ml) were cultured by hypoxia/SD medium for 24 h. (a) Fluorescence‐activated cell sorting (FACs) were performed to detect ROS levels in BMSCs. (b) MDA Assay Kit (TBA) and SOD Assay Kit (WST‐1) were applied to detect the levels of MDA and SOD in BMSCs, respectively. Student's t‐test or one‐way analysis of variance is used for comparison between two groups, and Tukey posttest is used for comparison between multiple groups. ##p < 0.01 versus Control group; *p < 0.05, **p < 0.01 versus Sham group. Error bars represent SD. Data represent…

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  FIGURE 4. ANGPTL4 up‐regulates metabolic energy of BMSCs inhibited by hypoxia/SD in a dose‐dependent manner. BMSCs, which were inoculated into six‐well plates at 1 × 104/well were randomly divided into six groups. Group 1, BMSCs were cultured by normoxia/serum supplement medium for 24 h. Group 2, BMSCs were cultured by hypoxia/SD medium for 24 h. Group 3, BMSCs treated with ANGPTL4 (1 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 4, BMSCs treated with ANGPTL4 (10 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 5, BMSCs treated with ANGPTL4 (100 ng/ml) were cultured by hypoxia/SD medium for 24 h. Group 6, BMSCs treated with ANGPTL4 (1000 ng/ml) were cultured by hypoxia/SD medium for 24 h. (a) ADP/ATP Ratio Assay Kit was applied to assess the levels of ATP/ADP in BMSCs. (b) Fluorescence‐activated cell sorting (FACs) were conducted to assess the ability of glucose uptake of BMSCs. Student's t‐test or one‐way analysis of variance is used for comparison between two groups, and Tukey posttest is used for comparison between multiple groups. ##p < 0.01 versus Control group; *p < 0.05, **p < 0.01 versus Sham group. Error bars represent SD. Data represent three independent…

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  FIGURE 5. ANGPTL4 treated‐BMSCs protect the cardiac function of rats with MI. Fifty males' rats were randomly divided into five groups, control group, sham group, MI model group (model group), model + BMSCs group, model + ANGPTL4 treated‐BMSCs group, 10 in each group. (a) TTC staining was performed to assess MI area. (b) Masson staining was conducted to evaluate the degree of cardiac fibrosis. (c) CD31 levels were detected by immunofluorescence. (d) reverse transcription polymerase chain reaction was performed to detect the levels of TGF‐β1, CTGF, PDGF, and VEGF. Student's t‐test or one‐way analysis of variance is used for comparison between two groups, and Tukey posttest is used for comparison between multiple groups. ##p < 0.01 versus Control group; **p < 0.01 versus Sham group. Error bars represent SD. Data represent three independent experiments; scale bar: 200 μm. Abbreviations: ANGPTL4, angiopoietin‐like protein 4; BMSC, bone marrow‐derived mesenchymal stem cell; CTGF, connective tissue growth factor; MI, myocardial infarction; PDFG, platelet‐derived growth factor; SD, serum deprivation; TGF‐β1, transforming growth factor‐beta 1; VEGF, vascular endothelial growth factor