Gastrin exerts a protective effect against myocardial infarction via promoting angiogenesis

Figures

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  Fig. 1. Comparison of plasma concentrations of gastrin at different days (day 1, 3, 5, 7, 11, and 13) in the myocardial infarction (MI) mice and in sham-operated mice. Data are expressed as the mean ± SD. n = 8. *P < 0.05, vs. sham-operated mice

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  Fig. 2. Gastrin ameliorates cardiac function and survival rate in myocardial infarction mice. a1 Representative M-mode images of hearts with the sham operation or myocardial infarction 28 days after the saline vehicle and gastrin infusion. a2–5 Represent left ventricle ejection fraction (EF), fractional shortening (FS), diastolic left ventricle internal diameter (LVIDd) and systolic left ventricle internal diameter (LVIDs) after MI. n = 10. *P < 0.05, vs. MI mice; #P < 0.05, vs. MI + Gastrin. b Gastrin improves Kaplan–Meier survival rate of mice subjected to MI. n = 10. Log-rank test P < 0.05

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  Fig. 3. Gastrin decreases cardiomyocyte apoptosis and fibrosis after myocardial infarction. a Representative images (a1) and quantification (a2) of the fibrotic area in infarct border zone by Masson trichrome staining at 28 days post-MI. b Representative images (b1) and quantification (b2) of terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive (TUNEL+) cardiomyocytes in infarct border zone at 2 days after MI. TUNEL+ nuclei are stained green, cardiac troponin T antibody (cTnT) is red, and 4'-6-diamidino-2-phenylindole (DAPI) is blue. b3 Western blot was used to measure the protein levels of Caspase 3 in the infarct border zone at 2 days after MI. Results are expressed as the ratio of cleaved caspased 3 to total caspase 3. c, Representative images (c1) and quantification (c2) of cardiomyocyte proliferation 28 days after myocardial infarction, through Ki67 immunostaining. Cardiac troponin T antibody (cTnT) is green, Ki67 is red, and 4'-6-diamidino-2-phenylindole (DAPI) is blue. a and c Scale bar = 50 µm. b Scale bar = 20 µm. n = 8. *P < 0.05, vs. MI mice. P = NS means no significant difference

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  Fig. 4. Gastrin induces angiogenesis after myocardial infarction. a1 Immunofluorescence staining with anti-cluster of differentiation 31 (CD31) antibody showed the microvessel densities in the infarcted border zone 14 days after MI. DAPI is blue, and CD31 is red. Scale bar = 50 µm. a2 Western blot was used to measure the protein levels of CD31 in the infarcted border zone of the heart 14 days after MI. Results are expressed as the ratio of CD31 to GAPDH. n = 8. *P < 0.05, vs. MI mice. b Human coronary artery endothelial cells (HCAECs) were incubated with gastrin at the indicated concentrations. Representative images (b1) and quantification (b2) of tube formation assay with HCAECs. Scale bar = 50 µm. n = 4. *P < 0.05, vs. Control

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  Fig. 5. PI3K/Akt/VEGF signaling pathway is involved in the pro-angiogenic effect of gastrin. Endothelial cells were incubated with gastrin at the indicated concentrations for 24 h. VEGFA (a), phospho-PI3K (b), and phospho-Akt (c) results were normalized by GAPDH, total PI3K, and Akt, respectively. Endothelial cells were treated with gastrin (100 nM, 24 h) in the presence or absence of a PI3K inhibitor, LY294003 (1000 nM). Phospho-Akt (d) and VEGFA (e) were normalized by GAPDH and total Akt, respectively. n = 4. *P < 0.05, vs. Control group; #P < 0.05, vs gastrin-treated group. f Representative images and quantification of endothelial cell migration detected by transwell migration assay. g, Representative images and quantification of endothelial cell migration detected by scratch wound-healing assay. f and g scale bar = 50 µm. n = 4. *P < 0.05, vs. Control group; #P < 0.05, vs. gastrin-treated group. VEGF, vascular endothelial growth factor; PI3K, phosphatidylinositol 3 kinase; Akt, protein kinase B; DU, density units; p, phospho

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  Fig. 6. CCK2R role in the protective effects of gastrin against myocardial infarction. Endothelial cells were treated with gastrin (100 nM) with or without the CCK2R inhibitor, CI988 (100 nM). a Western blot was used to detect the protein levels of vascular endothelial growth factor (VEGFA). Results are expressed as the ratio of VEGFA to GAPDH. n = 4. *P < 0.05, vs. Control; #P < 0.05, vs. gastrin-treated group. b Representative images and quantification of endothelial cell tube formation assay in vitro. Scale bar = 50 µm. n = 4. *P < 0.05, vs. Control; #P < 0.05, vs gastrin-treated group. Myocardial infarction mice were infused with gastrin or/and CI988. c1 Immunofluorescence staining with anti-cluster of differentiation 31 (CD31) antibody showed the microvessel densities in the infarcted border zone 14 days after MI. DAPI is blue, and CD31 is red. Scale bar = 50 µm. c2 Western blot was used to detect the protein levels of CD31 in the peri-infarct area of the heart 14 days after MI. Results are expressed as the ratio of CD31 to GAPDH. n = 8. *P < 0.05, vs. MI group; #P < 0.05, vs gastrin-treated MI mice. d1 Representative M-mode images of hearts with MI 28 days after gastrin or/and…