Gastrin mediates cardioprotection through angiogenesis after myocardial infarction by activating the HIF-1α/VEGF signalling pathway

Figure 1. Flow chart of animal Experiments

Figure 2. Gastrin improved the prognosis of myocardial infarction (MI). (A) The infarcted region was assessed based on TTC staining of heart sections, and the infarcted field was white. The MI + gastrin group showed a decreased infarcted area (*P < 0.05 vs. no gastrin, #P < 0.05 vs. the gastrin only group, n = 4). (B1) Representative M-mode images through echocardiography after acute MI for 2 and 4 weeks. Left ventricular wall motion in the MI + gastrin group was improved after AMI for 4 weeks. The corresponding B-mode images are presented in Supplementary Fig. 6 (Figure S6). (B2) Left ventricular ejection fraction (LVEF), (B3) Left ventricular fractional shortening (LVFS), (B4) left ventricular internal diameter in systole (LVIDs) and (B5) left ventricular internal diameter in diastole (LVIDd) were measured by echocardiography and were improved after treatment with gastrin (*P < 0.05 vs. no gastrin, #P < 0.05 vs. the gastrin only group, n = 10). (C) Survival charts for all groups of mice at 4 weeks. The survival rate was significantly improved in the MI + gastrin group (*P < 0.05 vs. sham, #P < 0.05 vs. no gastrin).

Figure 3. Gastrin optimized myocardial remodelling after MI. (A1) Representative Masson’s trichrome images of left ventricle (LV) sections in all groups showed decreased numbers of fibroblasts (blue cells) in the infarcted hearts treated with gastrin. (A2) The fibrotic area was significantly decreased by gastrin injection (*P < 0.05 vs. no gastrin, #P < 0.05 vs. the gastrin only group, n = 4). (B1) Immunofluorescent staining of CD31 was performed to detect angiogenesis levels in infarcted hearts 1 month post-MI. (B2) The comparison of CD31+ cells per area (10,000 μm2) among different groups of infarcted hearts (*P < 0.05 vs. no gastrin, #P < 0.05 vs. the gastrin only group, n = 4).

Figure 4. Gastrin increased the expression levels of HIF-1α and VEGF in the LV after MI for 4 weeks. The protein and mRNA expression levels of HIF-1a (B1 and B2) and VEGF (A1 and A2) were measured by immunoblotting and real-time PCR (*P < 0.05 vs. no gastrin, #P < 0.05 vs. the gastrin only group, n = 3). Full-length gels are presented in Supplementary Figs. 1—3 (Figures S1 – S3).

Figure 5. Effects of gastrin on capillary tube formation, proliferation and migration. HUVECs were seeded in Matrigel-coated wells in different groups. (A1) Representative photomicrograph of a tubular experiment, which showed an increase in tube formation in the gastrin group. Total tube length (A2), total mesh area (A3) and number of junctions (A4) were determined to represent the level of tube formation (*P < 0.05 vs. the control, #P < 0.05 vs. the gastrin only group, n = 4. (B) The absorbance and the number of cells were measured with the CCK-8 kit, which showed that gastrin increased the proliferation of HUVECs (*P < 0.05 vs. control, #P < 0.05 vs. the gastrin only group, n = 6). (C) Effects of gastrin and/or CI-988 on the Transwell migration of HUVECs and (D) increasing wound-healing in HUVECs (*P < 0.05 vs. the control, #P < 0.05 vs. the gastrin only group).

Figure 6. Identification of the pathway after siRNA treatment in vitro. (A1) Gastrin increased the mRNA transcription of HIF-1α in vitro, and CI-988 effectively reduced the transcription levels of HIF-1α. (A2) Gastrin also upregulated the VEGF content in the cell supernatant (*P < 0.05 vs. the control, #P < 0.05 vs. the gastrin only group, n = 8). (B) Through the knockdown of HIF-1α by siRNA, it was found that the regulatory effect of gastrin on VEGF was blocked (*P < 0.05 vs. the control, #P < 0.05 vs. the gastrin only group, n = 3). Full-length gels are presented in Supplementary Figs. 4 and 5 (Figures S4 and S5). (C) HIF-1α knockdown inhibited the tube formation of HUVECs after treatment with gastrin. Total tube length, total mesh area and number of junctions were determined to represent the level of tube formation (*P < 0.05 vs. the control, #P < 0.05 vs. the gastrin only group, n = 4).