Apelin/APJ relieve diabetic cardiomyopathy by reducing microvascular dysfunction

Figure 1. Effects of apelin on cardiac function. (A) The M-mode of echocardiography for each group. (B) Quantification of the cardiac output (CO: diabetes (P = 0.462), treatment (P = 0.051), interaction (P = 0.018)). (C) Quantification of the left ventricular ejection fraction (LVEF: diabetes (P = 0.161), treatment (P = 0.001), interaction (P = 0.031)). (D) Quantification of the left ventricular fractional shortening (LVFS: diabetes (P = 0.148), treatment (P = 0.141), interaction (P = 0.019)). (E) Quantification of the stroke volume (SV: diabetes (P = 0.020), treatment (P = 0.053), interaction (P = 0.022)). (F) Quantification of heart rate (diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.176)). n = 6 mice per group, *P < 0.05.

Figure 2. Apelin decreased cardiac hypertrophy and fibrosis in diabetic mice. (A) Representative H&E histology, periodic acid Schiff (PAS) staining, Masson Trichrome and Pico Sirius Red (PSR) staining in heart sections from C57 and kkAy mice with or without apelin/F13A treatment as quantified in (B, C, D and E) (interventricular septum thickness: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.009); left ventricular wall thickness: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.000); Masson staining: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.036); PSR staining: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.000); n = 6 mice per group, *P < 0.05). Scale bars represent 500 and 50 μm.

Figure 3. Apelin relived early inflammatory response in heart of diabetic mice. (A) Representative images of immunohistochemistry for CD68, mac-2 and E-cadherin in heart sections from C57 and diabetic mice with or without apelin/F13A treatment as quantified in (B, C and D) (CD68: diabetes (P = 0.001), treatment (P = 0.000), interaction (P = 0.578); mac-2: diabetes (P = 0.263), treatment (P = 0.002), interaction (P = 0.054); E-cadherin diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.000); n = 6 mice per group, *P < 0.05). Scale bars represent 50 μm.

Figure 4. Apelin increased angiogenesis and proliferation of endothelial cells in heart of diabetic mice. (A) Representative images of immunohistochemistry for CD31&BrdU double staining, CD31,VEGFR-2 and Tie-2 in heart sections from C57 and diabetic mice with or without apelin/F13A treatment as quantified in (B, C, D and E) (CD31&BrdU: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.025); CD31: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.000); VEGFR-2: diabetes (P = 0.160), treatment (P = 0.000), interaction (P = 0.000); Tie-2: diabetes (P = 0.477), treatment (P = 0.000), interaction (P = 0.039); n = 6 mice per group, *P < 0.05). Scale bars represent 25, 10, nd 50 μm.

Figure 5. Apelin decreased adhesion molecules in heart of diabetic mice. (A) Representative images of immunohistochemistry for VCAM-1 and ICAM-1 in heart sections from C57 and diabetic mice with or without apelin/F13A treatment as quantified in (B and C) (VCAM-1: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.000); ICAM-1: diabetes (P = 0.000), treatment (P = 0.000), interaction (P = 0.000); n= 6 mice per group, *P < 0.05). Scale bars represent 50 μm.

Figure 6. Apelin improved endothelial dysfunction in high-glucose environment. (A) Representative images of wound healing assay as quantified in (E). Scale bars represent 100 μm. (B) Representative images of Matrigel assay as quantified in (F) (n = 3, *P < 0.05). Scale bars represent 100 μm. (C) Representative images of the incorporation of BrdU as quantified in (G) (n = 3, *P < 0.05). Scale bars represent 50 μm. (D) Representative images of TUNEL assay as quantified in (H) (n = 3, *P < 0.05). Scale bars represent 100 μm. (I) Quantification of CCK-8 assay. n = 3, *P < 0.05.

Figure 7. Apelin relived endothelial dysfunction dependent on NFκB pathway. (A) Representative images of western blot for ICAM-1, VCAM-1, VEGFR-2, E-cadherin, p-NFκB, NFκB and GAPDH after HG and apelin treatment in NCMECs. (B, C, D, E and F) Quantification of ICAM-1, VCAM-1, VEGFR-2, E-cadherin, and p-NFκB/NFκB in (A). Values represent the mean ± s.d. n = 3, *P < 0.05.

Figure 8. Effects of apelin on cardiac function of APJΔEC mice. (A) The M-mode of echocardiography for each group. (B) Quantification of the cardiac output (CO). (C) Quantification of the left ventricular ejection fraction (LVEF). (D) Quantification of the left ventricular fractional shortening (LVFS). (E) Quantification of the stroke volume (SV). (F) Quantification of heart rate. n = 6 mice per group, *P < 0.05.

Figure 9. Effects of apelin on heart structure in diabetic APJΔEC mice. (A) Representative HE histology, Masson Trichrome and Pico Sirius Red (PSR) staining in heart sections from APJfl/fland APJΔEC mice with or without apelin treatment as quantified in (B, C, D and E) (n = 6 mice per group, *P < 0.05). Scale bars represent 50 μm.

Figure 10. Effects of apelin on endothelial dysfunction in diabetic APJΔEC mice. (A) Representative images of immunohistochemistry for CD31, CD68, VEGFR-2, Tie-2, ICAM-1, VCAM-1 and E-cadherin in heart sections from APJfl/fl and APJΔEC mice with or without apelin treatment as quantified in (B, C, D, E, F, G and H) (n = 6 mice per group, *P < 0.05). Scale bars represent 50 μm.