Stimulatory effects of the flavanol (-)-epicatechin on cardiac angiogenesis: additive effects with exercise

Abstract

The consumption of moderate amounts of cocoa products has been associated with reductions in the incidence of cardiovascular diseases. In animal studies, the flavanol (-)-epicatechin (Epi) yields cardioprotection. The effects may be partly due to its capacity to stimulate endothelial nitric oxide synthase (eNOS). The sustained activation of eNOS, as observed with exercise, can serve as a trigger of muscle angiogenesis via the activation of vascular endothelial growth factor (VEGF)-related events. Experiments were pursued to examine the potential of Epi to stimulate myocardial angiogenesis and determine the effects that its combined use with exercise (Ex) may trigger. Hearts obtained from a previous study were used for this purpose. Animals received 1 mg/kg of Epi or water (vehicle) via oral gavage (twice daily). Epi and/or Ex (by treadmill) was provided for 15 days. Results indicate that Ex or Epi significantly stimulate myocardial angiogenesis by ~30% above control levels. The use of Epi-Ex lead to further significant increases (to ~50%). Effects were associated with increases in protein levels and/or activation of canonical angiogenesis pathway associated events (HIF1a, VEGF, VEGFR2, PI3K, PDK, AKT, eNOS, NO, cGMP, MMP-2/-9, Src-1, and CD31). Thus, the use of Epi may represent a safe and novel means to stimulate myocardial angiogenesis.

Figures

Figure 1

Effects of exercise and/or Epi on myocardial capillary density. A) Capillary density was assessed using microphotographs of sections obtained from hearts from water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups, immunostained using an antibody against CD31 (as an endothelial marker) and counterstained with haematoxylin. Mean ± SE of at least 10 images (randomly chosen, 40X) from 3 nonconsecutive sections of 3-5 hearts per group. (B) CD31 levels were assessed by Western blots. *p

Figure 2

Effects of exercise and/or Epi on myocardial (A) Hypoxia inducible factor 1α HIF1α), (B) vascular endothelial growth factor (VEGF), (C, D) VEGF-R2 and p-VEGFR levels from water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Mean ± SE relative to W group (100%) values. *p

Figure 3

Effects of exercise and/or Epi on myocardial (A) Phosphoinositol kinase 3 (PI3K), p-PI3K and (B) PDK1, p-PDK1 levels from water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Mean ± SE relative to W group (100%) values. *p

Figure 4

Effects of exercise and/or Epi on myocardial (A) AKT, p-AKT and (B) Src, p-SRC levels from water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Mean ± SE relative to W group (100%) values. *p

Figure 5

(A) (B) Effects of exercise and/or Epi on myocardial HSP-90, AKT, CAM1 and Cav-1 protein levels from water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups. GAPDH was used as a loading. An antibody against total eNOS was used for the immunoprecipitation. (C) eNOS, p-Ser617, p-Ser1177 levels. Mean±SEM relative to W group (100%) values. *p < 0.05 vs. water; **significantly different from Ex or Epi, †significantly different vs. exercise only.

Figure 6

Effects of exercise and/or Epi on myocardial (A) NOx and, (B) cGMP levels as assessed by bioassays from water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups. Mean ± SE. *p < 0.05 vs. water; **significantly different from Ex or Epi, †significantly different vs. exercise only.

Figure 7

Effects of exercise and/or Epi on myocardial MMP-2 and MMP-9 protein (A) or activity (B) levels as assessed by Western blots and gelatin zymography, respectively. From water (W), exercise (Ex), epicatechin (Epi) and epicatechin plus exercise (Epi-Ex) groups. GAPDH was used as a loading control in the Western blot analysis. Mean ± SE relative to W group (100%) values. *p < 0.05 vs. water; **significantly different from Ex or Epi.

Figure 8

Possible pathways associated with exercise and/or Epicatechin angiogenic induced effects in tissues/organs