Characterization of the Antifibrotic Effect of (−)‐Epicatechin In Cardiac Fibroblasts.
Authors: Garate-Carrillo Elva Alejandra , Ramirez-Sanchez Israel , Gonzalez Julisa , Nyugen Justina , Ceballos Guillermo , Villarreal Francisco 
Published: April 2020

Background

Cardiac fibrosis is one of the hallmarks of pathological remodeling and can promote disease progression. In the setting of ongoing disease such as with hypertension or myocardial infarction, cardiac fibroblasts undergo phenotypical modifications giving rise to an active profibrotic “myofibroblast” type. Myofibroblasts acquire contractile activity through induction of genes such as α‐smooth muscle actin (αSMA), which allows these cells to physically remodel the extracellular matrix (ECM). A central orchestrator of the activated fibroblasts and profibrotic activity is Transformation Growth Factor (TGF)‐β1. This cytokine mediates cardiac fibrosis associated with processes such as mechanical overload and tissue injury. In clinical studies, patients with hypertrophic or dilated idiopathic cardiomyopathy present elevated levels of TGF‐β1 in the ventricles. The presence of type 2 diabetes mellitus (DM2) is associated with the development of cardiac hypertrophy and fibrosis. Hyperglycemia in DM2 patients is postulated as a key fibrotic instigator. There are very few effective treatment options for the treatment of tissue fibrosis. Preclinical and clinical studies indicate that the flavonol (−)‐epicatechin (Epi, which is abundant in cacao) has tissue protecting properties. Preliminary using animal models has identified an ability of Epi to decrease the amount of fibrosis in the myocardium of rodents undergoing cardiac remodeling. However, the underlying mechanisms responsible for these actions are not known. This study aims to characterize in CF cultured in HG the potential of Epi to inhibit a pro‐fibrotic phenotype and identify the mechanisms involved.

Methods

CF were isolated from 3‐month‐old male Sprague Dawley rats through enzymatic digestion of the heart. After isolation, CF were cultured in 10 cm dishes in normal glucose (NG = 5 mM) DMEM media. Passage two (P2) (figure
) cultures were then used. For treatment, CF underwent starvation (NG DMEM, no phenol red, 1% FBS) for 24 h. Subsequently, subgroups of CF were incubated in the following conditions: 1) NG, 2) HG, 3) HG + 1μM Epi. Changes in CF phenotype were characterized by ELISA, Western blots, wound healing (migration) and cell number (proliferation).

Results

CF cultured in HG express a profibrotic phenotype as demonstrated by a significant increases vs. NG cells in TGF‐β1 levels (+20%) (Fig.
), fibronectin (+15%) (Fig.
) and total collagen (+30%) level (Fig.
). In HG cells, the Smad pathway (Smad 2/3) appeared activated vs. NG. Cell proliferation and migration also increased with HG. This profibrotic phenotype was essentially reversed in HG cells when treated with Epi as shown by TGF‐β1/Smad, fibronectin and collagen levels as well as migration and cell numbers.

Conclusion

CF cultured in HG acquire a profibrotic phenotype which is blocked by Epi an effect likely mediated by reducing TGF‐β1 levels. The involvement of cells surface receptors in such responses is currently being pursued.

Support or Funding Information

Department of Defense DoD PR150090, National Institute of Health, NIH DK98717, AG47326, and VA I01BX3230

HG Stimulates TGFB1 synthesis (A), Fibronectin (B) expression and Collagen Content (C) in CF. Epi treatment can block such effects. Values are means ± SEM. *P<0.05.

Figure 1