Antifibrotic Effects of (−)-Epicatechin on High Glucose Stimulated Cardiac Fibroblasts

AUTHORS

Alejandra Garate-Carrillo, Israel Ramirez-Sanchez, Justina Nguyen, Julisa Gonzalez, Guillermo Ceballos, and Francisco Villarreal

ABSTRACT

Cardiac fibrosis is one of the hallmarks of a diabetic cardiomyopathy. When activated, cardiac fibroblasts (CFs) increase the production of extracellular matrix proteins. Transforming growth factor (TGF)-β1 is known to mediate cardiac fibrosis through the SMAD pathway. High glucose (HG = 25 mM) cell culture media can activate CFs using TGF-β1. There is a need to identify effective antifibrotic agents. Studies in animals indicate that treatment with (−)-epicatechin (Epi) appears capable of reducing myocardial fibrosis. Epi binds to G-protein coupled estrogen receptor (GPER) and activates downstream pathways. We evaluated the potential of Epi to mitigate the development of a profibrotic phenotype in HG stimulated CFs. CF primary cultures were isolated from young male rats and were exposed for up to 48 h HG media and treated with vehicle or 1 μM Epi. Relevant profibrotic end points were measured by the use of various biochemical assays. HG exposure of CFs increased TGF-β1 protein levels by ∼15%, fibronectin ∼25%, urea levels ∼60%, proline incorporation ∼70%, and total collagen ∼15%. Epi treatment was able to significantly block HG induced increases in TGF-β1, fibronectin, urea, proline, and total collagen protein levels. GPER levels were reduced by HG and restored in CFs treated with Epi an effect associated with the activation (i.e., phosphorylation) of c-Src. Epi treatment also reverted SMAD levels. Altogether, results demonstrate that CFs cultured in HG acquire a profibrotic phenotype, which is blocked by Epi an effect, likely mediated at least, in part, by GPER effects on the SMAD/TGF-β1 pathway.

Fibrinogen Mediates Cadmium-induced Macrophage Activation and Serves as a Predictor of Cadmium Exposure in Chronic Obstructive Pulmonary Disease

AUTHORS

Fu Jun Li, Ranu Surolia, Pooja Singh, Kevin G Dsouza, Crystal T Stephens, Zheng Wang

ABSTRACT

The etiologies of chronic obstructive pulmonary disease (COPD) remain unclear. Cadmium (Cd) causes both pulmonary fibrosis and emphysema, however the predictors for Cd exposure and the mechanisms by which Cd causes COPD remain unknown. We demonstrated that Cd burden was increased in lung tissue from subjects with COPD and this was associated with cigarette smoking. Fibrinogen levels increased markedly in lung tissue of smoked COPD patients, compared to never-smokers and control subjects. Fibrinogen concentration also correlated positively with lung Cd load, but inversely with the predicted % of FEV1 and FEV1/FVC. Cd enhanced the secretion of fibrinogen in a cdc2-dependent manner whereas fibrinogen further mediated Cd-induced peptidylarginine deiminase 2 (PAD2)-dependent macrophage activation. Using lung fibroblasts from CdCl2-treated toll-like receptor 4 (TLR4) wild type and mutant mice, we demonstrated that fibrinogen enhanced Cd-induced TLR4-dependent collagen synthesis and cytokine/chemokine production. We further showed that fibrinogen complexed with connective tissue growth factor (CTGF), which in turn promoted the synthesis of plasminogen activator inhibitor-2 (PAI-2) and fibrinogen, and inhibited fibrinolysis in Cd-treated mice. The amounts of fibrinogen were increased in the bronchoalveolar lavage fluid (BALF) of Cd-exposed mice. Positive correlations were observed between fibrinogen with hydroxyproline. Our data suggests that fibrinogen is involved in Cd-induced macrophage activation and increases in fibrinogen in patients with COPD may be used as a marker of Cd exposure and predict disease progression.

Antifibrotic Effects of (−)-Epicatechin on High Glucose Stimulated Cardiac Fibroblasts

AUTHORS

Alejandra Garate-Carrillo, Israel Ramirez-Sanchez, Justina Nguyen, Julisa Gonzalez, Guillermo Ceballos, and Francisco Villarreal

ABSTRACT

Cardiac fibrosis is one of the hallmarks of a diabetic cardiomyopathy. When activated, cardiac fibroblasts (CFs) increase the production of extracellular matrix proteins. Transforming growth factor (TGF)-β1 is known to mediate cardiac fibrosis through the SMAD pathway. High glucose (HG = 25 mM) cell culture media can activate CFs using TGF-β1. There is a need to identify effective antifibrotic agents. Studies in animals indicate that treatment with (−)-epicatechin (Epi) appears capable of reducing myocardial fibrosis. Epi binds to G-protein coupled estrogen receptor (GPER) and activates downstream pathways. We evaluated the potential of Epi to mitigate the development of a profibrotic phenotype in HG stimulated CFs. CF primary cultures were isolated from young male rats and were exposed for up to 48 h HG media and treated with vehicle or 1 μM Epi. Relevant profibrotic end points were measured by the use of various biochemical assays. HG exposure of CFs increased TGF-β1 protein levels by ∼15%, fibronectin ∼25%, urea levels ∼60%, proline incorporation ∼70%, and total collagen ∼15%. Epi treatment was able to significantly block HG induced increases in TGF-β1, fibronectin, urea, proline, and total collagen protein levels. GPER levels were reduced by HG and restored in CFs treated with Epi an effect associated with the activation (i.e., phosphorylation) of c-Src. Epi treatment also reverted SMAD levels. Altogether, results demonstrate that CFs cultured in HG acquire a profibrotic phenotype, which is blocked by Epi an effect, likely mediated at least, in part, by GPER effects on the SMAD/TGF-β1 pathway.

Effect of Fibroblast Growth Factor 2 on Extraocular Muscle Structure and Function

AUTHORS

Jolene C. Rudell; Linda K. McLoon

ABSTRACT

Purpose: Mutations in the fibroblast growth factor (FGF) receptor can result in strabismus, but little is known about how FGFs affect extraocular muscle structure and function. These were assessed after short-term and long-term exposure to exogenously applied FGF2 to determine the effect of enhanced signaling.

Methods: One superior rectus muscle of adult rabbits received either a series of three injections of 500 ng, 1 µg, or 5 µg FGF2 and examined after 1 week, or received sustained treatment with FGF2 and examined after 1, 2, or 3 months. Muscles were assessed for alterations in force generation, myofiber size, and satellite cell number after each treatment.

Results: One week after the 5 µg FGF2 injections, treated muscles showed significantly increased force generation compared with naïve controls, which correlated with increased myofiber cross-sectional areas and Pax7-positive satellite cells. In contrast, 3 months of sustained FGF2 treatment resulted in decreased force generation, which correlated with decreased myofiber size and decreased satellite cells compared with naïve control and the untreated contralateral side.

Conclusions: FGF2 had distinctly different effects when short-term and long-term treatments were compared. The decreased size and ability to generate force correlated with decreased myofiber areas seen in individuals with Apert syndrome, where there is sustained activation of FGF signaling. Knowing more about signaling pathways critical for extraocular muscle function, development, and disease will pave the way for improved treatment options for strabismus patients with FGF abnormalities in craniofacial disease, which also may be applicable to other strabismus patients.