P198Inhibition of TGF-beta pathway reverts extracellular matrix remodeling in T. cruzi-infected cardiac microtissues

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Abstract

Background and Purpose

Chagas disease, which is caused by the protozoan T. cruzi, affects over 10 million people worldwide and represents the leading cause of cardiac lesions in economically productive adults in endemic areas of Latin America. Chagasic cardiomyopathy is a progressive dysfunctional illness driven by fibrosis, myocarditis, and hypertrophy. TGF-β plays a central role in fibrosis, regulating extracellular matrix (ECM) molecules, such as fibronectin, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Therapeutic targets against fibrosis are of great pharmaceutical interest, since them would lead to functional improvement on injured tissue. This study aims to analyze the efficacy of a TGF-β pharmacological inhibitor, SB 431542, on fibrosis reversion using an in vitro model of cardiac microtissues infected with T. cruzi.

Methods

Primary cardiac-cell cultures were obtained from embryos of Swiss Webster mice through proceedings approved by Ethical Committee in Animal Use. Cardiac microtissues (3D cultures) were formed in agarose-coated 96-U-well plates. T. cruzi-infected (Y strain) and uninfected cardiac microtissues were treated or not with 10μM SB 431542 (Tocris Bioscience). The expression of ECM molecules (fibronectin, laminin, MMP-2, MMP-9 and TIMP-1) were observed by western blot and immunofluorescence assays.

Results

Our western blot analysis revealed that the expression of fibronectin and laminin were decreased by 2-fold when infected cardiac microtissues were treated with SB 431542. Confocal images indicated alterations in the distribution and also an increased staining of these two proteins in infected microtissues. SB 431542 treatment of infected microtissues resulted in a fibronectin staining pattern similar to control sample. Moreover, we observed by western blot that inhibition of TGF-β pathway led to a 1.5-fold increase in MMP-9, whereas MMP-2 expression was 3.6-fold decreased. TIMP-1 expression was decreased by 2.6-fold after treatment, suggesting that this molecule could be directly involved in MMP-9 regulation.

Conclusions

SB 431542 administration was able to reverse the observed higher levels of fibronectin and laminin, probably through positive regulation of MMP-9 and inhibition of TIMP-1 activity. Our study demonstrates that treatment of infected cardiac microtissues with a pharmacological inhibitor of TGF-β can restore ECM balance, indicating that its use could be considered for the treatment of chagasic cardiomyopathy.

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