Biglycan Involvement in Heart Fibrosis: Modulation of Adenosine 2A Receptor Improves Damage in Immortalized Cardiac Fibroblasts

Abstract

Cardiac fibrosis is a common pathological feature of different cardiovascular diseases, characterized by the aberrant deposition of extracellular matrix (ECM) proteins in the cardiac interstitium, myofibroblast differentiation and increased fibrillar collagen deposition stimulated by transforming growth factor (TGF)-β activation. Biglycan (BGN), a small leucine-rich proteoglycan (SLRPG) integrated within the ECM, plays a key role in matrix assembly and the phenotypic control of cardiac fibroblasts. Moreover, BGN is critically involved in pathological cardiac remodeling through TGF-β binding, thus causing myofibroblast differentiation and proliferation. Adenosine receptors (ARs), and in particular A_2AR, may play a key role in stimulating fibrotic damage through collagen production/deposition, as a consequence of cyclic AMP (cAMP) and AKT activation. For this reason, A_2AR modulation could be a useful tool to manage cardiac fibrosis in order to reduce fibrotic scar deposition in heart tissue. Therefore, the aim of the present study was to investigate the possible crosstalk between A_2AR and BGN modulation in an in vitro model of TGF-β-induced fibrosis. Immortalized human cardiac fibroblasts (IM-HCF) were stimulated with TGF-β at the concentration of 10 ng/mL for 24 h to induce a fibrotic phenotype. After applying the TGF-β stimulus, cells were treated with two different A_2AR antagonists, Istradefylline and ZM241385, for an additional 24 h, at the concentration of 10 µM and 1 µM, respectively. Both A_2AR antagonists were able to regulate the oxidative stress induced by TGF-β through intracellular reactive oxygen species (ROS) reduction in IM-HCFs. Moreover, collagen1a1, MMPs 3/9, BGN, caspase-1 and IL-1β gene expression was markedly decreased following A_2AR antagonist treatment in TGF-β-challenged human fibroblasts. The results obtained for collagen1a1, SMAD3, α-SMA and BGN were also confirmed when protein expression was evaluated; phospho-Akt protein levels were also reduced following Istradefylline and ZM241385 use, thus suggesting that collagen production involves AKT recruited by the A_2AR. These results suggest that A_2AR modulation might be an effective therapeutic option to reduce the fibrotic processes involved in heart pathological remodeling.

Keywords: A2A receptor; biglycan; cardiac fibrosis; collagen.