Dual Blockade of TGF- Receptor and Endothelin Receptor Synergistically Inhibits Angiotensin II-Induced Myofibroblast Differentiation: Role of AT₁R/G_q-Mediated TGF-1 and ET-1 Signaling

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-beta 1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-beta 1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-beta 1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (alpha-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by alpha-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and alpha-SMA synthesis and stress fiber formation through the AT(1)R/G(alpha q) axis in adult human cardiac fibroblasts (HCFs). Following AT(1)R stimulation, G(alpha q) protein, not G beta(?) subunit, was required for upregulation of TGF-beta 1 and ET-1. Moreover, dual inhibition of TGF-beta and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT(1)R/G(alpha q) cascade transduced signals to TGF-beta 1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ETAR), leading to increases in collagen I and alpha-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-beta receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-beta 1 and ET-1 are major effectors of AT(1)R/G(alpha q) cascade, and therefore, negative regulation of TGF-beta and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.