Dual Blockade of TGF-β Receptor and Endothelin Receptor Synergistically Inhibits Angiotensin II-Induced Myofibroblast Differentiation: Role of AT1R/Gαq-Mediated TGF-β1 and ET-1 Signaling

Abstract

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-β1) 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-β1 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-β1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT₁R/G_αq axis in adult human cardiac fibroblasts (HCFs). Following AT₁R stimulation, G_αq protein, not G_βγ subunit, was required for upregulation of TGF-β1 and ET-1. Moreover, dual inhibition of TGF-β and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT₁R/G_αq cascade transduced signals to TGF-β1, 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 (ET_AR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-β receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-β1 and ET-1 are major effectors of AT₁R/G_αq cascade, and therefore, negative regulation of TGF-β and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.

Keywords: TGF-β1; angiotensin II; angiotensin receptor blocker (ARB); cardiac fibrosis; endothelin receptor antagonist (ERA); endothelin-1; myofibroblast differentiation; α-SMA.

Figure 1

Blockade of AT₁R and G_αq protein antagonizes Ang II-induced myofibroblast differentiation. Serum-starved adult HCFs were pretreated with valsartan (valsar; 1 µM), FR900359 (G_αq inh; 1 µM), or gallein (G_βγ inh; 10 µM) for 1 h before stimulation with 200 nM Ang II for 6 h (A) or 24 h (B,C). (A) Relative mRNA levels of fibrotic markers, α-SMA and collagen I, were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). After treatment, immunofluorescence staining was used to determine α-SMA expression (green) (B) and stress fiber formation (red) (C). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Data are expressed as the mean ± SEM (n = 3). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. Ang II.

Figure 2

The synergistic function of TGF-β and ET-1 is required for Ang II-induced myofibroblast differentiation. Serum-starved adult HCFs were pretreated with valsartan (valsar; 1 µM), LY2109761 (TGF-β inh; 5 µM), bosentan (1 µM), or LY2109761 plus bosentan for 1 h before stimulation with 200 nM Ang II for 6 h (A) or 24 h (B,C). (A) Relative mRNA levels of fibrotic markers, α-SMA and collagen I, were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). After treatment, immunofluorescence staining was used to determine α-SMA expression (green) (B) and stress fiber formation (red) (C). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Data are expressed as the mean ± SEM (n = 3). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. Ang II.

Figure 3

ET-1 is a downstream effector of TGF-β1, and blockade of either Smad or ERK1/2 activities attenuates profibrotic effects of TGF-β1. (A–C) Serum-starved adult HCFs were pretreated with LY2109761 (TGF-β inh; 5 µM), bosentan (1 µM), or valsartan (valsar; 1 µM) for 1 h before treatment with 1 ng/mL TGF-β1 for 6 h (A) or 24 h (B,C). (D–F) Serum-starved adult HCFs were pretreated with 1 µM Smad inhibitor (Smad inh), 1 µM FR180204 (ERK inhibitor; ERK inh), 1 µM SB203580 (p38 MAPK inhibitor; p38 inh), or Smad inh plus ERK inh for 1 h before treatment with 1 ng/mL TGF-β1 for 6 h (D) or 24 h (E,F). (A,D) Relative mRNA levels of fibrotic markers, α-SMA and collagen I, were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). After treatment, immunofluorescence staining was used to determine α-SMA expression (green) (B,E) and stress fiber formation (red) (C,F). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Data are expressed as the mean ± SEM (n = 3). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. TGF-β1.

Figure 4

ET-1 acts as a downstream effector of Ang II and TGF-β1, which induces α-SMA and collagen I expression and stress fiber formation through the ET_ARs. (A–C) Serum-starved adult HCFs were incubated with LY2109761 (TGF-β inh; 5 µM), bosentan (1 µM), or valsartan (valsar; 1 µM) for 1 h before stimulation with 20 nM ET-1 for 6 h (A) or 24 h (B,C). (D–F) Serum-starved adult HCFs were pretreated with 1 µM ambrisentan (ambri; ET_AR antagonist) or 1 µM BQ788 (ET_BR antagonist) for 1 h before stimulation with 20 nM ET-1 for 6 h (D) or 24 h (E,F). (A,D) Relative mRNA levels of fibrotic markers, α-SMA, and collagen I were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). After treatment, immunofluorescence staining was used to determine α-SMA expression (green) (B,E) and stress fiber formation (red) (C,F). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Data are expressed as the mean ± SEM (n = 3). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. ET-1.

Figure 5

Ang II induces the secretion and synthesis of TGF-β1 and ET-1 via the AT₁R/G_αq axis in adult HCFs. Serum-starved adult HCFs were incubated with valsartan (valsar; 1 µM), PD123319 (1 µM), FR900359 (G_αq inh; 1 µM), or LY2109761 (TGF-β inh; 5 µM) for 1 h before stimulation with 200 nM Ang II for 24 (A) or 6 h (B). (A) After Ang II treatment, the culture medium was collected. The amounts of TGF-β1 and ET-1 secreted into the culture medium were determined by ELISA. (B) Relative mRNA levels of TGF-β1 and ET-1 were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. Ang II.

Figure 6

TGF-β1 induces the secretion and production of ET-1 via Smad- and ERK1/2-dependent pathways. Serum-starved adult HCFs were pretreated with 1 µM Smad inhibitor (Smad inh), 1 µM FR180204 (ERK inhibitor; ERK inh), or 1 µM SB203580 (p38 MAPK inhibitor; p38 inh) for 1 h before treatment with 1 ng/mL TGF-β1 for 24 (A) or 6 h (B). (A) After TGF-β1 treatment, the culture medium was collected. The amounts of ET-1 secreted into the culture medium were determined by ELISA. (B) Relative mRNA levels of ET-1 were analyzed by qRT-PCR. Data are expressed as the mean ± SEM (n = 4). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. TGF-β1.

Figure 7

Restorative effects of ARB, TGF-β inhibitor, and ERA on Ang II-induced myofibroblast differentiation. (A) Experimental design and timeline of the treatment in cultured adult HCFs. (B,C) Fibroblasts were treated with 200 nM Ang II for 24 h to activate fibroblast-to-myofibroblast transformation. After 24 h, cells were replaced with media containing Ang II with different inhibitors, including valsartan, LY2109761, bosentan, and LY2109761 plus bosentan for up to 3 days. Analysis was performed on days 1 or 3 after cotreatment. At the end of treatment, immunofluorescence staining was used to determine α-SMA expression (green) (B) and stress fiber formation (red) (C). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Data are expressed as the mean ± SEM (n = 3). * p < 0.05 vs. vehicle; ^# p < 0.05 vs. Ang II.

Figure 8

Schematic representing the TGF-β1, ET-1 and Ang II networking on myofibroblast differentiation. In adult HCFs, Ang II stimulation of AT₁Rs lead to an upregulation of TGF-β1 and ET-1 and activation of their effectors through the AT₁R/G_αq axis. The activated AT₁Rs transduce the signal through TGF-β1 cascade, which in turn upregulates ET-1 through the Smad-dependent and ERK1/2-dependent pathways. The elevated ET-1 then binds to and activates ET_ARs, leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Interestingly, antagonism of AT₁Rs and dual blockade of TGF-β1 receptors and ETRs exhibit antifibrotic effects by prevention and restoration of myofibroblast differentiation induced by Ang II. α-SMA: α-smooth muscle actin; Ang II: angiotensin II; AT₁R: angiotensin II receptor type 1; ARB: angiotensin II receptor blocker; ET-1: Endothelin-1; ERAs: endothelin receptor antagonists; TβR I/II: TGF-β receptor type I and II.