Reverse right ventricular structural and extracellular matrix remodeling by estrogen in severe pulmonary hypertension

Abstract

Chronic pulmonary hypertension (PH) leads to right-ventricular failure (RVF) characterized by RV remodeling. Ventricular remodeling is emerging as an important process during heart failure and recovery. Remodeling in RVF induced by PH is not fully understood. Recently we discovered that estrogen (E2) therapy can rescue severe preexisting PH. Here, we focused on whether E2 (42.5 μg·kg(-1)·day(-1), 10 days) can reverse adverse RV structural and extracellular matrix (ECM) remodeling induced by PH using monocrotaline (MCT, 60 mg/kg). RV fibrosis was evident in RVF males. Intact females developed less severe RV remodeling compared with males and ovariectomized (OVX) females. Novel ECM-degrading disintegrin-metalloproteinases ADAM15 and ADAM17 transcripts were elevated ∼2-fold in all RVF animals. E2 therapy reversed RV remodeling in all groups. In vitro, E2 directly inhibited ANG II-induced expression of fibrosis markers as well as the metalloproteinases in cultured cardiac fibroblasts. Estrogen receptor-β agonist diarylpropionitrile (DPN) but not estrogen receptor-α agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was as effective as E2 in inhibiting expression of these genes. Expression of ECM-interacting cardiac fetal-gene osteopontin (OPN) also increased ∼9-fold in RVF males. Intact females were partially protected from OPN upregulation (∼2-fold) but OVX females were not. E2 reversed OPN upregulation in all groups. Upregulation of OPN was also reversed in vitro by E2. Plasma OPN was elevated in RVF (∼1.5-fold) and decreased to control levels in the E2 group. RVF resulted in elevated Akt phosphorylation, but not ERK, in the RV, and E2 therapy restored Akt phosphorylation. In conclusion, E2 therapy reverses adverse RV remodeling associated with PH by reversing fibrosis and upregulation of novel ECM enzymes ADAM15, ADAM17, and OPN. These effects are likely mediated through estrogen receptor-β.

Fig. 1.

Experimental protocols. A: in vivo. Three experimental groups: control (CTRL) received no monocrotaline (MCT) injection at day 0. RVF received MCT injection at day 0 and was left untreated to develop severe right ventricular failure. E2 received 10-day estrogen (E2) treatment beginning day 21 post-MCT injection, and left for an additional 12 days after the end of therapy. B: in vitro. Neonatal rat ventricular myocytes (NRVMs) and fibroblasts were cocultured, starved for 24 h, and then treated with ANG II (ANG II group); ANG II with E2, estrogen receptor-β agonist diarylpropionitrile (DPN), or estrogen receptor-α agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) (ANG II + E2, ANG II + DPN, and ANG II + PPT groups, respectively); or left untreated (CTRL).

Fig. 2.

E2 restores RV function and reverses RV fibrosis associated with pulmonary hypertension (PH). A: RV pressure in CTRL, RVF, and E2. B: trichrome staining of RV cross sections. Red indicates cardiomyocytes and blue shows collagen deposition (fibrosis). C: quantification of %RV fibrosis in CTRL, RVF, and E2. D and E: RV lysil oxidase (D) and collagen I relative transcript expression (E) in CTRL, RVF, and E2. **P < 0.05 vs. CTRL; ##P < 0.05 vs. RVF.

Fig. 3.

E2 reverses RV ADAM15 and ADAM17 expression induced by PH. A: ADAM15 and ADAM17 relative transcript expression in the RV in CTRL, RVF, and E2. B: Western blot showing ADAM17 (bottom band) and vinculin (top band) representative samples from CTRL, RVF, and E2 groups. C: quantification of ADAM17 relative protein expression normalized to vinculin and CTRL from Western blot in CTRL, RVF, and E2 groups. **P < 0.05 vs. CTRL; ##P < 0.05 vs. RVF.

Fig. 4.

Estrogen therapy directly inhibits the expression of extracellular matrix (ECM) remodeling enzymes in vitro in cocultured NRVMs and fibroblasts. Relative transcript expression of ECM remodeling enzymes TGF-β (A), collagen I (B), ADAM15 (C), and ADAM17 (D) in cocultured NRVMs and fibroblasts in CTRL, ANG II, ANG II + E2, ANG II + DPN, and ANG II + PPT groups. **P < 0.05 vs. CTRL; ##P < 0.05 vs. ANG II.

Fig. 5.

Osteopontin (OPN) expression is elevated in the RV of RVF group and is reversed with E2-therapy. A: OPN relative transcript expression measured in CTRL, RVF, and E2. **P < 0.05 vs. CTRL; ##P < 0.05 vs. RVF. B: Western blot showing 4 different length isoforms of OPN (bottom 4 bands, 32 kDa, 44 kDa, 50 kDa, and 66 kDa) and vinculin (top band) representative samples. C: quantification of OPN protein expression normalized to appropriate CTRL and vinculin protein from Western blot in CTRL, RVF, and E2 groups. **P < 0.05 vs. corresponding CTRL; ##P < 0.05 vs. corresponding RVF. D: staining of RV cross sections double labeled with L-type calcium channel α1c (green) and OPN (red). E: OPN plasma levels measured by sandwich-ELISA, normalized to CTRL in CTRL, RVF, and E2. **P < 0.05 vs. CTRL; ##P < 0.05 vs. RVF. F: OPN transcript expression in NRVM/fibroblast culture in CTRL, ANG II treated, and ANG II + E2 treated. **P < 0.05 vs. CTRL; ##P < 0.05 vs. ANG II.

Fig. 6.

RV failure is associated with elevated Akt phosphorylation, but not ERK, in the RV. E2 therapy restores RVF-induced Akt phosphorylation. A: Western blot showing phospho-Akt (pAkt; top band) and Akt (bottom band) representative samples from CTRL, RVF, and E2 groups. B: quantification of relative pAkt/Akt and protein expression from Western blots. **P < 0.05 vs. CTRL; ##P < 0.05 vs. RVF. C: Western blot showing phospho-ERK1/2 (pERK1/2) (top two bands) and ERK1/2 (bottom two bands) representative samples from CTRL, RVF, and E2 groups. Quantification of relative pERK1/ERK1 (D) and pERK2/ERK2 protein expression (E) from Western blots. **P < 0.05 vs. CTRL; ##P < 0.05 vs. RVF.

Fig. 7.

Intact females are partially protected from PH-induced RV remodeling while ovariectomized (OVX) females are not. A: RV pressure in OVX and intact females. Relative transcript expression of TGF-β (B), OPN (C), ADAM15 (D), and ADAM17 (E). **P < 0.05 vs. corresponding CTRL; ##P < 0.05 vs. corresponding RVF; ∧∧P < 0.05 vs. corresponding OVX group.

Fig. 8.

Hypothetical scheme of PH-induced adverse RV remodeling and its reversal by estrogen. In a healthy right ventricle, the ECM is well-ordered and ECM degradation is tightly controlled. Severe PH leads to an increase in RV Akt signaling, and also elevated RV fibrosis, a degradation of general RV ECM proteins and integrins, and a reexpression of ADAMs and fetal gene OPN in the RV. Some OPN leaks into the plasma where it can be used as a biomarker. E2 therapy reduces Akt and ERK signaling and reverses the changes in the ECM, returning the RV to a healthy state.