β-Arrestin–Mediated Angiotensin II Type 1 Receptor Activation Promotes Pulmonary Vascular Remodeling in Pulmonary Hypertension

Figures

• 

  Figure 1. Hemodynamic Effects of Targeting AT1R With Acute or Chronic Infusions in Healthy and MCT PH Rats At 5-minute intervals, either increasing doses (1, 10, and 100 μg/kg) of AngII or increasing doses (10, 100 μg/kg, 1 mg/kg) of TRV023 were administered followed by treatment with 100 μg/kg losartan via the left internal jugular vein. Acute infusion of AngII in healthy Sprague-Dawley rats resulted in significant increases in left ventricular (LV) (A) pressure and (B) contractility (dP/dt – change in pressure over change in time), whereas TRV023 did not. AngII infusion also increased right ventricular (RV) (C) pressure but not (D) contractility, whereas TRV023 did not have a significant effect on either. “Blocker” denotes losartan. With chronic infusion in MCT PH rats, 1 week after MCT injection a (E) 2-week infusion of AngII, TRV023, losartan, or vehicle did not result in significant changes in (F) RV systolic pressure (RVSP) or (G) RV hypertrophy as assessed by Fulton index (RV mass/LV + septum (S) mass). Assessing survival by Kaplan-Meier in MCT PH rats (H), 2 weeks after MCT injection (I) an infusion of losartan had a significant effect on survival, whereas the AngII, TRV023, and…

• 

  Figure 2. Effect of TRV023 on Cellular Proliferation in the Lungs of MCT Rats Lungs from MCT rats treated with AngII, TRV023, losartan, or vehicle were inflation-fixed in 10% formalin, paraffin-embedded, and then sectioned for staining. (A) Endothelial cell staining with vWF (green) and α-smooth muscle cell actin (red) demonstrated (B) no significant differences in gross pulmonary vascular remodeling. For immunohistochemical staining with Ki-67 and cleaved caspase-3, the number of Ki-67/cleaved caspase-3–positive (brown) and Ki-67/cleaved caspase-3–negative cells (blue) were counted in pulmonary arteries and the ratio of positive/total cells was calculated. Representative micrographs showing that (C) Ki-67 and (E) cleaved caspase-3–positive cells were stained in brown and the negative cells in blue. Quantitative assessment of (D) Ki-67– and (F) cleaved caspase-3–positive cells from MCT rats. n = 5 rats for each group. N = 5 PA for each rat. Each bar represents mean ± SEM (n = 25). ∗P < 0.05; ∗∗P < 0.01. Scale = 50 μm. (G) Immunofluorescence demonstrating colocalization of Ki-67 (green) and α-smooth muscle actin (red) along with 4′,6-diamidino-2-phenylindole (DAPI) (blue) in AngII- and…

• 

  Figure 3. RNA Sequencing of Lungs From AT1R Ligand–Treated MCT Rats Demonstrates That TRV023 and AngII Regulate Pathways Important in Proliferation and Metabolism Total RNA from lung tissues from MCT rats treated with or without AngII, TRV023, losartan, and vehicle were isolated, and RNA sequencing was performed (n = 3 rats per group). (A) Volcano plot of differentially expressed genes between losartan, TRV023, and AngII from vehicle. (B) Gene ontology (GO) pathways from those differentially expressed genes for losartan, TRV023, and AngII, respectively. (C) Venn diagram of genes that were differentially expressed relative to vehicle in the losartan, TRV023, and AngII groups. (D) Hierarchical clustering of differentially expressed genes between AngII, TRV023, losartan, and vehicle. (E) Heat map showing selected differentially expressed genes shared between AngII- or TRV023-treated MCT rats. (F) Increased messenger RNA (mRNA) expression of MAPK signal regulating candidates Trem2 and Gdf15 by RNA sequencing. Decreased mRNA expression of negatively MAPK regulating candidates Cxcl14 and Arpp19 by RNA sequencing. Pathway enrichment performed by DAVID and GO enrichment tool. Statistical analysis…

• 

  Figure 4. AngII and TRV023 Activate MAP Kinases in PASMCs Isolated From Patients With PH PASMCs isolated from patients with PH were cultured and stimulated with AngII or TRV023 for analyzing phosphorylation of ERK and p38 at 10 and 20 minutes, respectively. (A) Representative immunoblot demonstrating AngII- and TRV023-induced phosphorylation of ERK and p38 activation in PASMCs isolated from patients with PH. Quantitation of (B) phospho-ERK and (C) phospho-p38 MAP kinases from n = 3 biological repeats of patients with PH ([AngII] = 500 nM, [TRV023] = 5 μM). Real-time PCR of targets important in regulation of cell migration demonstrate that AngII and TRV023 both increase levels of (D) MMP2 with no significant expression of (E) TIMP1 expression. ∗P < 0.05 by 1-way analysis of variance from vehicle-treated samples. ERK = extracellular signal-regulated kinase; FBS = fetal bovine serum; MAP = mitogen-activated protein; MMP = matrix metalloproteinase; PASMC = pulmonary artery smooth muscle cell; PCR = polymerase chain reaction; pERK = phospho-ERK; p-p38 = phospho-p38; TIMP = tissue inhibitor of metallopeptidases; Veh = vehicle; other abbreviations as in Figure 1.

• 

  Figure 5. AngII and TRV023 Promote PH-PASMC Proliferation and Migration (A) AngII-mediated proliferation of PASMCs at 24 hours. ∗P < 0.05 versus vehicle. (B) TRV023-mediated proliferation of PASMCs at 48 hours. ∗P < 0.05 versus vehicle. Quantification of (C) FBS-induced (∗P < 0.05 versus vehicle) and (D) AngII- and TRV023-induced PASMC migration at 12 hours (∗P < 0.05 versus vehicle). AngII- and TRV023-mediated migration was blocked by losartan (∗P < 0.05 versus AngII and TRV023 with no losartan). (D) Representative live cell imaging of AngII and TRV023 induced PASMC migration at 0 and 12 hours. Manual cell outlines are shown in red. Statistical analysis was performed by 1-way analysis of variance. n = 3 biological repeats of PASMCs from patients with PH. Abbreviations as in Figures 1 and 4.