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

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SUMMARY
Pulmonary arterial hypertension (PAH) is a disease of abnormal pulmonary vascular remodeling whose medical therapies are thought to primarily act as vasodilators but also may have effects on pulmonary vascular remodeling. The angiotensin II type 1 receptor (AT 1 R) is a G protein-coupled receptor that promotes vasoconstriction through heterotrimeric G proteins but also signals via b-arrestins, which promote cardioprotective effects and vasodilation through promoting cell survival. We found that an AT1R b-arrestin-biased agonist promoted vascular remodeling and worsened PAH, suggesting that the primary benefit of current PAH therapies is through pulmonary vascular reverse remodeling in addition to their vasodilation.  increased levels of AngII are associated with PAH progression and mortality (4)(5)(6)(7). Blocking AT 1 R signaling is beneficial in rats with PAH, including restored RV dysfunction, decreased pulmonary vascular remodeling, and delayed PAH progression (4,8).
G protein-coupled receptors (GPCRs), such as the AT 1 R, signal canonically through heterotrimeric G proteins and multifunctional b-arrestin adapter proteins, which in addition to signaling, also promote receptor internalization and desensitization of G protein signaling (9). Subsequently, G protein-dependent (10) and/or b-arrestin-dependent (11) signaling is transduced by the activation of downstream mediators, such as Ca 2þ -dependent protein kinase C, mitogen-activated protein kinase signaling, and GPCR-mediated transactivation of receptor tyrosine kinases (12,13). G proteinmediated AT 1 R signaling has been found to lead to hypertension, myocardial hypertrophy, and cardiac dysfunction (13,14), whereas b-arrestin-mediated signaling has been shown to promote cardioprotective effects and decrease systemic and renovascular resistance in systolic heart failure (15)(16)(17)(18). Recently, "biased" AT 1 R ligands were shown to engage and stabilize distinct active conformations of AT 1 R (19) and activate only a subset of receptor-mediated signaling pathways (15,20), such as only through G proteins ("G proteinbiased") or b-arrestins ("b-arrestin-biased") (21,22). It is not known whether the benefit of current therapies is primarily mediated through vasodilation or reverse remodeling of the pulmonary vasculature, as patients on long-standing PAH therapies can still display severe vascular pathology (23). We hypothesized that we could address this question through the selective activation of AT 1 R b-arrestin-mediated signaling with a b-arrestin-biased agonist, which would be predicted to act as an acute vasodilator while promoting AT 1 R b-arrestin-mediated signaling.    Signaling), p38 (8690S; Cell Signaling), and phospho-  Figures 1A and 1B). Similarly, AngII increased RV pressure, whereas TRV023 did not ( Figure 1C), and neither drug had a significant effect on RV contractility ( Figure 1D). Notably, neither AngII or TRV023 had a significant effect on LV filling pressure (data not shown). These findings are consistent with G protein-  Figure 1F). Similarly, there were no significant differences in most hemodynamic parameters from PV loop analysis (Supplemental Figure 1).
Since there was no effect of chronic infusion of AngII, TRV023, or losartan on RV hemodynamics in MCT PAH rats, we next assessed RV and LV hypertrophy.
We found that LV hypertrophy was increased in rats that received TRV023 or AngII compared with losartan (Supplemental Figure 2), consistent with the significant effect of AngII on LV hemodynamics, whereas there were no differences in RV hypertrophy among treatment groups ( Figure 1G). Although this  Figure 3A). We performed gene ontology enrichment to identify specific cellular processes associated with these differentially regulated genes, which were  Figure 1. The expression and activation of MMPs can also be of significant importance in vascular remodeling (8,39).
To determine whether AT 1 R activation directly involves in the activation of MMPs, consistent with our previous results, we found that AngII and TRV023 increased the messenger RNA expression of MMP-2 ( Figure 4D) but not TIMP-1 in PH PASMCs ( Figure 4E), consistent with an effect that would promote vascular remodeling. We did not observe any other significant differences between groups in expression of similar markers (Supplemental   AT 1 R antagonist telmisartan decreased mean arterial pressure and cardiac contractility (15). TRV120027 has also been shown cause a neonatal-specific sustained positive inotropic effect without increasing heart rate, an effect thought to be mediated by its activation of L-type calcium channels (50). TRV023 has been shown to increase LV contractility and promote cell survival in mice, the effects of which are dependent on b-arrestin (16). Taken together, conventional and b-arrestin-biased AT 1 R agonists are pharmacologically distinct in vivo.
In preclinical models of left heart failure,   fibroblast activation that results in pulmonary vascular obstruction and right heart failure. The beneficial effect of current therapies for PAH is thought to be based primarily on their role as vasodilators, although any longterm benefit of these therapies is likely related to their promotion of pulmonary vascular reverse remodeling. At this time, there are few data to support a role for these therapies in promoting long-term pulmonary vascular reverse remodeling. To address this knowledge gap, we took advantage of "biased agonists" of the AT 1 R, a receptor that is known to play a role in pulmonary vascular remodeling. Although the AT 1 R is not targeted clinically in PAH, other GPCRs, such as the type A endothelin and prostacyclin receptor, are drug targets in PAH. We found that the AT 1 R "biased agonist" TRV023, which acts as a pulmonary vasodilator but also promotes cellular proliferation, promoted similar levels of abnormal pulmonary vascular remodeling as the vasoconstrictor AngII, effects that could be blocked by the antagonist losartan. Thus, this study suggests that the beneficial effect of current PAH therapies is through their long-term effects on pulmonary vascular reverse remodeling, although they may also have a short-term benefit from promoting pulmonary vasodilation as well.