AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis
Authors: Chow Bryna S. M. , Kocan Martina , Shen Matthew , Wang Yan , Han Lei , Chew Jacqueline Y. , Wang Chao , Bosnyak Sanja , Mirabito-Colafella Katrina M. , Barsha Giannie , Wigg Belinda , Johnstone Elizabeth K. M. , Hossain Mohammed A. , Pfleger Kevin D. G. , Denton Kate M. , Widdop Robert E. , Summers Roger J. , Bathgate Ross A. D. , Hewitson Tim D. , Samuel Chrishan S. 
Published: September 2019

Significance Statement
Studies have shown that the hormone serelaxin, which has organ-protective actions mediated via relaxin family peptide receptor 1 (RXFP1), its cognate G protein–coupled receptor, requires the angiotensin II type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. In this study, the authors describe a functional interaction between RXFP1, AT2R, and the angiotensin II type 1 receptor (AT1R), all of which are expressed on extracellular matrix–producing myofibroblasts, the cellular basis of progressive fibrosis. The crosstalk between these G protein–coupled receptors allows antagonists acting at each receptor to directly or allosterically block the antifibrotic actions of agonists acting at AT2R or RXFP1. These findings have significant therapeutic implications for a mechanistic understanding of the concomitant use of drugs acting at each receptor.

Background
Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein–coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown.

Methods
We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo.

Results
The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin’s antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan’s inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R–RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21).

Conclusions
These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.