Piezo1-driven Synovial Fibrosis in Post-Traumatic Osteoarthritis

Abstract Investigators: Easton Farrell (PhD Student), Tristan Maerz, PhD (Sponsor), Kurt Hankenson, DVM, PhD, MS (Co-sponsor). Contributors: Brendon Baker, PhD; Carla Scanzello, MD, PhD; Robert Mauck, PhD Context: Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis induced by traumatic injury. We study the pathological contributions of synovium – the inner joint lining – in PTOA. Increased deposition of extracellular matrix (ECM) proteins by PTOA-enhanced synovial fibroblasts (SFs) raises synovial stiffness and contributes to an exacerbatory cycle of stromal-immune crosstalk and ECM deposition, orchestrating fibrosis. Overactive Canonical Wnt/β-catenin signaling (cWnt) has emerged as a potential inducer of synovial fibrosis, but upstream cWnt effectors are not characterized. Piezo1, a mechanosensitive membrane ion channel, is a promising candidate for mediating cWnt signaling and concurrent fibrosis. Under mechanical stimulation (e.g., loading), Piezo1 translocates Yap1 to the nucleus, which perturbs cWnt signaling and drives fibrosis; however, the Piezo1-Yap1-cWnt axis and its role in promoting PTOA has not been established in SFs. The central objective of these proposed studies is to identify the role of Piezo1 in the emergence of pathologic SF phenotypes that interpret and drive fibrosis. The guiding hypothesis is that Piezo1 mediates cWnt signaling and promotes the pro-inflammatory and pro-fibrotic SF phenotypes observed in PTOA. Specific Aims: 1) Characterize the bi-directional regulation of cWnt and Piezo1 mechanotransduction in SFs; 2) Characterize the pro-fibrotic and pro-inflammatory role of Piezo1. Research Plan: Aim 1) cWnt activation will be measured under gain and loss of Piezo1 and Yap1, followed by evaluation of fibrosis-related and cWnt-target genes, as well as β-catenin localization to the nucleus (primary cWnt readout). To establish a reciprocal requirement of cWnt on active Piezo1, SFs will be cultured under gain and loss of cWnt. Bulk RNA-seq will confirm decreased Piezo1 and Yap1 transcripts, as well as identify perturbed biological processes related to mechanosensitivity, ion channel function, and ECM remodeling. Protein expression of Piezo1 and the functional ability of the Piezo1 channels to uptake calcium will also be evaluated. Aim 2) We will assess pro-fibrotic SF phenotype downstream of Piezo1 with and without inhibited Yap1. A SF- specific Piezo1 knockout mouse model (Pdgfrɑ-CreERT2;Piezo1fl/fl) will be utilized to study the differential progression of PTOA with or without intact Piezo1, based on our multifaceted “PTOA severity assessment.” Pain sensitization, protease production, bone remodeling, histological inflammation scoring, and IHC of fibrotic and inflammatory indicators will be compared between WT and Piezo1 knockout mice. Single cell RNA-seq will unbiasedly assess transcriptional changes, emergence of pro-fibrotic SFs, and inflammatory crosstalk patterns dependent on Piezo1 in PTOA.