α10hi MSCs decrease synovial membrane long term expression of TIMP-2 and NFκB following articular injury

Abstract

Purpose: Mesenchymal stem cell (MSC)-based therapies can limit the progression of focal cartilage lesions and prevent ongoing cartilage degeneration after joint injury by modulating the joint environment and/or contributing to repair. Integrin α10β1-selected mesenchymal stem cells (α10hi MSCs) are immunomodulatory, and display improved adhesion to osteochondral defects. In a previous study, intra-articular administration of α10hi MSCs four days post-articular impact surgery had protective effects against posttraumatic osteoarthritis (PTOA) in equine talocrural joints. To gain insight into mechanism, the current study aimed to determine if there was differential gene expression between treated and untreated joints. A NanoString custom codeset and nCounter SPRINT profiler was utilized to analyze mRNA extracted from formalin fixed paraffin embedded (FFPE) synovial membrane samples. Methods: Adult horses (2-5 years, n=7) were anesthetized and three focal cartilage injuries were arthroscopically delivered on both tali. In each horse, joints were randomized to receive treatment with 20 x 106 α10hi MSCs (treated), or vehicle only (control). FFPE synovial biopsies from both tali were obtained from each horse at 3 timepoints: Time 0 (initial surgery), six weeks (second look arthroscopy), and six months post-injury (euthanasia). RNA was extracted from each sample using High Pure FFPET RNA Isolation Kit (Roche) and qualified for NanoString using NanoDrop, gel electrophoresis, and a BioAnalyzer smear analysis. To detect expression of 39 genes associated with early posttraumatic osteoarthritis (PTOA), a custom NanoString codeset was developed. For each sample, 200ng of extracted RNA was run against the codeset on an nCounter SPRINT profiler. NanoString nSolver 2.6 Analysis Software (NanoStringTechnologies) was used to process raw data. Background threshold was calculated from the raw data as the mean ±2SD mRNA count across all the negative controls. Data were normalized against three housekeeping genes (GAPDH, HPRT1, and UBC). Normalized log transform data were exported in JMP Pro 13 and analyzed using paired t-test, with p < 0.05 as significant. Results: We designed a custom NanoString gene expression panel, including a subset of 39 genes selected to describe innate and adaptive immune system signaling, GPCR downstream signaling, extracellular matrix organization and metabolism of protein. Six months after injury, synovial membrane gene expression did not display a specific pattern associated with the treatment (Fig 1a), but CCL-5 expression was increased in the treated joints (p=0.028, negative binomial regression; Fig 1b). Interestingly, PRG4 expression had a trend toward higher expression in treated versus control joints (p=0.09). Paired t-test analysis (to account for the dependency of treated and control joints within the same horse) revealed that treatment with α10hi MSCs resulted in decreased expression of TIMP-2 (p=0.028) and NFκB (p=0.031) and increased expression of CCL-5 (p=0.049; Fig 2). Conclusions: Evidence suggests that after traumatic injury, cartilage homeostasis is disrupted, and oxidative and inflammatory stresses deregulate gene expression involving metalloproteinase production leading to joint destruction. Abnormal NF-κB activation provokes production of pro-catabolic mediators inducing cartilage degradation, while early suppression TIMPs seems predictive of therapeutic outcome. In the current study, decreased expression NF-κB and TIMP-2 in joints treated with α10hi MSCs may be indicative of earlier recovery from the posttraumatic pro-inflammatory and catabolic state. MSCs are known to be a source of the chemokine CCL-5 in culture. The increased expression of CCL-5 in joints treated with MSCs could indicate persistence of such cells, and further investigation including fate tracking of intraarticular α10hi MSCs is warranted. A trend toward higher PRG4 expression may explain the previously reported increase in lubricin localized to areas of cartilage injury in α10hi MSC-treated joints. In conclusion, post-traumatic injection of α10hi MSCs could represent a therapeutic aid to modulate the signaling pathways activated with trauma which lead to chronic cartilage degradation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)