Proteomic landscape of IGF-I gene transfer in osteoarthritis-derived human bone marrow stromal cells undergoing chondrogenesis

Abstract

Osteoarthritis (OA) is a progressive joint disease marked by the breakdown of cartilage and impaired repair capacity. Gene therapy applied to human bone marrow-derived adherent stromal cells (BM-MSCs) with recombinant adeno-associated virus (rAAV) vectors represents a promising strategy for cartilage regeneration. However, the molecular mechanisms underlying its effects remain largely unexplored. In this study, we investigated the proteomic changes induced by rAAV-mediated insulin-like growth factor I (IGF-I) gene transfer during the chondrogenic differentiation of BM-MSCs derived from OA patients. BM-MSCs from OA donors were transduced with recombinant rAAV vectors encoding either IGF-I or the lacZ gene (β-galactosidase) as a control and cultured in three-dimensional micromass systems under chondrogenic conditions for 21 days. Histological, immunohistochemical (IHC) and immunofluorescence (IF) analyses were performed to characterize the chondrogenic phenotype of the model and to provide orthogonal validation of selected proteomic findings. Mass spectrometry-based proteomic profiling identified protein detection patterns and quantitative changes associated with IGF-I overexpression. Pathway and protein-protein interaction (PPI) analyses were carried out to explore the functional implications of differentially expressed proteins. A total of 4,284 proteins were identified, of which 112 showed notable changes in detection patterns or abundance associated with IGF-I. Functional enrichment analyses showed involvement of pathways related to metabolism, endoplasmic reticulum (ER) homeostasis, apoptosis regulation, cell proliferation, glycosylation, and the endosomal sorting complex required for transport (ESCRT) machinery. Histological and IHC analyses provided supportive evidence of cartilage-related matrix deposition. Orthogonal validation by IHC and IF confirmed the IGF-I-mediated upregulation of β-catenin and exportin-1 (XPO1). Further analyses provided exploratory evidence of increased active β-catenin and predominantly extranuclear distribution of sirtuin-1, a known XPO1 substrate, in IGF-I-transduced micromasses. This study provides the first proteomic characterization of rAAV-mediated IGF-I gene therapy in OA-derived BM-MSCs undergoing chondrogenesis. The IGF-I gene transfer was associated with broad proteomic changes involving metabolic and protein processing pathways, and enhanced cartilage-specific matrix deposition, highlighting molecular mechanisms of potential relevance to cartilage repair in OA.