Exosomal delivery of GrpE-like 1 from synovial mesenchymal stem cells activates PINK1-mediated mitophagy for cartilage repair in osteoarthritis

Abstract

GrpE-like 1 (GRPEL1)-carrying exosomes derived from synovial mesenchymal stem cells (SMSC) prevent mitochondrial dysfunction associated with osteoarthritis (OA) by activating PINK1-mediated mitophagy, restoring chondrocyte function, and preserving the extracellular matrix both in vitro and in vivo. Bioinformatics analysis of human OA datasets identified GRPEL1 as a mitophagy-related gene that is downregulated in OA. Exosomes enriched with GRPEL1 derived from SMSCs enhanced mitochondrial membrane potential and ATP production, reduced lipid peroxidation and reactive oxygen species, increased mitophagy markers (PINK1, Parkin, LC3-II/I), decreased p62 levels, and alleviated cartilage degeneration in a rat destabilization model. A causal role for mitophagy is supported by co-immunoprecipitation experiments confirming a GRPEL1-PINK1 interaction, and by PINK1 knockdown, which diminishes the protective effects of GRPEL1. These findings suggest that exosomes enriched with GRPEL1 derived from SMSCs represents a promising disease-modifying approach for OA by targeting mitochondrial quality control.

Key Words: Osteoarthritis; Exosomes; Synovial mesenchymal stem cell; GrpE-like 1; PINK1; Mitophagy; Mitochondrial quality control; Cartilage repair

Core Tip: This study identifies GrpE-like 1 (GRPEL1) as a mitophagy-related biomarker that is suppressed in osteoarthritis and demonstrates that exosomes derived from synovial mesenchymal stem cells can be engineered to deliver GRPEL1 to chondrocytes. Delivery of GRPEL1 via these exosomes restores mitochondrial homeostasis through PINK1-dependent mitophagy, reduces oxidative damage, preserves the extracellular matrix, and improves histological outcomes in a rat model of osteoarthritis. This represents a translationally promising, cell-free strategy for disease modification in osteoarthritis.

TO THE EDITOR

A study by Xiang et al[1] demonstrates that exosomes derived from synovial mesenchymal stem cells (SMSCs) carrying GrpE-like 1 (GRPEL1) activate PINK1-mediated mitophagy, thereby protecting chondrocytes and promoting cartilage repair in osteoarthritis (OA). This paper presents a compelling chain of evidence linking exosomal GRPEL1 to mitochondrial quality control (QC) and cartilage protection by integrating bioinformatics analysis of human OA cartilage, rigorous in vitro assays using CHON-001 cells, mechanistic co-immunoprecipitation studies, loss-of-function PINK1 experiments, and a rat destabilization OA model treated with intra-articular exosome injections. These findings expand the therapeutic rationale for mitochondria-targeted, exosome-based treatments in degenerative joint diseases. Mesenchymal stem cell-exosomal GRPEL1 activates PINK1-mitophagy to restore mitochondrial function and repair cartilage in OA - highlighting a cell-free, organelle-targeted strategy with disease-modifying potential for joint preservation.

Experimental approach and analytics

Bioinformatics: Differential expression analysis on Gene Expression Omnibus datasets GSE169077 and GSE114007 identified GRPEL1 (plus EFEMP1, SERPINA5, BNIP3 L) as mitophagy/mitochondria linked differentially expressed genes and correlated GRPEL1 downregulation with inflammatory markers and KL grade.

Exosome engineering & characterization: Human SMSCs were transfected (oe/shGRPEL1) and exosomes isolated (reagent-based protocol), then validated by TEM, NTA and exosomal markers (CD63/CD9/TSG101); GRPEL1 levels in exosomes were confirmed by quantitative reverse-transcription polymerase chain reaction and western blot.

Cellular models: CHON-001 chondrocytes challenged with interleukin (IL)-1β were treated with exosomes (control, oeGRPEL1, shGRPEL1) and assayed for viability (CCK-8), migration (Transwell), extracellular matrix (ECM) proteins (collagen II, aggrecan, matrix metalloproteinase-13, a disintegrin and metalloproteinase with thrombospondin motifs 5), reactive oxygen species (ROS), malondialdehyde, mitochondrial membrane potential (JC-1), mitochondrial DNA, ATP, and autophagy/mitophagy markers (PINK1, Parkin, p62, LC3).

Mechanistic tests: Co-immunoprecipitation demonstrated GRPEL1-PINK1 association; PINK1 knockdown experiments tested dependency of GRPEL1 effects.

In vivo: Rat destabilization of medial meniscus OA model; intra-articular injection of 1 × 10¹¹ particles/mL exosomes every 3 days for 4 weeks; macroscopic scoring, hematoxylin and eosin and Safranin O-Fast Green histology, and OARSI scoring were used to quantify cartilage repair.

Major insights and biological significance

GRPEL1 levels are decreased in the synovial fluid and cartilage of OA patients and negatively correlate with inflammatory markers, supporting its potential as a biomarker. Exosomal delivery of GRPEL1 restores its expression in chondrocytes and reverses IL-1β-induced functional impairments. Treatment with Exo-GRPEL1 reduces total and mitochondrial ROS, lowers malondialdehyde levels, restores mitochondrial membrane potential (ψm) and ATP production, and increases mitochondrial DNA content compared to injured controls. Exo-GRPEL1 also upregulates PINK1 and Parkin expression and increases the LC3-II/I ratio while decreasing p62 levels. Co-immunoprecipitation and PINK1 knockdown experiments demonstrate that the effects of GRPEL1 are PINK1-dependent. Intra-articular administration of Exo-GRPEL1 significantly improves macroscopic and histological cartilage scores, restores proteoglycan content and ECM proteins, and suppresses matrix-degrading enzymes in vivo[1].

Emerging trends and opportunities

Beyond microRNA, direct protein delivery - such as GRPEL1 - is an emerging, modular strategy to modulate intracellular organelle biology[1]. Engineering parental SMSCs to overexpress protective mitochondrial proteins or loading exosomes post-isolation enables targeted restoration of organelle homeostasis in degenerative tissues[2,3]. Integrating exosome therapy with biomaterial scaffolds, sustained-release hydrogels, or standard OA interventions (e.g., platelet-rich plasma, mechanical unloading) could enhance retention and repair efficacy[4].

Translational hurdles and regulatory considerations

Robust good manufacturing practices (GMP)-grade production, purification, and scalable yield of homogeneous SMSC-derived exosomes (SMSC-Exos) are essential for clinical translation. Due to heterogeneity in exosome cargo, validated potency assays and stringent batch QC are required[5,6]. The optimal dose and frequency, biodistribution following intra-articular injection, off-target effects, and long-term safety - including immune responses and tumorigenicity risk - must be established in larger animal models before proceeding to human trials[7,8]. Exosome therapeutics currently face unclear regulatory classification - whether as biologics, cell-free biologics, or nanoparticles - necessitating early engagement with regulatory authorities and standardized documentation of manufacturing processes and characterization[9].

Exosome-enabled interventions and delivery platforms

Local intra-articular injections[1] provide high joint exposure but exhibit rapid clearance, solutions include hydrogel carriers, exosome-loaded scaffolds, or surface-modified exosomes to enhance retention[10]. Engineering targeting ligands on exosome membranes (peptides, antibodies)[11] or loading with dual cargo (GRPEL1 + regenerative microRNAs) may improve selectivity and multi-pathway modulation[12,13]. Scalable exosome mimetics/exosome-inspired vesicles that mimic the functional cargo of exosomes could be alternatives for consistent manufacturing and regulatory simplicity[14]. From a clinical standpoint, the use of SMSC-Exos engineered to deliver GRPEL1 represents a paradigm shift - moving beyond symptomatic relief toward targeted molecular repair. The demonstrated activation of PINK1-mediated mitophagy addresses a core pathological feature of OA: Mitochondrial dysfunction in chondrocytes. Restoration of mitochondrial membrane potential, reduction in ROS, and preservation of ECM components such as collagen II and aggrecan are all clinically meaningful endpoints that correlate with improved joint function and reduced progression. This cell-free approach circumvents the regulatory and logistical complexities of stem cell transplantation, while retaining the regenerative benefits of mesenchymal stem cells. Moreover, the identification of GRPEL1 as a biomarker with negative correlation to inflammatory markers (C-reactive protein, IL-6) and KL grading opens avenues for patient stratification and monitoring therapeutic response. If validated in human trials, this strategy could complement existing OA treatments, delay the need for joint replacement, and offer a personalized therapeutic option for patients with mitochondrial-driven cartilage pathology. The integration of exosome-based therapeutics into orthopedic practice may redefine the clinical management of OA in the coming decade.

Roadmap for future studies

Dissect the sequence of GRPEL1 action, including mitochondrial targeting, steps of PINK1 activation, autophagosome formation, lysosomal flux, and extracellular clearance of mitochondrial debris. Conduct systematic dose-response, biodistribution, and toxicology studies in large animal OA models; define the minimum effective dose and therapeutic window. Develop GMP-compatible production pipelines and potency assays linked to mitophagy, along with standardized cargo profiling. Validate GRPEL1 levels in synovial fluid and cartilage as disease and prognostic biomarkers in prospective cohorts, correlating these findings with clinical endpoints. Evaluate Exo-GRPEL1 combined with sustained-release carriers or other regenerative modalities to enhance retention and functional repair. Once preclinical safety and manufacturing processes are established, design a first-in-human phase I trial incorporating robust biomarker, imaging, and functional endpoints.

Limitations and translation gaps

Batch consistency and potency require standardized assays to ensure uniform GRPEL1 content and exosome quality. Exposure and biodistribution studies should define intra-articular kinetics, tissue targeting, and retention of exosomes. Evaluations of long-term safety must address immunogenicity, off-target effects, and tumorigenicity of exosomes in large-animal models. Regulatory classification and CMC documentation should be clarified alongside the establishment of GMP-grade manufacturing with validated QC.

CONCLUSIONS

Xiang et al[1] provide compelling preclinical evidence that SMSC-Exos carrying GRPEL1 restore mitochondrial QC through PINK1-dependent mitophagy, reduce oxidative damage and ECM degradation, and promote cartilage repair in OA models. These findings broaden the therapeutic logic of exosome-based, organelle-targeted interventions and nominate GRPEL1 both as a mechanistic biomarker and a cargo for engineered exosome therapies. To translate this promising approach, focused efforts are required on scalable GMP manufacturing, potency and safety profiling, optimized delivery platforms, and prospective clinical validation.