Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast-Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

Abstract

Osteoarthritis (OA), the prevalent debilitating joint disorder, is accelerated by dysregulated intercellular crosstalk, yet the role of fibroblast-like synoviocyte (FLS)-derived extracellular vesicles and particles (EVPs) in disease progression remains to be elucidated. Here, integrative analysis of clinical specimens, animal models, and publicly available datasets revealed significant alterations in exosomal pathways within OA synovium. Proteomic profiling revealed distinct molecular signatures in EVPs derived from inflammatory and senescent FLSs, reflecting the pathophysiological status of their parent cells. We demonstrated that FLSs under inflammatory and senescent states in OA secreted pathogenic EVPs that propagated joint degeneration by disrupting chondrocyte homeostasis, polarizing macrophages towards a pro-inflammatory phenotype, and impairing chondrogenesis of mesenchymal stem cells. To therapeutically target these pathogenic EVPs, we engineered an adeno-associated virus 9 (AAV9) vector fused with a synovium-affinity peptide (HAP-1) to deliver shRNA against Rab27a, a key regulator of EVP secretion. Intra-articular administration of the engineered AAV9 in a murine OA model induced by destabilization of the medical meniscus significantly reduced synovial hyperplasia, cartilage degradation and inflammatory responses, while demonstrating satisfactory systemic biosafety. Our findings establish FLS-derived EVPs as critical mediators of OA pathogenesis and propose a targeted strategy to block their secretion, offering a promising disease-modifying therapeutic avenue for OA.

Keywords: RAB27A; adeno‐associated virus; extracellular vesicles; fibroblast‐like synoviocytes; osteoarthritis; proteomics.

FIGURE 1

FLS pathology was accompanied by increased EVP secretion in OA. (A) Gene ontology (GO) cellular component enrichment analysis of differentially expressed genes between normal and OA synovium. (B, C) Violin plots for the expression levels of the exosome secretion marker (RAB27A), senescence markers (CDKN1A, CDKN2A) and inflammation‐related markers (PTGS2, IL6, MMP13), along with Pearson correlation analyses between the expression levels of these markers and RAB27A. Data was normalized as fragments per kilobase of exon model per million mapped fragments (FPKM). (D–G) Multiplex immunohistochemical (mIHC) staining of normal and OA synovium, with magnified views of the boxed areas showing individual colour channels. Scale bar = 100 µm. Line graphs display the relative intensity and co‐localization of each fluorescence along the line in the magnified images. (H, I) Representative RAB27A fluorescence staining images and quantification in the synovial region of sham‐operated mice and mice at 4 and 8 weeks post‐DMM surgery. Scale bar = 25 µm. (J) Schematic diagram of in vitro EVP collection. Briefly, FLSs were pre‐treated with IL‐1β (10 ng/mL) and Bleomycin (25 µg/mL) for 24 h and changed into fresh medium without inducers. EVPs were then isolated from the conditioned medium through ultracentrifugation. (K, L*) Representative MMP13 fluorescence staining and SA‐β‐gal staining images for FLSs after inflammation and senescence induction. (M) Diameter distribution of EVPs from control and pathological FLSs by NTA, with screenshots of the particle flow. (N, O) Representative TEM images for EVPs and Western blot gels for EVP protein markers. Scale bar = 200 µm. (P, Q) Quantification and protein concentration of EVPs isolated from different culture medium, both of which were normalized to original cell counts. ** Indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, versus the indicated groups, one‐way ANOVA.

FIGURE 2

Proteomic profiling of pathogenic EVPs reflected the pathological changes of the source cells. (A) Schematic diagram for proteomics analysis of EVPs isolated from FLSs after inducing inflammatory and senescent phenotypes, created with Figdraw (www.figdraw.com). (B) SDS‐PAGE gel electrophoresis images of proteins lysed from abovementioned EVPs. (C) Number of proteins identified by mass spectrometry in EVPs secreted from control FLSs and FLSs induced with inflammation and senescence. (D, E) Principal component analysis plot and Pearson's Correlation Coefficient heatmap of the protein composition in EVPs from three groups (n = 3 samples per group). The gradient colours and annotated values represent the Pearson correlation coefficients. (F) Number of differentially expressed proteins in EVPs between the three groups, with screening criteria set at an adjusted p value < 0.01 and a fold change >2 or <0.5. (G) Circos plot visualization of the overlaps among significantly altered proteins that overlap in Inf‐EVP and Sen‐EVP, with lines connecting the commonly altered proteins. (H) Pathway heatmaps of significantly differentially expressed proteins in Inf‐EVP and Sen‐EVP compared to Ctr‐EVP, enriched using Metascape. (I, J) Representative gene set enrichment analysis of proteins in Inf‐EVP or Sen‐EVP relative to those in Ctr‐EVP.

FIGURE 3

Inf‐EVP and Sen‐EVP contributed to OA progression through multifaceted mechanisms. (A, B) Bubble plots of GO enrichment analysis for differentially expressed proteins in Sen‐EVP and Inf‐EVP compared to Ctr‐EVP, highlighting inflammation‐ and cartilage‐related pathways. (C–E) Venn diagrams showing the overlap between differentially expressed proteins in Inf‐EVP and Sen‐EVP (vs. Ctr‐EVP) and genes associated with (C) OA cartilage degeneration, (D) chondrogenic differentiation of mesenchymal stem cells and (E) macrophage M1 polarization. Protein‐protein interaction networks were constructed using the top 20 hub proteins ranked by the MCC algorithm of the CytoHubba plugin in Cytoscape.

FIGURE 4

Pathogenic FLS EVPs disrupt chondrocyte and macrophages homeostasis in vitro. (A) Representative fluorescence for the internalization of EVPs by mouse chondrocytes. Scale bar = 20 µm. The red fluorescence represents EVPs labelled with the MemGlow fluorescent dye, and the blue fluorescence represents nuclei stained with DAPI. (B, C) Representative images and quantification of SA‐β‐gal staining for mouse chondrocytes after co‐culturing with different EVPs for 48 h, blue arrow indicting the SA‐β‐gal positive cells. Scale bar = 200 µm. (D) Representative Western blot images showing the senescence‐associated markers P16 and γ‐H2AX in chondrocytes treated with three types of EVPs for 48 h. (E) mRNA expression for OA‐related genes of mouse chondrocytes after co‐culturing with different EVPs for 24 h. (F–K) Representative images and quantification of COL2A1 and TUNEL staining for mouse chondrocytes, as well as EdU staining for ATDC5 cell line after stimulation with different EVPs for 48 h. Scale bar = 200 µm. (L) Internalization of EVPs by RAW264.7 macrophages. Scale bar = 200 µm. (M) mRNA expression for senescence marker (Cdkn1a), M1 polarization‐related genes (Il6, Tnf, Nos2 and Ptgs2), and M2 polarization‐related genes (Arg1, Cd163 and Cd206) of RAW264.7 macrophages after co‐culturing with different EVPs for 24 h. (N–Q) Representative images and quantification of iNOS and P16 fluorescence staining for RAW264.7 macrophages after stimulation with different EVPs for 48 h. Scale bar = 50 µm. (R, S) Concentration of TNF‐α and IL‐6 in the cell culture supernatant of EVP‐stimulated RAW264.7 macrophages. ** Indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, versus the indicated groups, one‐way ANOVA.

FIGURE 5

Pathogenic FLS EVPs impair chondrogenic differentiation of mesenchymal stem cells. (A) Schematic diagram of EVP stimulation on mouse BMSCs isolated from the femoral bone marrow cavity of mice. (B) Internalization of EVPs by BMSCs. Scale bar = 50 µm. (C) Expression of chondrogenic differentiation‐related genes in chondrogenesis‐induced BMSCs after 7 days of treatment with different EVPs. (D) Representative Western blot images of COL10A1, the marker of chondrocyte hypertrophy. (E, F) Representative images of alcian blue staining and SA‐β‐gal staining in BMSCs after 7 days of stimulation with different EVPs. Scale bar = 1 mm and 200 µm separately. (G) Schematic diagram of section staining observation after inducing BMSCs to form chondrocyte pellets for 21 days while simultaneously stimulating with different EVPs. (H, I) Representative images of SOX9 fluorescence staining, safranin O (SO) staining, alcian blue (AB) staining and toluidine blue (TB) staining of BMSC‐differentiated chondrocyte pellet sections. Scale bar = 50 µm. (J) Schematic diagram of EVP treatment on mouse ADSCs isolated from the iWAT of mice. (K) Internalization of EVPs by ADSCs. Scale bar = 50 µm. (L, M) Representative images of SA‐β‐gal staining and alcian blue staining of ADSCs after 7 days of chondrogenic induction and treatment with different EVPs. (N) Representative images of SOX9 staining in sections of chondrocyte pellets formed by ADSC after 21‐day induction.

FIGURE 6

Intra‐articular injection of FLS‐targeting AAV for delivering Rab27a‐shRNA to specifically reduce EVP secretion. (A) Rab27a expression levels in FLSs after transfection with control shRNA and Rab27a knockdown shRNA. (B, C) EVP size distribution and quantification after transfection with control shRNA and Rab27a knockdown shRNA. ** Indicates p < 0.01, versus the indicated groups, student’s t‐test. (D) Representative Western blot images for positive and negative surface markers of EVPs at equal concentration after shRNA transfection. (E) Schematic diagram of constructing a virus targeting FLS to inhibit Rab27a for intra‐articular injection. The synovium‐affinity peptide HAP‐1 is fused with the AAV9 viral capsid viral protein 2 (VP2), and the AAV9 vector is designed to simultaneously carry the gene encoding the mScarlet fluorescent protein and an expression cassette for shRNA. (F) Representative images of mScarlet fluorescence in the synovium and cartilage regions of mouse knee joints. Scale bar = 200 µm and 100 µm separately. (G) Representative images of mScarlet fluorescence in sections of multiple mouse organs. Scale bar = 200 µm. (H) Representative images and quantitative results of RAB27A expression in the synovial region after intra‐articular AAV injection. ** Indicates p < 0.01, versus the indicated groups, two‐way ANOVA.

FIGURE 7

Inhibition of FLS EVPs Alleviated OA in a Murine Model Without Toxicity. (A) Representative images of SO staining, HE staining, TB staining, COL2A1 immunohistochemistry staining and MMP13 immunofluorescence staining in the articular cartilage region after DMM operation and AAV virus injection. (B) Representative images of HE staining, and iNOS, IL‐1β and MMP13 immunofluorescence staining in the synovial region after DMM operation and AAV virus injection. (C–E) OARSI scores, synovitis scores and quantitative heatmaps of the immunostaining shown in (A) and (B), with six mice per group. ** Indicates p < 0.01, versus the indicated groups, two‐way ANOVA. (F) Levels of liver (ALT, AST), kidney (BUN, CREA) and cardiac injury (CK‐MB) markers in the serum of mice from each group after DMM operation and AAV injection (n = 6 per group). ns indicates p > 0.05, two‐way ANOVA.

FIGURE 8

Damaging effects of pathogenic FLS EVPs on multiple joint cell types and alleviation of OA via targeted R a b 2 7 a inhibition using genetically‐modified AAV (created in biorender.com).