Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study

Abstract

Objective: The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA).

Animals: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA.

Methods: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes.

Results: Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA.

Clinical relevance: We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Keywords: equine; exosomes; joint disease; miRNA; small non-coding RNA.

Figure 1—

Representative images from radiocarpal, middle carpal, and metacarpophalangeal joints classified as advanced post-traumatic osteoarthritis (PTOA) based on the modified Osteoarthritis Research Society International scores/grades for articular cartilage erosions (green arrowheads, green box), wear lines (black arrows), and palmar arthroses of the third metacarpal condyles (white asterisks).

Figure 2—

Nanoparticle tracking analysis, western blot, and transmission electron microscopy characterization of extracellular vesicles (EVs) from plasma and synovial fluid obtained from horses without (control) and with PTOA. Nanoparticle tracking analysis EV concentration and subtype data were evaluated using a mixed-effects model with horse as a random effect. There was no significant (ns) difference between plasma (A) or synovial fluid (B) EV concentration between control horses and horses with PTOA. There was a significant increase in plasma (C) and synovial fluid (D) exosomes in horses with PTOA compared to control and a significant decrease in plasma (C) and synovial fluid (D) microvesicles in horses with PTOA compared to control. (E) Western blot analysis of specific EV surface markers using antibodies CD9, CD81, TSG101, and calnexin confirmed the expression of CD9, CD81, and TSG101 and the absence of calnexin. (F) Transmission electron microscopy characterization of EVs isolated from plasma and synovial fluid detected a heterogeneous population of EVs (green arrowheads). Ultrastructural assessment confirmed expected EV size distribution and membrane integrity.

Figure 3—

Ingenuity Pathway Analysis (IPA)-derived functions of differentially expressed (DE) plasma EV microRNAs (miRNAs). A—IPA cellular functions, including fibrosis, differentiation of mesenchymal stem cells, proliferation of chondrocytes, and inflammation, were correlated with plasma EV DE miRNAs. B—Synovial fluid DE miRNAs were associated with IPA cellular functions, including fibrosis, differentiation of mesenchymal stem cells, differentiation of chondrocytes, cell viability, angiogenesis, and inflammation. Figures generated are graphical representations of molecules identified in our data in their respective networks. Molecule color indicates upregulation (red) or down-regulation (green). Cellular function color denotes predicted activation (orange), inhibition (blue), or those which could not be predicted (gray) in PTOA. Intensity of color is directly proportionate to fold change. Legends to the main features in the networks are shown. Scale bar, 500 nm.

Figure 4—

Bioinformatics analysis of plasma (A) and synovial fluid (B) EV DE miRNAs following small RNA sequencing and their putative mRNA targets. Treemap of the top 100 gene ontology (GO) terms. Hierarchical level is represented by a different colored rectangle (branch), each containing smaller rectangles (leaves). The size of the space inside each rectangle is based on the measured value. GO biological processes associated with dysregulated miRNA targets were identified following the TargetScan filter module in IPA. ToppGene was used to perform a functional enrichment analysis of predicted miRNA targets to highlight biological processes most significantly affected by dysregulated miRNA-mRNA interactions. GO terms (false discovery rate < 0.05) were summarized and visualized using REViGO and Cytoscape. The allowed similarity setting in REViGO is medium. Boxes represent the top biological processes that were significantly influenced by dysregulated miRNAs between horses with and without PTOA.