Therapeutic Potential of Mesenchymal Cell-Derived miRNA-150-5p-Expressing Exosomes in Rheumatoid Arthritis Mediated by the Modulation of MMP14 and VEGF

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial tissue inflammation and joint destruction associated with the activation of angiogenesis. Exosomes, which play a role in cell-to-cell communication as carriers of genetic information, transfer microRNAs (miRNAs or miRs) between cells and have been studied as delivery vehicles for therapeutic molecules. The aim of the current study was to investigate the therapeutic effect of mesenchymal stem cell (MSC)-derived miR-150-5p exosomes on joint destruction in RA. The expression and secretion of miR-150-5p, matrix metalloproteinase (MMP) 14, and vascular endothelial growth factor (VEGF) in RA patients and fibroblast-like synoviocytes (FLS) were examined by quantitative RT-PCR, ELISA, and Western blotting. Immunohistochemistry was used to assess angiogenesis. MSCs were transfected with an miR-150-5p expression plasmid, and MSC-derived exosomes were harvested. The effect of MSC-derived miR-150-5p exosomes (Exo-150) on MMP14 and VEGF expression was examined. The effects of Exo-150 on cell migration and invasion in cytokine-stimulated FLS from RA patients were examined by HUVEC tube formation and transwell assays. The effect of Exo-150 in vivo was examined in a collagen-induced arthritis mouse model. Exo-150 decreased migration and invasion in RA FLS and downregulated tube formation in HUVECs by targeting MMP14 and VEGF. Injection of Exo-150 reduced hind paw thickness and the clinical arthritic scores in collagen-induced arthritis mice. Exo-150 reduced joint destruction by inhibiting synoviocyte hyperplasia and angiogenesis. Exosomes facilitate the direct intracellular transfer of miRNAs between cells and represent a potential therapeutic strategy for RA.

FIGURE 1.

Expression of miR-150-5p, MMP14, and VEGF in the serum, synovial tissue, and FLS of patients with RA. (A) Quantification of MMP14 and VEGF expression in serum from OA and RA patients by qRT-PCR. (B) MMP14 and VEGF expression in OA and RA synovial tissues. (C) Quantification of MMP14 and VEGF protein levels in serum from of OA and RA patients by ELISA. (D) miR-150-5p, MMP14, and VEGF expression in OA and RA FLS cells. (E) Lymphocyte infiltration was analyzed using H&E (HE) staining (original magnification × 200). VEGF, MMP14, and CD31 immunohistochemistry in OA and RA synovial tissue sections (original magnification × 400). **p < 0.01. CD31, cluster of differentiation 31.

FIGURE 2.

Identification of MSCs. (A) Von Kossa staining in MSCs cultured in osteogenic differentiation medium for 21 d. (B) Alcian blue staining in MSCs cultured in chondrogenesis differentiation medium for 21 d. (C) Oil Red O staining in MSCs cultured in adipogenesis differentiation medium for 14 d. (D) FCM analysis of the surface markers in MSCs.

FIGURE 3.

Exo-150 reduces the expression of RA markers. (A) MMP14 and VEGF expression in OA FLS and RA FLS measured by qRT-PCR. (B) Endogenous miR-150-5p expression in OA FLS and RA FLS. (C) miR-150-5p expression in the Exo-150, Exo-67, miR-150-5p–transfected MSCs, and cel-miR-67–transfected MSCs. (D) Exosomes extracted from MSCs were visualized by TEM and dynamic light scattering. Scale bar, 100 nm. (E) RA FLS were incubated with Exo-150 and labeled with DiO. Fluorescence photomicrographs show Hoechst RA FLS nuclei (blue) and DiO-labeled exosomes (green). Merge picture shows exosome uptake into RA FLS. (F) Western blot showing CD9 and CD63 expression in MSC-derived exosomes. (G) MMP14 and VEGF mRNA expression and protein expression in Exo-150 RA FLS cells. CD63, cluster of differentiation 63; CD9, cluster of differentiation 9; MSC, adipose-derived MSC. **p < 0.01, ***p < 0.001.

FIGURE 4.

Inhibition migration and angiogenesis of by Exo-150 in vitro. (A) Migration and invasion assays in RA FLS cells. Administration of Exo-150 inhibited the migration and invasion of RA FLS in vitro. Original magnification ×100. (B) Tube formation by HUVECs was reduced by Exo-150 treatment in vitro. Original magnification ×100. **p < 0.001.

FIGURE 5.

miR-150-5p suppresses MMP14 and VEGF expression in RA FLS by directly binding to the 3ʹ-UTRs. (A) Potential miR-150-5p binding sites on the 3′-UTR of MMP14 and VEGF. (B) FLS were transfected with firefly luciferase reporter constructs with wild-type or mutated binding sites and miR-150-5p mimics or inhibitor, and luciferase activity was measured at 48 h after transfection. (C) Western blot analysis of MMP14 and VEGF protein expression in miR-150-5p–treated FLS. (D) qRT-PCR analysis of MMP14 and VEGF mRNA expression in miR-150-5p mimics or inhibitor-treated cells. **p < 0.001.

FIGURE 6.

Effect of Exo-150 on the migration and invasion of proinflammatory cytokine–treated RA FLS mediated by MMP14 and VEGF. Proinflammatory cytokines upregulated MMP14 (A) and VEGF (B) expression in RA FLS. The effect of Exo-150 on MMP14 and VEGF expression is shown. (C) Effect of Exo-150 on the migration of RA FLS treated with proinflammatory cytokines. (D) Effect of Exo-150 on the invasion of RA FLS treated with proinflammatory cytokines. (E) Proinflammatory cytokines upregulated VEGF expression in HUVECs by cocultured RA FLS and HUVEC supernatants. (F) Tube formation by HUVECs was reduced by Exo-150 treatment in vitro under treatment of supernatants with proinflammatory cytokine–treated RA FLS and HUVECs coculture system. **p < 0.01.

FIGURE 7.

Therapeutic effect of Exo-150 in mice with established CIA. CIA mice received injection with either Exo-150 or Exo-67. (A) Immunofluorescence assay of DiO-labeled MSC exosomes in the CIA model articular cavity. Original magnification ×100. (B) Quantification of MMP14 and VEGF expression in joint tissue by qRT-PCR. Hind paw thicknesses (C) and clinical arthritis scores (D) were assessed on the indicated days after primary immunization. (E) H&E (HE) staining and Safranin O staining of the joint tissue from CIA mice on day 35. Original magnification ×100. (F) VEGF and CD31 immunohistochemistry and (G) MMP14 immunohistochemistry of mouse synovial tissue sections prepared on day 35. Original magnification ×400. Values represent the mean and SEM of three independent experiments (n = 10 mice per group). **p < 0.01. CD31, cluster of differentiation 31.