Attenuated Joint Tissue Damage Associated With Improved Synovial Lymphatic Function Following Treatment With Bortezomib in a Mouse Model of Experimental Posttraumatic Osteoarthritis

Abstract

Objective: To investigate the roles of the synovial lymphatic system in the severity and progression of joint tissue damage and functional responses of synovial lymphatic endothelial cells (LECs) to macrophage subsets, and to evaluate the therapeutic potential of the proteasome inhibitor bortezomib (BTZ) in a mouse model of experimental posttraumatic osteoarthritis (OA).

Methods: C57BL/6J wild-type mice received a meniscal ligamentous injury to induce posttraumatic knee OA. Lymphangiogenesis was blocked by a vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody. Synovial lymphatic drainage was examined by near-infrared imaging. Joint damage was assessed by histology. RNA-sequencing and pathway analyses were applied to synovial LECs. Macrophage subsets in the mouse synovium were identified by flow cytometry and immunofluorescence staining. M1 and M2 macrophages were induced from mouse bone marrow cells, and their effects on LECs were examined in cocultures in the presence or absence of BTZ. The effects of BTZ on joint damage, LEC inflammation, and synovial lymphatic drainage were examined.

Results: Injection of a VEGFR-3 neutralizing antibody into the joints of mice with posttraumatic knee OA reduced synovial lymphatic drainage and accelerated joint tissue damage. Synovial LECs from the mouse OA joints had dysregulated inflammatory pathways and expressed high levels of inflammatory genes. The number of M1 macrophages was increased in the knee joints of mice with posttraumatic OA, thereby promoting the expression of inflammatory genes by LECs; this effect was blocked by BTZ. Treatment with BTZ decreased cartilage loss, reduced the expression of inflammatory genes by LECs, and improved lymphatic drainage in the knee joints of mice with posttraumatic OA.

Conclusion: Experimental posttraumatic knee OA is associated with decreased synovial lymphatic drainage, increased numbers of M1 macrophages, and enhanced inflammatory gene expression by LECs, all of which was improved by treatment with BTZ. Intraarticular administration of BTZ may represent a new therapy for the restoration of synovial lymphatic function in subjects with posttraumatic knee OA.

© 2018, American College of Rheumatology.

Figures

Figure 1.. Inhibition of lymphangiogenesis via VEGFR3 blockade exacerbates cartilage destruction in PTOA joints.

(A) Schematic illustration of the experimental design in which WT C57BL/6J mice received MLI or sham surgery, and on day 3 post-surgery were randomized to treatment with anti-VEGFR3 neutralizing antibody (R3-Ab, 0.8 mg/kg, i.p. 3 times/week) or IgG (placebo) for 6 weeks. (B) Mice were subjected to NIR-ICG imaging to quantify synovial lymphatic drainage, and representative images of the ICG remaining in the knee 24hr post-injection (circled region) are shown to illustrate the lack of lymphatic clearance in OA + R3-Ab treated mice. (C) Paraffin sections of knee joints were stained with AB/OG for OARSI scores, and representative micrographs are shown at 10× (bar = 0.1mm). The synovitis score (D) and osteophyte numbers (E) are presented as the mean +/− SD, n=10 mice per group (*p

Figure 2.. Lymphatic endothelial cells in PTOA synovium have an inflammatory phenotype.

Mice received MLI or sham surgery as in Fig.1. (A) LECs were isolated from synovium at 6 weeks post-surgery with anti-PDPN antibody, and the enrichment of this PDPN+ population was confirmed by flow cytometry (n=6 mice). (B) RNAseq was performed on the PDPN+ cells, which revealed about 1,000 differentially expressed genes in PTOA vs. sham synovial LECs. Pathway analysis indicated 12 dysregulated pathways (# indicates pathways involving NF-κB signaling; n=4–5 mice/group). (C) Synovial LECs from a different cohort of PTOA mice at 6 weeks post-surgery were subjected to qPCR. Data are mean + SD in which the Sham expression level = 1 (n=5 mice/group; *p

Figure 3.. Increased M1 macrophages in synovium of PTOA joints. Mice received MLI or sham surgery as in Fig.1, and knee joints were harvested at 6 weeks post-surgery for flow cytometry and histology.

(A) Total synovial cells were subjected to flow cytometry to determine the percentage of B cells (B220+), T cells (CD3+) and macrophages (F4/80+) (*p

Figure 4.. M1 macrophage accumulation adjacent to lymphatic vessels in PTOA synovium.

Frozen sections (30 μm thick) of knees at 5 weeks post-MLI were immuno-stained with antibodies against PDPN (red) for lymphatic vessels (a), F4/80 (green) for pan macrophages (b), iNOS or CD206 (purple) for macrophage subsets (c). M1s (A) were defined as F4/80/iNOS+ (a-d), and M2s (B) were defined as F4/80/CD206+ cells (g-l). Confocal microscopy was used for z-section imaging to obtain 20–25 consecutive images (e and k) with a step-width of 1μm. PDPN+ lymphatic vessels (red) and M1s or M2s (purple) were detected by Amira to generate 3D images (f and l) in a SurfaceGen module. Arrows in f indicate M1 cells near lymphatic vessels in a 3D image (n=4 mice).

Figure 5.. M1 macrophages promote expression of inflammatory genes by LECs, which is inhibited by Bortezomib.

(A) WT BMMs were treated with IL-1 or IL-4 to induce M1s or M2s, respectively. Cells were cultured for 24 hours to generate conditioned media (CM). LECs from a murine LEC cell line were treated with different concentrations of CM for 24 hours. Expression of inflammatory genes in LECs was determined by qPCR (data are mean +/− SD in which Ctl = 1; *pt test; n=3). (C) M1s were pre-treated with Btz for 8 hours, and then co-cultured with LECs for 24 hours. Expression of inflammatory genes in LECs was determined by qPCR (data are mean +/− SD in which PBS = 1; *p<0.05 via Unpaired t test; n=4). (D) LECs were treated with Btz for 8 hours. Expression of inflammatory genes was determined by qPCR (data are mean +/− SD in which PBS = 1; n=3).

Figure 6.. Bortezomib attenuates tissue damage in PTOA joints, improves synovial lymphatic function, and reduces LEC inflammation.

(A) WT mice were used. Different doses of Btz or 1% DMSO (Veh) were i.a. injected to knee joint. Levels of total ubiquitinated proteins in synovial tissues were examined by Western blot at 4 hours post-injection. Cortical bone from femur of the same joint was used as a control. (B) Schematic illustration of the experimental design in which WT C57BL/6J mice received MLI surgery, and 4 weeks later were randomized to DMSO or Btz (1× per week intraarticular injection for 7 weeks). (C) Ultrasound images of B-mode and 3D reconstruction indicate synovial volume at the end of treatment. (D) Changes of synovial volume (mm3) pre- and post-treatment. (E) ICG signal intensity of knee joints 6 hours post-ICG administration. (F) ICG clearance (%). (G) AB/OG stained sections. (H-J) OARSI score, synovitis score, and osteophyte numbers. Data are mean + SD. n=8–10 mice/group. Unpaired t test. *p