Halofuginone attenuates osteoarthritis by inhibition of TGF-β activity and H-type vessel formation in subchondral bone

Abstract

Objectives: Examine whether osteoarthritis (OA) progression can be delayed by halofuginone in anterior cruciate ligament transection (ACLT) rodent models.

Methods: 3-month-old male C57BL/6J (wild type; WT) mice and Lewis rats were randomised to sham-operated, ACLT-operated, treated with vehicle, or ACLT-operated, treated with halofuginone. Articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. Immunostaining, flow cytometry, RT-PCR and western blot analyses were conducted to detect relative protein and RNA expression. Bone micro CT (μCT) and CT-based microangiography were quantitated to detect alterations of microarchitecture and vasculature in tibial subchondral bone.

Results: Halofuginone attenuated articular cartilage degeneration and subchondral bone deterioration, resulting in substantially lower OARSI scores. Specifically, we found that proteoglycan loss and calcification of articular cartilage were significantly decreased in halofuginone-treated ACLT rodents compared with vehicle-treated ACLT controls. Halofuginone reduced collagen X (Col X), matrix metalloproteinase-13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS 5) and increased lubricin, collagen II and aggrecan. In parallel, halofuginone-attenuated uncoupled subchondral bone remodelling as defined by reduced subchondral bone tissue volume, lower trabecular pattern factor (Tb.pf) and increased thickness of subchondral bone plate compared with vehicle-treated ACLT controls. We found that halofuginone exerted protective effects in part by suppressing Th17-induced osteoclastic bone resorption, inhibiting Smad2/3-dependent TGF-β signalling to restore coupled bone remodelling and attenuating excessive angiogenesis in subchondral bone.

Conclusions: Halofuginone attenuates OA progression by inhibition of subchondral bone TGF-β activity and aberrant angiogenesis as a potential preventive therapy for OA.

Keywords: Arthritis; Osteoarthritis; Treatment.

Figure 1

Halofuginone preserves articular cartilage after anterior cruciate ligament transection (ACLT). (A) Safranin O and fast green staining (top). Solid arrows indicate proteoglycan loss and cartilage destruction at 30 and 60 days post operation. Scale bar, 500 µm. H&E staining (bottom) where calcified cartilage (CC) and hyaline cartilage (HC) thickness are marked by double-headed arrows. Scale bars, 200 µm. (B–E) Immunostaining and quantitative analysis of lubricin (B-top, C), matrix metalloproteinase (MMP) 13 (B-middle, D) and COL X (B-bottom, E) in articular cartilage at 30 days post operation. Scale bar, 100 µm. (F) Osteoarthritis Research Society International–modified Mankin scores of articular cartilage at 0, 14, 30 and 60 days after surgery. Sham=sham-surgery. Vehicle=ACLT-surgery treated with vehicle. HF=ACLT-surgery treated with halofuginone. n=6 per group. *p<0.05 compared with sham or as denoted by bar, **<0.01 compared as denoted by bar; ^#p<0.05 compared with the vehicle.

Figure 2

Halofuginone normalises subchondral bone after anterior cruciate ligament transection (ACLT). (A) Representative three dimensional micro-CT images of sagittal views of subchondral bone medial compartment at 30 and 60 days after sham operation or ACLT surgery. Scale bar, 500 µm. (B–D) Quantitative micro-CT analysis of tibial subchondral bone of total tissue volume (TV) (B), trabecular pattern factor (Tb.pf) (C) and subchondral bone plate thickness (D). (E and F) Tartrate-resistant acid phosphatase (TRAP) staining (E) and quantitative analysis (F) at 14 days after surgery. Scale bar, 50 µm. (G and H) Immunohistochemical staining (G) and quantification (H) of osterix-positive cells (brown) in subchondral bone 30 days after surgery. Scale bar, 100 µm. Sham=sham-surgery. Vehicle=ACLT-surgery treated with vehicle. HF=ACLT-surgery treated with halofuginone. n=6 per group. *p<0.05 compared with sham or as denoted by bar, **<0.01 compared as denoted by bar; ^#p<0.05 compared with the vehicle.

Figure 3

Halofuginone suppresses Th17 cells after anterior cruciate ligament transection (ACLT). (A) Representative immunofluorescence double staining for CD4 (green), interleukin (IL) 17 (red) and merged images (colocalisation=yellow) 14 days after surgery. Scale bar, 50 µm. (B) Quantitative analysis of the number of CD4+IL17+ (Th17) cells per bone marrow (BM) area (mm²). (C) Flow cytometry analysis of double staining Th17 (CD4+IL17+) cells in subchondral BM 14 days post surgery. The green line represents CD4+ cells in an unstained control. (D) Flow cytometry quantitative analysis of the percentage of Th17 cell in subchondral BM and peripheral blood plasma 14 days after surgery. (E and F) Immunofluorescence staining (E) and quantification (F) for receptor activator of nuclear factor κ B ligand (RANKL) in subchondral bone 14 days post surgery. Sham=sham-surgery. Vehicle=ACLT-surgery treated with vehicle. HF=ACLT-surgery treated with halofuginone. n=6 per group. *p<0.05, compared with the sham; ^#p<0.05 compared with the vehicle.

Figure 4

Halofuginone restores coupled bone remodelling after anterior cruciate ligament transection (ACLT). (A) Immunofluorescence staining for nestin in sagittal sections of subchondral bone medial compartment 30 days post surgery. Scale bar, 100 µm. (B) Quantitative analysis of the number of nestin-positive cells per bone marrow (BM) area of tibial subchondral bone. (C) Western blot analysis of the phosphorylation of Smad2 in cultured mesenchymal/stromal stem cells treated with the combination of recombinant human TGF-β1 and increasing doses of halofuginone after 4, 8 or 24 h. (D and E) Immunohistochemistry staining (D) and quantification (E) of pSmad2/3-positive cells in sagittal sections of subchondral bone medial compartment 14 days post surgery. Sham=sham-surgery. Vehicle=ACLT-surgery treated with vehicle. HF=ACLT-surgery treated with halofuginone. n=6 per group. Scale bar, 50 µm. *p<0.05 compared with the sham and ^#p<0.05 compared with the vehicle.

Figure 5

Halofuginone attenuates aberrant angiogenesis in subchondral bone of anterior cruciate ligament transection (ACLT) mice. (A–C) Three dimensional CT-based microangiography of medial tibial subchondral bone (A) 30 days post surgery, with a quantification of vessel volume relative to tissue volume (VV/TV) (B) and vessel number (VN) (C). Scale bar, 500 µm. (D and F) Representative immunofluorescence double staining (D) and quantification (F) of CD31 (green) and endomucin (red) positive cells 1 month after surgery. Scale bar, 50 µm. (E and G) Immunofluorescence double staining (E) and quantification (G) of Ki67 (green) and endomucin (red) positive cells 1 month after surgery. Scale bar, 50 µm. (H) Quantification of MMP-2 positive cells in subchondral bone marrow (BM) 1 month post surgery. Sham=sham-surgery. Vehicle=ACLT-surgery treated with vehicle. HF=ACLT-surgery treated with halofuginone. n=6 per group. *p<0.05 compared with sham and ^#p<0.05 compared with vehicle.