24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats

Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25 (OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3] accumulates in articular cartilage following injection of [3 H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OAlike environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis

Qingfen_Pan_Dissertation

Osteoarthritis (OA) is a degenerative disease characterized by joint inflammation and cartilage degeneration due to matrix degradation and chondrocyte apoptosis. Previously, drug therapies have been developed that aim to ease pain and reduce local inflammation. Currently, no effective drug exists that has no significant side effects. Therefore, an unmet medical demand exists for development of tissue-engineering strategies to promote articular cartilage repair and regeneration to treat OA. 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] is an attractive option for articular cartilage repair because of its anti-inflammatory and anti-apoptotic properties. 24R,25(OH)2D3, which is a naturally occurring metabolite of vitamin D3, also has not been shown to cause toxic side effects. Results from the study demonstrate that 24R,25(OH)2D3 can inhibit chondrocyte apoptosis and suppress the production of catabolic factors that result in cartilage degeneration in the in vitro model. Furthermore, although 24R,25(OH)2D3 regulates components of TGF-β1 pathway, the effect of 24R,25(OH)2D3 is not mediated through TGF-β1 signaling. In vivo delivery of 24R,25(OH)2D3 prevented cartilage degeneration and disease progression. In addition, intraarticular injection of 24R,25(OH)2D3 had an effect on cytokines and growth factors production both locally and systemically. Human articular chondrocytes responded to 24R,25(OH)2D3 treatment in both sex and maturation dependent manner. Collectively, results from this study suggest that 24R,25(OH)2D3 ccould be used as a clinical therapy for knee OA.Ph.D

Additional file 3: Figure S2. of Characterization of osteoarthritic human knees indicates potential sex differences

Phenotypic characteristics of female and male primary osteoblasts isolated from osteoarthritic knees. First-passage osteoblasts were treated with 10−8 M DHT. Alkaline phosphatase-specific activity (A) was measured in whole cell lysates. Protein levels of osteocalcin (B) and osteoprotegerin (C) were measured in conditioned media. Data show treatment compared with vehicle control ratios of the responses of 6 male and 6 female patients. The dashed line represents the vehicle control (dashed line = 1). * p < 0.05 vs. control; † p < 0.05 vs. male. (TIF 1003 kb

Additional file 2: Figure S1. of Characterization of osteoarthritic human knees indicates potential sex differences

Phenotypic characteristics of female and male chondrocytes isolated from osteoarthritic knees. First-passage chondrocytes were treated with 10−7 M 24R,25(OH)2D3 (A-D) or 10-8 M DHT (E–H). Alkaline phosphatase-specific activity (A, D) was measured in whole cell lysates. mRNAs for chondrocyte genes aggrecan (B, F), type-II collagen (C, G), and cartilage oligomeric matrix protein (D, H) were measured using real-time qPCR. Data show treatment compared with vehicle control ratios of the responses of 6 male and 6 female patients. The dashed line represents the vehicle control (dashed line = 1). * p < 0.05 vs. control; † p < 0.05 vs. male. (TIF 1363 kb

Additional file 1: Table S1. of Characterization of osteoarthritic human knees indicates potential sex differences

Primers for real-time PCR. (DOCX 16 kb

Cross talk between 24R,25(OH)₂D₃ and TGF-β1 signaling.

<p>First passage rat articular chondrocytes were treated with 10 ng/ml IL-1β for 12 hours. Then, the medium was exchanged and cells incubated with 10 ng/ml IL-1β containing 0, 10⁻⁹, 10⁻⁸, and 10⁻⁷ M 24R,25(OH)₂D₃. (<b>A-C</b>) After 12 hours, mRNA for TGF- β1 signaling molecules Smad 2, Smad3, and receptor Tgfbr2 were measured. * p<0.05 vs. 24R,25(OH)₂D₃ control; # p<0.05 vs. IL-1β control. (<b>D, E</b>) After 24 hours, active and latent TGF-β1 levels in the conditioned media were measured and normalized to DNA content in the cell lysate for each sample. * p<0.05 vs. IL-1β control; # p<0.05 vs. 24R,25(OH)₂D₃ control; $ p<0.05 vs. 10⁻⁹ M 24R,25(OH)₂D₃.</p

Primer sequences used in real-time qPCR analyses.

<p>Primer sequences used in real-time qPCR analyses.</p

Dose-dependent effect of 24R,25(OH)₂D₃ treatment.

<p>First passage rat articular chondrocytes were treated with 10 ng/ml IL-1β for 12 hours. Then, the medium was exchanged and cells incubated with 10 ng/ml IL-1β containing 0, 10⁻⁹, 10⁻⁸ and 10⁻⁷ M 24R,25(OH)₂D₃. (<b>A</b>-<b>C</b>) After 24 hours, NO, MMP-13 and PGE2 levels in the conditioned media were measured and normalized to total DNA content of each sample. (<b>D</b>-<b>F</b>) After 12 hours of treatment, mRNA levels of Acan, Col2a1, and Comp were measured and normalized to Gapdh. * p<0.05 vs. IL-1β control; # p<0.05 vs. 24R,25(OH)₂D₃ control; $ p<0.05 vs. 10⁻⁸ M; ^ p<0.05 vs. 10⁻⁷ M.</p

Assessment of catabolic, apoptotic, and chondrocytic mRNA levels in rat articular chondrocytes stimulated with IL-1β.

<p>First passage articular chondrocytes were treated with IL-1β for 24 hours. (<b>A</b>) NO production was measured in the conditioned media using a fluorometric assay. (<b>B</b>, <b>C</b>) MMP-13 activity and PGE2 production were measured in conditioned media using ELISA. (<b>D</b>, <b>E</b>) Apoptosis factors were measured in cell lysates using fluorometric assay and ³H-thymidine labeling. Amount normalized to total DNA. First passage articular chondrocytes were treated with IL-1β for 12 hours. (<b>F</b>-<b>J</b>) Expression of mRNA for chondrocyte genes was measured by real-time qPCR and normalized to Gapdh mRNA. * p<0.05 vs. control; # p<0.05 vs. 1 ng/ml IL-1β; $ p<0.05 vs. 5 ng/ml IL-1β.</p