| 1, BackgroudOsteoarthritis (OA), characterized by joint pain and functional impairment, is the most common degenerative joint disorder for middle aged and older people. Joint pain is the main symptom. The aim of most treatment is just to alleviate pain and improve joint function limited.There is no effective disease modifying treatment for OA until the end stage of disease necessitating joint replacement.Due to its incurable, costly and poor response to therapy, OA represents an enormous societal burden that elevates greatly alongwith the aging population.Although some risk factors like mechanical, metabolic or genetic factors have been identified, the cell signaling mechanisms that mediate joint degeneration leading to OA remain an active area of investigation as targets for preventive and disease-modifying therapies are greatly needed.It becomes clear that integrity of the subchondral bone is the key for preserving the function of the entire osteochondral unit. Increasing evidence shows that articular cartilage and subchondral bone act in concert as a functional unit. Subchondral bone provides mechanical support for overlying articular cartilage and undergoes constant adaptation to respond the changes in the mechanical environment by modeling or remodeling. Our previous studies have revealed, during bone remodeling, bone resorption and formation occur following a well-defined bone remodeling cycle and undergo at specific anatomical sites, while active TGFal released during osteoclast bone resorption couple bone resorption and formation by recruiting mesenchymal stem cells (MSCs) to form the new bone at the resorption area. We also found TGFa1 was aberrantly elevated in OA subchondral bone in both animal ACLT models and human specimen, and the high level of TGFa1 in the subchondral bone induce the formation of bone marrow osteoid islets during the onset of OA. What’s more, the progression of OA was attenuated when we block TGFal signal pathway by using TGFa1 antibody or knockout of TGFa type Ⅱ receptor in bone marrow MSCs.There are some common viewpoints on the onset of OA:1, abnormal mechanical loading plays a major role for the development of OA; 2, articular cartilage and subchondral bone act in concert as a functional unit to receive and dissipate the mechanical loading imposed on the joints; 3, the alteration for subchondral bone and articular cartilage is a dynamic, reciprocal and accumulated process.The small molecule halofuginone (HF), a presently FDA-approved drug for the treatment of scleroderma, is the active principal derived from ancient Chinese herbal medicine, which has been used to treat malarial fevers for more than 2000 years. HF has shown therapeutic promise in clinic trials for scleroderma and chronic graft-versus-host disease by inhibiting TGF-β mediated collagen type I synthesis as well as TGF-β-dependent phosphorylation of Smad2/3. HF has also been shown to induce anti-angiogenic effect in preclinical study at several essential stages of angiogenesis. Additionally, HF has been reported to inhibit Th17 differentiation via activating the amino acid response pathway, which is through suppressing prolyl-transfer RNA synthetase (ProRS) to induce uncharged tRNA accumulation within cells. And further study clarified that the inhibition of HF on ProRS is in need of the existence of unhydrolysed ATP, which directly connect two parts of HF onto two different substrate binding sites of ProRS. Taken together, we hypothesized that HF would attenuate OA progression by not only inhibiting the TGF-β-dependent phosphorylation of Smad2/3, but also reducing the release of TGF-β through suppressing Th17 induced osteoclastic bone resorption in subchondral bone. Our findings confirm that HF is competent to inhibit TGF-β-dependent smad2/3 phosphorylation and Thl7 differentiation, and the systemic injection and local embedding of HF normalized subchondral bone microarchitecture and ameliorated the degeneration of articular cartilage in anterior cruciate ligament transaction (ACLT) mouse and rat model.2, Materials and Methods(1) The effect of halofuginone on the subchondral bone and articular cartilage1) Animal model3-month-old male C57BL/6J (WT) Mice were purchased from Charles River. We then created the ACLT model. The mice were anesthetized with Ketamine and xylazine and after that the anterior cruciate ligament (ACL) were transected to induce abnormal mechanical loading-associated osteoarthritis on the left knee. Sham operation was done by opening the joint capsule and then suturing the incision in the left knee of independent mice. We divided the mice into 3 groups:sham-operated, vehicle-treated and halofuginone treated groups (n=18 pre group). Second day after surgery, we injected either halofuginone (sc-211579, Santa Cruz Biotechnology) with the dose of lmg/kg body weight or equivalent volume of vehicle (DMSO and PBS) intraperitoneally every other day for 30 d. And the mice will be sacrificed at 14,30 and 60 d post operation. For rats, we purchased 3-month-old male Lewis rats from Charles River. ACLT surgery was conducted as described as above. After ALCT surgery, a canal located in the medial plateau was made using a 20G needle. Halofuginone (2.5 ng) embedded in the alginate bead will be implanted in subchondral bone canal. The canal was then closed with bone wax. The rats were euthanized at 3 months after surgery (n=6 per group). All mice and rats were maintained in the Animal Facility of the Johns Hopkins University School of Medicine. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University, Baltimore, MD, USA.2) Detection of the marks of articular cartilageAt the time of euthanasia, we dissected the knee joint of mice and rats and fixed them in 10% buffered formalin for 48 h. After that, the knee joint were decalcified in 10% EDTA (pH 7.4) for 3 weeks and embedded in the paraffin or optimal cutting temperature (OCT) compound (Sakura Finetek).4-μm-thick longitudinal-oriented section of the knee joint medial compartment were cut and processed for H&E and safanin O and fast green staining.Sagittal sections of knee joint medial compartment were incubated with primary antibodies against MMP13 (Abcam,1:40, ab3208), collagen X (Abcam,1:80, ab58632) and Lubricin (Santa Cruz Biotechnology Inc.,1:50, sc-98454) at 4 ℃ overnight. And then for immunohistochemical staining, we used a horseradish peroxidase-streptavidin detection system(Dako) to detect immunoreactivity, which was followed by counterstaining with hematoxylin (Dako) or methyl green (Sigma-Aldrich). For immunofluorescent staining, we added second antibody conjugated with fluorescence to the sample sections, which were incubated for 1 h at room temperature (RT) while avoiding light. We then microphotographed slices to perform histomorphometric measurements on the entire area of the tibia subchondral bone (Olympus DP71). And quantitative analysis was conducted in a blinded fashion with OsteoMeasureXP software (OsteoMetrics, Inc.). The number of positive stained cells was counted in the subchondral bone or cartilage area from five sequential sections of per sample in each group. And the OAR.SI scores were calculated as previously described.3) Detection of the marks of subchondral boneMicro-CT analysis. We acquired the knee joints of mice and rats and dissected them free of soft tissue, fixed them in 70% ethanol overnight. We then scanned the specimen using high-resolution micro-CT (SkyScan 1172) and reconstructed the scanned images by image reconstruction software (NRecon v1.6). The data were analyzed using data analysis software (CTAn v1.9) and three-dimensional model visualization software (μ CTVol v2.0) regarding to the parameters of the tibial subchondral bone in the epiphysis. We set the scanner at a voltage of 50 k Vp, a current of 200 μA and a resolution of 5.8 μm per pixel. Three-dimensional histomorphometric analysis was performed using longitudinal images of the tibial subchondral bone. Three-dimensional structural parameters analyzed included:TV (total tissue volume; contains both trabecular and cortical bone), BV/TV (trabecular bone volume per tissue volume), Tb.Th (trabecular thickness), Tb.Sp (trabecular separation), SMI (structure model index), Conn.Dn (connectivity density) and Tb.Pf (trabecular pattern factor). The region of interest was defined to cover the whole tibial subchondral bone medial compartment, and a total of five consecutive images from the medial tibial plateau were used for three-dimensional reconstruction and analysis.Tartrate-resistant acid phosphatase (Trap) staining was conducted using a standard protocol (Sigma-Aldrich).Sagittal sections of knee joint medial compartment were incubated with primary antibodies against osterix (Abeam,1:600, ab22552) at 4 ℃ overnight. And then for immunohistochemical staining, we used a horseradish peroxidase-streptavidin detection system(Dako) to detect immunoreactivity, which was followed by counterstaining with hematoxylin (Dako) or methyl green (Sigma-Aldrich). We then microphotographed slices to perform histomorphometric measurements on the entire area of the tibia subchondral bone (Olympus DP71). And quantitative analysis was conducted in a blinded fashion with OsteoMeasureXP software (OsteoMetrics, Inc.). The number of positive stained cells was counted in the subchondral bone or cartilage area from five sequential sections of per sample in each group.(2) The effect of halofuginone on the Th17 cells in subchondral bone of the early stage of OA1) Detection of Th17 cells in subchondral boneAt the time of euthanasia, we dissected the knee joint of mice and rats and fixed them in 10% buffered formalin for 48 h. After that, the knee joint were decalcified in 10% EDTA (pH 7.4) for 3 weeks and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek. Sagittal sections of knee joint medial compartment were incubated with primary antibodies against CD4 (Santa Cruz Biotechnology Inc.,1:50, sc-19642), IL-17 (Santa Cruz Biotechnology Inc.,1:50, sc-7927) at 4 ℃ overnight. For immunofluorescent staining, we added second antibody conjugated with fluorescence to the sample sections, which were incubated for 1 h at room temperature (RT) while avoiding light.Flow cytometry. The C57BL/6J mice were assigned into 3 groups:sham operated, vehicle-treated (DMSO and PBS) and halofuginone-treated (sc-211579, Santa Cruz Biotechnology) groups. At 2 weeks and lmonth post surgery, the mice were euthanized and the tibial subchondral bone marrow and peripheral blood (heart puncture) were harvested to detect the alteration of the number of Th17 cells in subchondral bone marrow and peripheral blood separately. We lysed red blood cells using commercial ACK lysis buffer (Quality Biological, Inc.) and centrifuged the cells with a velocity of 1,200 r.p.m.,5min at room temperature. And then the cell pallet was resuspended and fixed with 4% paraformaldehyde. After that, the cells were washed with 0.1% BSA in PBS and counted, on average,3 x 106 cells were obtained from per specimen in each group. We then permeabilized the cells in 0.1% Triton X-100 before blocking in 3% FACS buffer (PBS,3%FBS and 0.1% NaN3 sodiumazide) for 30 min on ice. All staining was performed with fluorophore-conjugated primary and isotype control antibodies. The cells were incubated with Alexa Fluor 488-conjugated antibody against CD4 (eBioscience Inc.,1:100,53-0041) and eFluor 660-conjugated antibody against IL-17 (eBioscience Inc.,1:50,50-7177) or isotype control antibody for 1 h at 37 ℃ in dark room and then washed twice with 0.1% BSA in PBS. The cells were acquired immediately after washing with 3% FACS buffer. And the data were obtained using CellQuest software on a FACS Calibur flow cytometer (Becton Dickinson). Data were analyzed and all flow cytometry contour plots (with outliers) were generated using FlowJo software (TreeStar).2) Detection of RANKL expression in subchondral boneSagittal sections of knee joint medial compartment were incubated with primary antibodies against RANKL (Santa Cruz Biotechnology Inc.,1:50, sc-7628) at 4 ℃ overnight. For immunofluorescent staining, we added second antibody conjugated with fluorescence to the sample sections, which were incubated for 1 h at room temperature (RT) while avoiding light. We then microphotographed slices to perform histomorphometric measurements on the entire area of the tibia subchondral bone (Olympus DP71). And quantitative analysis was conducted in a blinded fashion with OsteoMeasureXP software (OsteoMetrics, Inc.). The number of positive stained cells was counted in the subchondral bone or cartilage area from five sequential sections of per sample in each group.(3) The effect of halofuginone on the smad2/3-dependent TGF-β signal pathway in MSCs1) Cell culture and Western Blot in vitroThe mouse adult MSCs were given from the Texas A&M Health Science Center College of Medicine Institute (College Station). We then cultured cells (Passage 3-5) in Iscove’s Modified Dulbeccco’s Medium (Invitrogen), which were supplemented with 10% FBS (Atlanta Biologicals),10% horse serum (Thermo Scientific) and 1% penicillin-streptomycin (Mediatech). The cell density in each well of six-well plate was 1.8 x 105 cells. And before the Western blot, the cultured MSCs were pretreated with halofuginone for 4,8 and 12 h separately and then examined following the treatment of TGF-β 1 for 30 min. We conducted the Western Blot to detect the protein of lysates from MSCs cultured in vitro. We centrifuged the cell lysates and separated the supernatants using SDS-PAGE and blotted them on a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories). And then, the proteins were analyzed by the incubation in specific antibodies and visualized by an enhanced chemiluminescence Kit (Amersham Bioscience). Smad2 (Cell Signaling Technology Inc.,1:1,000,3103) and pSmad2 (Cell Signaling Technology Inc.,1:1,000,3108) were identified by relative antibodies to measure the protein concentrations in the cell lysates.2) Detection of the alteration of MSCs and pSmad2/3 in vivoWe also created the animal model of ACLT and harvest joint tissue at the relative time points. At the time of euthanasia, we dissected the knee joint of mice and rats and fixed them in 10% buffered formalin for 48 h. After that, the knee joint were decalcified in 10% EDTA (pH 7.4) for 3 weeks and embedded in the paraffin or optimal cutting temperature (OCT) compound (Sakura Finetek). Sagittal sections of knee joint medial compartment were incubated with primary antibodies against Nestin (Aves Labs, Inc.,1:300, lot NES0407) at 4 ℃ overnight. And then for immunohistochemical staining of pSmad2/3, we used a horseradish peroxidase-streptavidin detection system(Dako) to detect immunoreactivity, which was followed by counterstaining with hematoxylin (Dako). For immunofluorescent staining of Nestin, we added second antibody conjugated with fluorescence to the sample sections, which were incubated for 1 h at room temperature (RT) while avoiding light. We then microphotographed slices to perform histomorphometric measurements on the entire area of the tibia subchondral bone (Olympus DP71). And quantitative analysis was conducted in a blinded fashion with OsteoMeasureXP software (OsteoMetrics, Inc.). The number of positive stained cells was counted in the subchondral bone or cartilage area from five sequential sections of per sample in each group.(4) The effect of halofuginone on the angiogenesis of subchondral bone1) AngiographyAngiography of microphil-perfused bones was performed to image blood vessels in bone. The mice were euthanized and then we opened the thoracic cavity and transected the inferior vena cava. The vascular system was flushed with 0.9% normal saline solution containing heparin sodium (100U ml-1) through a needle inserted into the left ventricle. We then pressure fixed the specimen with 10% neutral buffered formalin and washed formalin from blood vessels by heparinized saline solution. After that, radiopaque silicone rubber compound containing lead chromate (Microfil MV-122, Flow Tech) were injected into vascular system through the same needle inserted into the left ventricle to label the vasculature. We stored the specimens at 4℃ overnight for contrast agent polymerization. We then dissected and harvested the mouse knee joint and soaked them in 10% neutral buffered formalin for 4 d to make sure complete tissue fixation. The bone specimens were decalcified in a formic acid-based solution (Cal-Ex II) for 48 h to facilitate image threshold of the vasculature from the surrounding tissues. Images were acquired using a high-resolution micro-CT imaging system (Skyscan 1172). The scanner was set at a resolution of 9 μ m isotropic voxel size. We choose a threshold of 60 initially based on visual interpretation of threshold two-dimensional tomograms.2) Double staining of CD31 and Endomucin in subchondral boneKnee joints were dissected, fixed in 10% buffered formalin for 48 h, and decalcified in 10% EDTA (pH 7.4) for 3 weeks. Specimens were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek. Sagittal sections of knee joint medial compartment were incubated with primary antibodies against CD31 (Abcam,1:100, ab28364), endomucin (Santa Cruz, V.7C7,1:50), Ki67 (Novus Biologicals, NB500-170,1:50), MMP-2 (Santa Cruz Biotechnology Inc.,1:50, sc-10736)at 4 oC overnight. For immunofluorescence staining, second antibodies conjugated with fluorescence were incubated for 1 h at room temperature (RT) while avoiding light. We then microphotographed slices to perform histomorphometric measurements on the entire area of the tibia subchondral bone (Olympus DP71). Quantitative analysis was conducted in a blinded fashion with OsteoMeasureXP software (OsteoMetrics, Inc.).3, Results1, Halofuginone protects articular cartilage in ACLT mice by normalizing subchondral bone microarchitectureTo investigate the protective effect of halofuginone on the articular cartilage of osteoarthritis (OA) affected joint, we employed the anterior cruciate ligament transaction (ACLT) OA mice model in the current study. By intraperitoneally injection of halofuginone to the ACLT mice every other day for 1 month after surgery, we observed protective effect of halofuginone on the articular cartilage of ACLT mice. Specifically, proteoglycan loss and calcification of articular cartilage were significantly retarded in halofuginone-treated mice compared to vehicle treated controls. Immunostaining for collagen X (ColX), which reflects chondrocytes hypertrophy, and matrix metalloproteinase (MMP-13) also revealed subsidence of these two molecules after halofuginone treatment compared to the vehicle-treated controls. We also examined the lubricin levels in the cartilage, and we found increased lubricin-positive chondrocytes in the halofuginone-treated mice compared to the vehicle-treated control. In addition, Osteoarthritis Research Society International (OARSI)-modified Mankin scores were employed to quantitatively rate the severity of cartilage degeneration. Consistently, we found lower OARSI scores in halofuginone-treated mice compared with that in vehicle-treated control. All these data clearly demonstrate a protective effect of halofuginone treatment on the articular cartilage of OA.Our recent study on the pathogenesis of OA has indicated that de novo aberrant bone formation in subchondral bone plays a critical role in the onset of cartilage degeneration in OA. To investigate whether the attenuated cartilage degeneration by halofuginone treatment is attributed to the normalization of subchondral bone microarchitecture underneath, we used microcomputed tomography (μCT) to analyze the structure of tibial subchondral bone. Data obtained from μCT revealed normalization of subchondral bone microarchitecture of ACLT mice after systemic administration of halofuginone. Specifically, halofuginone treatment significantly reduced the tibial subchondral bone tissue volume (TV). And lower trabecular pattern factor (Tb.pf) was found in the halofuginone-treated mice compared to the vehicle-treated control, which reflects the disruption of connectivity and microarchitecture of subchondral trabecular bone, In addition, halofuginone treatment significantly increased the thickness of subchondral bone plate (SBP), which dynamically interacts with articular cartilage above. Taken together, we believe that halofuginone protects articular cartilage in ACLT mice by normalizing subchondral bone microarchitecture.2, Halofuginone suppresses Th17 cells in the subchondral bone of ACLT miceImmnofluorescent staining of Thl7 specific markers (CD4 and IL17) revealed a significant increase of Th17 cells in the subchondral bone marrow of ACLT mice from as early as 2 weeks after ACLT surgery, whereas systemic administration of halofuginone to the ACLT mice significantly suppressed these cells aggregated in the subchondral bone marrow. We further employed flow cytometry to enumerate Thl7 cells present in the bone marrow and peripheral blood of ACLT mice treated with either halofuginone or vehicle. In consistence with findings from immnofluorescent staining, we observed dramatic increase of the number of Th17 cells in the ACLT mice bone marrow 14 days after surgery, and halofuginone treatment effectively suppressed the Thl7 cells number in the bone marrow. However, the amount of Thl7 cells in the peripheral blood was unchanged regardless of ACLT surgery or treatment.Th17 cells are believed to be one of the major contributors to the RANKL-mediated osteoclastogenesis via the secretion of IL-17 in the bone marrow. In parallel with the increased aggregation of Thl7 cells in the subchondral bone marrow of ACLT mice, excessive expression of RANK ligand and aggregation of TRAP positive cells were observed in the subchondral bone of ACLT mice. More importantly, halofuginone treatment significantly suppressed the RANK ligand and TRAP positive cells compared to the vehicle-treated controls. Taken together, these data have demonstrated that halofuginone is able to suppress Th17 cells, consequently alleviate the RANKL load and inhibit osteoclasts maturation, and thus attenuates the elevated bone remodeling process observed in the early stage of ACLT OA model.3, Halofuginone inhibits Smad2/3-dependent TGF-β signaling pathway in bone marrow MSCsOur previous study on ACLT-induced OA model has demonstrated that elevated osteoclastic activity releases in the subchondral bone excessive active TGF-β，which will recruit nestin-positive MSCs to the subchondral bone marrow to form aberrant bone, and eventually lead to articular cartilage degeneration in OA. Thereby, we also examined the TGF-β signaling in the subchondral bone of ACLT mice in the current study. Immnohistochemical staining for phosphorylated Smad 2/3 (Psmad2/3) revealed significant activation of canonical TGF-β signaling pathway, indicating elevated activation of TGF-β ligand along with the increased osteoclastogenesis in the subchondral bone of ACLT mice. Interestingly, halofuginone treatment significantly decreased Psmad2/3 positive cells in the ACLT subchondral bone, indicating that halofuginone attenuates the TGF-β signaling either by directly acting on bone marrow cells or by indirectly suppressing the osteoclast-mediated TGF-β ligand activation.To investigate if halofuginone can directly act on bone marrow MSCs which are believed to be a major player at the onset of ACLT-mediated OA, we further performed western blot assays to determine the effect of halofuginone on the magnitude of TGF-p signaling in MSCs. We found that pre-treatment of MSCs with halofuginone can significantly inhibited the expression of Psmad2 in a dose-and time-dependent manner. Although these findings can not rule out the possibility that halofuginone may indirectly attenuate TGF-β signaling by suppression of Th17 cell-mediated osteoclastogenesis and the consequential activation of TGF-P ligand, our data have clearly shown that halofuginone is able to directly act on bone marrow MSCs and attenuate their response to the TGF-P ligand, which is excessively activated in the subchondral bone of ACLT animal models.4, Halofuginone relocates osteoprogenitors to the bone resorption sites and attenuates excessive angiogenesis.Our previous study has shown that modulation of TGF-P signaling in nestin+ MSCs re-couples the uncoupled bone remodeling phenotype in the ACLT subchondral bone, and thus protects the articular cartilage from degeneration. In the current study, we investigated whether suppression of excessive TGF-P signaling by halofuginone could redirect the bone marrow MSCs and osteoprogenitors to the bone resorbing sites to couple the bone remodeling process. By using immunofluorescent staining for nestin protein, we observed significant increase of nestin-positive cells in the subchondral bone marrow of ACLT mice 30 days after surgery, indicating an excessive recruitment of MSCs possibly through TGF-P signaling. In addition, osterix-positive osteoprogenitors were also significantly increased and aggregated in the subchondral bone marrow of both ACLT animal models, suggesting the osteoblastic differentiation of nestin-positive MSCs and potential de novo aberrant bone formation. By systemic administration of halofuginone to the ACLT mice significantly diminished nestin-positive cells present in the tibial subchondral bone, indicating a retarded recruitment of nestin+ MSCs by halofuginone. Importantly, osterix-positive osteoprogenitors were relocated to the subchondral bone surface after either systemic or local administration of halofuginone compared to vehicle-treated controls.These data indicate that halofuginone "re-couples" bone remodeling process in ACLT-induced OA by directing osteoprogenitors to the bone resorption sites. In addition to the relocation of osteoprogenitors, we also observed significant inhibition of angiogenesis in the subchondral bone of ACLT mice after administration of halofuginone. This was demonstrated by decrease in endothelial progenitor cells maker CD31, Endomucin and blood vessels visualized by microfil contrast-enhanced iCT-based microangiography. Because excessive angiogenesis is closely associated with de novo bone formation, we believe that the suppressive effect of halofuginone on the angiogenesis in the subchondral bone of ACLT mice will contribute to the inhibition of aberrant bone formation, thus being beneficiary to the articular cartilage.4, Conclusions1, Halofuginone protects articular cartilage in ACLT mice by normalizing subchondral bone microarchitecture;2, The effect of halofuginone on the Thl7 cells in subchondral bone of the early stage of OA3, Halofuginone inhibits Smad2/3-dependent TGF-β signaling pathway in bone marrow MSCs;4, Halofuginone relocates osteoprogenitors to the bone resorption sites and attenuates excessive angiogenesis. |
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