The mechanism study of THBS3 in regulating cartilage vascularization/bone coupling via the TGF-β/Smad2/3 pathway in osteoarthritis

The mechanism study of THBS3 in regulating cartilage vascularization/bone coupling via the TGF-β/Smad2/3 pathway in osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The mechanism study of THBS3 in regulating cartilage vascularization/bone coupling via the TGF-β/Smad2/3 pathway in osteoarthritis Jingyao Yan, Yanping Zhao, Xiaoying Zhu, Hanya Lu, Yanli Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4167008/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Vascularization and osteogenesis coupling is observed in osteoarthritis (OA) cartilage. THBS-3 belongs to the extracellular matrix (ECM) proteins and is highly expressed in cartilage tissue. The effect of THBS-3 on OA is unclear. This study aims to explore the mechanistic role of THBS-3 in OA. Design: Expressions of THBS-3 was detected by Western blot (WB) and RT-qPCR. WB was employed to measure the expression levels of synthesis and degradation metabolism, as well as vascularization/ossification coupling. Migration and tube formation experiments were conducted to assess the migratory and tube-forming abilities of HUVECs influenced by THBS-3. Micro-CT was utilized for 3D imaging in mice. Immunohistochemistry was employed to detect the expression of synthesis, degradation metabolism, and vascularization/ossification coupling-related markers. Additionally, WB was utilized to assess the transforming growth factor-beta (TGF-β) signaling pathway. Results Proteinomics sequencing has revealed a higher expression level of THBS-3 in OA cartilage. Chondrocytes from OA joints exhibited significantly higher expression of THBS-3 relative to healthy individuals. In experiments conducted both in vivo and in vitro, THBS-3 exhibited a dual impact by enhancing catabolic metabolism, suppressing synthetic metabolism, and fostering the coupling of vascularization and osteogenesis within the cartilage. THBS-3 activated the TGF-β signaling pathway, and blockade of the TGF-β signaling pathway resulted in increased p-Smad2/3 expression in OA cartilage cells and decreased expression of vascularization /ossification coupling. Conclusion THBS-3 can promote the vascularization/ossification coupling of cartilage cells by activating the TGF-β/Smad2/3 signaling pathway, providing new insights and targets for the treatment of OA. Osteoarthritis THBS-3 vascularization ossification Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction OA is an extremely common and multiarticular degenerative disease that leads to chronic joint pain and physical disability, resulting in a primary financial burden and extensive impairments in quality of life[ 1 – 2 ]. Accumulating studies have revealed that cartilage degradation, osteophyte formation, bone remodeling, neoangiogenesis, and synovial inflammation that lead to the pathogenesis of OA[ 3 ].But so far, the underlying mechanisms of OA remain abstruse. Normal articular cartilage is avascular and aneural, but accumulating studies have suggested that increased angiogenesis at the bone-cartilage junction and in non-calcified cartilage in OA.The invasion of blood vessels across the osteochondral junction could potentially lead to cartilage calcification or ossification[ 4 ]. Within this context, the process of ossification within the cartilage contributes to the formation of bone spurs, exacerbating the progression of osteoarthritis[ 5 ]. In the context of osteoarthritis, the formation of new blood vessels plays a role in sustaining persistent inflammation. This occurs through the continuous provision of oxygen and nutrients to endothelial cells. These neo-vessels facilitate the entry of pro-inflammatory cytokines into the surrounding local microenvironment, further exacerbating the diseases processes[ 6 ]. Angiogenesis is regulated by the balance of proangiogenic and antiangiogenic factors. However, pathological neovascularization in OA represents a breaking of these normal homeostatic mechanisms. Additionally, the process of vascularization is accompanied by sensory nerve growth, which may also be a contributing factor to the pain experienced by OA patients[ 7 ]. Osteogenesis and angiogenesis are coupled in space-time and interconnected manner. In order to maintain bone homeostasis, osteoblasts secrete angiogenic factors to promote growth of vessels and regulate their functions, while endothelial cells release angiocrine signals to regulate bone remodeling and repair. The bidirectional cross-talk between osteogenesis and angiogenesis is complex and several factors such as VEGF, FGFs, PDGF-B, BMP-2 have been involved in this interplay[ 4 ]. Therefore, regulating cartilage vascularization/bone coupling may contribute to the structural damage and pain associated with osteoarthritis and offer potential targets for novel treatments. THBS-3 belongs to the thrombospondin (THBS) protein family, which is a part of the ECM protein family. THBS form a family of five secreted, multimeric, modular matricellular glycoproteins[ 8 ]. They exhibit specific spatio-temporal expression patterns in both embryonic and adult tissues and are involved in a wide range of essential processes, including angiogenesis, wound healing, cell proliferation, migration, as well as the organization of connective tissues[ 9 ]. THBS-3 can also regulate postnatal skeletal maturation, influence endochondral ossification within cartilage, and participate in bone formation[ 10 ]. However, the exact relationships between THBS-3 and OA are still to be clarified, and there have been no reported studies on this connection. Further research is needed to understand the role of THBS-3 in OA and its implications for the disease. Materials and Methods 1.In vitro experiments 1.1. Isolation and culture of chondrocytes from OA patients Cartilage tissue was obtained from discarded knee joints of osteoarthritis patients (n=10) who underwent total knee replacement surgery at the First Affiliated Hospital of Harbin Medical University. Cartilage tissues were washed three times with phosphate-buffered saline (PBS), and then cut into 1 mm 2 , digested in 2mg/ml collagenase type II (Sigma, St. Louis, MO, USA) 37℃ overnight. After that, the cells were filtered through a cell strainer. Primary cells were used from the P0-P1 passage. 1.1.2 Culture of human normal chondrocytes Human normal articular chondrocytes were obtained from the C28/I2 human normal chondrocytes line(Otwo Biotech, Inc, Cat#HTX2308;China)and cultured in DMEM medium with the addition of 10% FBS and 0.5% penicillin-streptomycin, at 37 °C and 5% CO 2 . 1.1.3 Culture of HMVEC Human microvascular ECs (HUVECs) (OTWO, Inc, Cat#HTX1922;China), were grown in endothelial cell medium (ECM)(ScienCell, Cat#1001; USA) supplemented with 25ml fetal bovine serum (FBS)(ScienCell, Cat#0025; USA), 5 ml Endothelial Cell growth supplement (ECGS)(ScienCell, Cat#1052; USA), 5 ml penicillin/streptomycin solution(P/S)(ScienCell, Cat#0503; USA), and used for tube formation and migration experiments. HUVECs were cultured to confluence, followed by incubation in serum-reduced ECM (1% FBS) for 24 hours prior to stimulation with THBS-3 for an additional 24 hours. 1.2 Small interfering RNA and transfection experiment The small interfering RNAs (siRNAs) and transfection reagent were purchased from RIBOBIO Co., Ltd. (RiboBio, China)according to manufacturer’s instructions. Transfection target sequence (Table 1). 1.3 Western blotting Following a triple wash with PBS, cells underwent protein extraction. Proteins were separated on 10% or 12% SDS-PAGE gels. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes (Thermo-Fisher, Hampton, NH) and probed with primary antibodies against THBS-3 (Proteintech, Cat#19727-1-AP;USA), Aggrecan (Abbkine, Cat#ABP54013;China), ADAMTs-5 (Abcam, Cat#AB41037;USA), MMP-13(Bioword Technologyl, Cat#BS1231;China),BMP-2(Abbkine, Cat#ABP0179;China), FGF-2 (Abbkine, Cat#ABP53280;China), ANG-2(Abcam, Cat#AB155106;USA), VEGF-A (Abcam, Cat#AB214424;USA), PDGF-B(Abcam, Cat#AB178409;USA), TGF-β1 (Abcam, Cat#AB179695;USA), p-smad2/3 (Abbkine, Cat#ABP0048;China)and GAPDH (ABclonal, Cat#A19056;China),overnight at 4°C. The blots were then subjected to incubation with a secondary antibody (LI-COR, Cat#926-32211;USA) at room temperature for 1 hour. Visualization was achieved using a LI-COR Imaging System (Biosciences, USA), and subsequent quantification was carried out with Image-Pro Plus software. 1.4 Real-time quantitative PCR. Total RNA from chondrocytes were extracted with Trizol reagent (Invitrogen, Cat#R1100; USA) according to manufacturer’s instructions. cDNA was then synthesized from 1 µg of total RNA using the 4*DN Master Mix with gDNA Remover and 5*RT Master Mix II ( TOYOBO ,Cat#FSQ-301; Japan) of the resultant cDNA, 25 ng was used for qRT-PCR. The qRT-PCR mix contains 5 μl iTaq Universal (BIO-RAD, Cat#1725124; ,USA), 0.5 μl of each primer, cDNA 1ul and Nuclease-free H 2 O with a total volume of 10 μl. Samples were amplified for 40 cycles using the Applied Biosystems® 7500 Real-Time PCR System (ThermoFisher Scientific). The 2^ -ΔΔCT method was employed for calculating relative gene expression levels, with normalization against the housekeeping gene GAPDH. Primer sequences(Table 2) 1.5 Tube Formation Assay Endothelial cells were seeded at a density of 2 × 10*4 cells per well in μ-Slide Angiogenesis (ibidi, Cat#81506; GER) pre-coated with 50 μL of Matrigel in each well (Corning, Cat#354230; USA). The plates were subsequently incubated at 37°C under various treatment conditions. After 6 hours of incubation, the cells were imaged and analyzed using an inverted microscope (Leica) and Image-Pro Plus software. 1.6 Migration assay The wound scratch assay was employed to assess the migration of BMECs under various concentrations of human recombinant THBS-3 proteins. In brief, 5.0 × 10*5 cells were seeded per well in a Six-well culture plate and incubated for 24 hours at 37°C until reaching confluence. Subsequently, a scratch was created using a sterile p200 pipette tip, and the cell debris was washed three times with PBS. Following this, the cells were incubated in DMEM supplemented with 5% fetal bovine serum (FBS; Gibco, USA) under the various concentrations of human recombinant THBS-3 proteins. Images of the wounds were captured immediately after scratching and again 24 hours later. Image J software was used to measure the change in the scratched areas. The migration rate was calculated as follows: migration area (%) = (A0 – An)/A0 × 100, where An and A0 represent the remaining area and initial area of the wound, respectively. 2 In vivo experiments 2.1 VII type collagenase-induced OA mouse models For the collagenase-induced OA model, 12-week-old male C57BL/6 (Animal Center of Harbin Medical University) mice were anesthetized, and then injected the VII type collagenase on the left knee. 2’OMe+5’Chol modified THBS-3 siRNA labeled with 5FAM were purchased from Ribo Company (RiboBio, China). Mice(n=18) were randomly divided into the following groups: Control(n=6), CIOA(n=6), CIOA+siTHBS-3(n=6). At 7 days post-injection, 5nM siTHBS-3 (OA + siTHBS-3 group) were injected intra-articularly once a week into the mice knee joints. Mice were killed at week 7 for bone and cartilage analysis. All the animals were kept in a controlled environment that ensured they were free from specific pathogens. They were subjected to a 12-hour light and 12-hour dark cycle and were provided with a standard chow diet. All animal experiments were approved by the Harbin Medical University Committee on the Use and Care of Animals. 2.2 Micro-CT Subchondral bone explants from the mouse specimens were initially fixed overnight in a 10% formalin solution. Subsequently, these explants were examined using Microcomputed Tomography (Micro-CT) imaging (Quantum GX, Perkin Elmer, Waltham, USA). 2.3 Histology. Knee joints were fixed in 4% paraformaldehyde for 24 hours and then decalcified over an 8-week period using a 10% EDTA solution at pH 7.4. The samples were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek) or paraffin. The medial portion of the samples was longitudinally oriented and sectioned into 4 μm slices. The knee joint's medial compartment was subjected to staining procedures, including hematoxylin-eosin (H&E staining), Safranin O and fast green staining as well as immunohistochemistry. Images were captured using a microscope (Olympus). 4.Statistical Analysis The data is presented as the mean ± standard error of the mean (SEM). For comparisons between two groups, a two-tailed Student's t-test was employed. In the case of multiple group comparisons, a one-way analysis of variance (ANOVA) was utilized. Initially, the homogeneity of variance was assessed, followed by post hoc multiple comparisons to evaluate differences between groups. Statistical significance was defined as p < 0.05. Results 1.THBS-3 expression is elevated in OA cartilage, causing a reduction in synthetic metabolism and an increase in catabolic metabolism. Proteomic analysis revealed a significant difference in THBS-3 levels between OA-induced mice and normal mice cartilage (Fig1A). To investigate the involvement of THBS-3 in OA, we first analyzed the THBS-3 expression in human cartilage. In OA samples(n=10) from patients undergoing total knee arthroplasty (TKA), expressions of THBS-3 is significantly up-regulated (p=0.0236)(Fig1B) and RT-qPCR(p=0.0002) (Fig1C) compared with healthy group(n=10). To explore a deeper understanding of the influence of THBS-3 on the catabolism and synthesis metabolism within chondrocytes, we exposed normal chondrocytes to 50, 100, 200nM concentrations of human recombinant THBS-3 protein for a 24-hour period. We assessed the expression of MMP-13, ADAMTs-5 and Aggrecan. Our findings indicated that THBS-3 actively promoted expression of MMP-13 and ADAMTs-5 while simultaneously suppressing expression of Aggrecan (Fig1D). To investigate whether IL-1β induces an increase in THBS-3 expression in normal chondrocytes, we examined THBS-3 expression in human normal chondrocytes after 24 hours of IL-1β (10 ng/ml) treatment. We designed three THBS-3 siRNAs (S1-S3) and selected the most efficient THBS-3 siRNA based on WB and RT-qPCR analysis for subsequent experiments. The WB analysis indicated a significant increase in THBS-3 expression in response to IL-1β (p=0.0138). In WB (p<0.0001) and RT-PCR (p<0.0001), the expression of THBS-3 is lowest in the S3 group (Fig1E). For a more in-depth examination of the influence of THBS-3 on IL-1β-induced chondrocyte extracellular matrix (ECM) degradation, chondrocytes were subjected to transfection with THBS-3 siRNA as well as a scrambled siRNA control. The THBS-3 siRNA resulted in a substantial reduction in the IL-1β-induced upregulation of MMP13 and ADAMTs 5 in chondrocytes. On the contrary, the THBS-3 siRNA led to an elevated expression of Aggrecan compared to normal chondrocytes treated by IL-1β alone (Fig1F). 2. THBS-3 promotes the vascularization and osteogenesis coupling of chondrocytes. To further investigate the role of THBS-3 in chondrocytes, we stimulated NC chondrocytes with 50, 100, 200nM concentrations of recombinant human THBS-3 protein. Subsequently, we conducted WB analysis to examine markers associated with angiogenesis and osteogenesis. Our research findings indicate that THBS-3 promotes the crosstalk between angiogenesis and osteogenesis processes (Fig2A, E). On the contrary, the THBS-3 siRNA led to a decline expression of angiogenesis and osteogenesis (FigB, E). The pivotal phase in angiogenesis is the migration of endothelial cells, which facilitates the extension of blood vessels. We prepared HUVEC cells that were either treated with THBS-3 (50,100,200nM) or left untreated, and we assessed the impact on angiogenesis through analysis of endothelial cell migration and tube formation. It was observed that THBS-3 significantly enhanced the migration of HUVEC cells compared to the control group. With the elevation of THBS-3 concentration, the migration area expands proportionally, reaching its most pronounced extent at a concentration of 100nM (p=0.0040) (Fig2C). The results of tube formation indicated that THBS-3 at concentrations of 50 nM (p=0.0036) and 100 nM (p<0.0001) significantly augmented tube formation compared to the NC group (Fig2D). 3. THBS-3 siRNA application demonstrated effective inhibition of bone spur formation and mitigated pathological changes in cartilage tissue among CIOA mice. The in vitro data encouraged us to further investigate the therapeutic effectiveness of THBS-3 inhibition in an experimental OA mice model induced by Type VII collagenase. Intra-articular injections of THBS-3 siRNA (5nM) were administered once a week for a duration of 7 weeks. Upon analyzing the results from each group, we observed that THBS-3 siRNA treatment led to a reduction in cartilage damage in OA mice through Micro-CT (Fig3A). Safranin O-Fast Green staining illustrated a decrease in cartilage damage in the knees of OA mice treated with THBS-3 siRNA when compared to control group (Fig3C). Furthermore, HE staining revealed that the tidemark, which had moved upwards in OA mice, was reversed by THBS-3 siRNA treatment (Fig3B). Evaluate the knee joint tissues of each group of mice using the OARSI scoring system (Fig3D). Compared to the Control group, the CIOA group showed a significant increase in scores. In comparison to the CIOA group, the CIOA+siTHBS-3 group exhibited a decrease in scores. In other words, the inhibition of THBS-3 effectively counteracted the thickening of the calcified cartilage zone during the progression of OA. The inhibition of THBS-3 resulted in the restoration of Aggrecan expression, suppressed MMP-13 and ADAMTs-5 expression, as evaluated through immunohistochemistry in THBS-3 siRNA treated OA mice THBS-3 siRNA has inhibited vascularization /osteogenesis coupling in the articular cartilage tissue of CIOA mice(Fig3E-F). 4.THBS-3 promotes the vascularization and osteogenesis coupling of chondrocytes through the TGF-β/Smad2/3 signaling pathway. To investigate whether THBS-3 plays a role in OA through the TGF-β pathway, we made an intriguing discovery. After introducing 100nM of THBS into normal chondrocytes, we observed a significant upregulation of TGF-β expression within the 1-6 hour timeframe, peaking at 6 hours (p=0.0353) (Fig4A). Our current study revealed that the phosphorylation levels of SMAD2/3(p-SMAD2/3), which play a critical role in regulating the canonical TGF-β signaling pathway, were significantly lower in both the NC+IL-1β group (p<0.0001) and the NC+THBS-3 group (p=0.0004) when compared to the NC group. Furthermore, in the NC + THBS-3 +TGF-β inhibitor group, the expression level of p-SMAD2/3 was significantly reduced compared to the NC+THBS-3 group (p=0.0070)(Fig4B). Previous studies have already pointed to the involvement of THBS-3 in vascularization and osteogenesis coupling within the context of OA. What's particularly interesting is that when we pre-treated normal chondrocytes with a TGF-β inhibitor 10uM(SB-431542, MCE, China) and then introduced THBS-3, we observed a marked reduction in the expression of BMP-2 (p=0.0276), FGF-2 (p=0.0052), ANG-2 (p=0.0379), VEGF-A (p=0.0093) and PDGF-B (p=0.0065) associated with vascularization and osteogenesis coupling (Fig4C). Discussion OA is a prevalent form of arthritis and a leading cause of chronic, non-traumatic disability among the elderly population. The results of this study can be succinctly summarized as follows: First, we confirmed that OA resulted in up-regulated expressions of THBS-3. THBS-3 actively promoted degradation metabolism while simultaneously suppressing synthesis metabolism. Second, THBS-3 promotes the crosstalk between angiogenesis and osteogenesis processes in chondrocytes. Third, we demonstrated that THBS-3 in chondrocytes through activating TGF-β /smad2/3 signaling pathways and promoted angiogenesis and osteogenesis coupling in vitro, which may lead to vascular up-growth in OA. Previous studies have found that in a meta-analysis of gene expression in OA and non-OA chondrocytes, there was a significant increase in the expression of THBS-3 and COMP in OA[ 11 ]. Our findings also highlighted a notable increase in the expression of THBS-3 in the OA. Posey's research discovered that mice lacking extracellular matrix proteins, THBS-1, THBS-3, THBS-5, and type IX collagen, exhibited skeletal abnormalities. THBS-3, THBS-5, and type IX collagen all play a direct role in regulating linear growth in growth plate tissue [ 12 ]. Additionally, Hankenson's study revealed that THBS-3 regulates postnatal bone maturation, influencing endochondral ossification. In cases of THBS3 deficiency, there was an accelerated ossification of the calcified cartilage in the femoral head[ 13 ]. These findings provide compelling evidence for the involvement of THBS-3 in the regulation of bone development, especially in the context of endochondral ossification during postnatal skeletal maturation. Nevertheless, the specific mechanism behind this phenomenon remains elusive at present. The exact pathogenic mechanism of OA remains elusive, but it is generally acknowledged that an imbalance in the synthesis and degradation metabolism of chondrocytes is a primary factor contributing to the development of OA. We observed that THBS-3 promotes chondrocyte degradation metabolism while inhibiting synthesis metabolism. Furthermore, research indicates that angiogenesis/osteogenesis coupling is involved in the progression of OA[ 4 ]. This coupling phenomenon is observed in the synovium, cartilage, and subchondral bone within the context of OA. In a study exploring the influence of THBS-3 on the biological behavior and survival of osteosarcoma patients, THBS-3 gene exhibited significant differential expression in osteosarcoma. Functioning as a potent stimulant for tumor progression, heightened levels of THBS-3 were found to actively stimulate angiogenesis[ 14 ]. Our research indicates that THBS-3 promotes the expression of VEGF, ANG-2, FGF-2, BMP-2 and PDGF-BB in osteoarthritis chondrocytes, thereby enhancing vascularization/bone coupling generation within the cartilage cells. In vivo experiments, immunohistochemical analysis yielded results consistent with the cellular experiments. Compared to the CIOA group, the CIOA + siTHBS-3 group showed a reduction in indicators related to vascularization/osteogenesis coupling in cartilage tissue. TGFβ1 has been a subject of extensive investigation in the context of osteoarthritis and chondrocytes for many years. TGF-β1 is a crucial growth factor for the development, maintenance, and repair of articular cartilage[ 15 ]. Previous studies have found that in in vitro culture of endothelial cells and in vivo vascular generation model experiments, THBS-4 promotes angiogenesis through TGF-β1 mediation[ 16 ]. Suzuki Takahisa's research discovered an elevated expression of THBS-1 in synovial tissues of rheumatoid arthritis, and TGF-β1 significantly increased the expression of THBS-1 at both mRNA and protein levels in the synovium[ 17 ]. THBS-1 may be involved in the pathogenesis of rheumatoid arthritis through the TGF-β1/THBS-1 pathway. Kimberly BD's study revealed that THBS-1 inhibits the osteogenic differentiation of human mesenchymal stem cells by activating TGF-β[ 18 ]. Thus, it is evident that THBS-1 and 4 can promote osteogenesis and angiogenesis through TGF-β, although there is currently no research on THBS-3. Further, our findings indicate that the expression of TGF-β1 in chondrocytes stimulated with THBS-3 (100nM) progressively increased over time, with the peak expression observed at the 6-hour.These observations suggest that proper endogenous production of TGF-β1 is essential for maintaining chondrocyte homeostasis, whereas excessive or inadequate activation of TGF-β1 can be detrimental [ 19 ] The elevated TGF-β1 levels prompted us to further explore the regulatory mechanisms. Given the observed alterations in the expression levels of TGF-β signaling receptors in the context of OA, it is reasonable to expect changes in the activity of their signal mediators, Smad2/3. Research has provided compelling evidence of reduced Smad2 phosphorylation levels in cartilage during OA progression, evident in both spontaneous (STR/Ort) and collagenase-induced mouse models of OA [ 20 ]. Furthermore, it has been observed that Smad2 phosphorylation diminishes in cartilage in older mice when compared to their younger counterparts [ 21 ]. While Smad3 phosphorylation was not specifically examined in these models, a recent study has reported decreased Smad3 phosphorylation levels in Smurf-2 transgenic mice that spontaneously develop an OA-like phenotype[ 22 ]. These collective findings strongly suggest that OA is associated with a compromised TGF-β/Smad2/3 signaling pathway. Our findings are consistent with previous research. In the chondrocyte osteoarthritis (OA) model induced by IL-1 stimulation, a reduction in p-Smad2/3 expression was observed. To delve deeper into the pathways associated with THBS-3, stimulation of chondrocytes with THBS-3 revealed an inhibitory effect on p-Smad2/3 expression. Notably, the addition of a TGF inhibitor reversed the expression of p-Smad2/3, underscoring the intricate regulatory role of THBS-3 in this context. Conclusions Our conclusion can be summarized as follows: THBS3 in regulating cartilage vascularization/bone coupling via the TGFB1/Smad2/3 pathway in OA. Furthermore, this presents novel therapeutic targets for addressing OA. Abbreviations ECGS Endothelial Cell growth supplement ECM extracellular matrix FBS fetal bovine serum H&E staining hematoxylin-eosin HUVECs Human microvascular ECs Micro-CT Microcomputed Tomography OA osteoarthritis OCT optimal cutting temperature P/S penicillin/streptomycin solution PBS phosphate-buffered saline THBS thrombospondin WB Western blot Declarations Ethics approval and consent to participate The procedures of this study were approved by the Institutional Ethics Committee of the First Affiliated Hospital of Harbin Medical University. (NO:2023JS57) Consent for publication （ Not applicable ） Availability of data and material All data generated or analysed during this study are included in this published article [and its supplementary information files]. Acknowledgements （ Not applicable ） Funding This work was supported by grants from the National Natural Science Foundation of China to Zhiyi Zhang (NSFC 82271826) and Shuya Wang (NSFC 82202020), and partially by the First Afliated Hospital of HMU Merit Review Frontiers grant to Shuya Wang (HYD2020YQ0008). Authors` contributions JY.Y and SY.W contributed to the conception and design of the study. HY.L and XY.Z performed the experiments and contributed to the analysis and interpretation of data. YP. Z and ZY.Z contributed to draft manuscript. All approved the submitted manuscript. Competing interests No conflicts of interest were declared. References Abramoff B, Caldera FE. Osteoarthritis: Pathology, Diagnosis, and Treatment Options. Med Clin North Am. 2020,104(2):293-311. Quicke JG, Conaghan PG, Corp N, Peat G. Osteoarthritis year in review 2021: epidemiology & therapy. Osteoarthritis Cartilage. 2022, 30(2):196-206. Bonnet CS, Walsh DA. Osteoarthritis, angiogenesis and inflammation. 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Dalla-Torre CA, Yoshimoto M, Lee CH, et al. Effects of THBS3, SPARC and SPP1 expression on biological behavior and survival in patients with osteosarcoma. BMC Cancer. 2006, 6:237. Blaney Davidson EN, van der Kraan PM, van den Berg WB. TGF-beta and osteoarthritis. Osteoarthritis Cartilage. 2007, 15(6):597-604. Muppala S, Xiao R, Krukovets I. Thrombospondin-4 mediates TGF-β-induced angiogenesis. Oncogene. 2017, 36(36):5189-5198. Suzuki T, Iwamoto N, Yamasaki S. Upregulation of Thrombospondin 1 Expression in Synovial Tissues and Plasma of Rheumatoid Arthritis: Role of Transforming Growth Factor-β1 toward Fibroblast-like Synovial Cells [published correction appears in J Rheumatol. 2017 Jan;44(1):131]. J Rheumatol. 2015, 42(6):943-947. Hunter DJ, Bierma-Zeinstra S. Osteoarthritis. Lancet. 2019, 393(10182):1745-1759. Zhen G, Guo Q, Li Y, et al. Mechanical stress determines the configuration of TGFβ activation in articular cartilage. Nat Commun. 2021;12(1):1706. Blaney Davidson EN, Vitters EL, van der Kraan PM, van den Berg WB. Expression of transforming growth factor-beta (TGFbeta) and the TGFbeta signalling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: role in cartilage degradation, chondrogenesis and osteophyte formation. Ann Rheum Dis. 2006;65(11):1414-1421. Blaney Davidson EN, Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB. Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity. Arthritis Res Ther. 2005;7(6):R1338-R1347. Wu Q, Huang JH, Sampson ER, et al. Smurf2 induces degradation of GSK-3beta and upregulates beta-catenin in chondrocytes: a potential mechanism for Smurf2-induced degeneration of articular cartilage. Exp Cell Res. 2009;315(14):2386-2398. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.jpg Table2.jpg Figure1B.tif Figure1D.png Figure1E.png Figure1G.png figure2A2E.png Figure2B.png Figure2F.png Figure4A.png Figure4B.png Figure4C.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4167008","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287170083,"identity":"c05b24d2-151a-4621-9664-f46a25fe839b","order_by":0,"name":"Jingyao Yan","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingyao","middleName":"","lastName":"Yan","suffix":""},{"id":287170084,"identity":"87329414-1d06-49c8-bc93-4193e59aabc2","order_by":1,"name":"Yanping Zhao","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanping","middleName":"","lastName":"Zhao","suffix":""},{"id":287170085,"identity":"730f0d86-33db-465a-a7aa-b3cce6e6659c","order_by":2,"name":"Xiaoying Zhu","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Zhu","suffix":""},{"id":287170086,"identity":"8dcfde67-2791-4de9-a03b-ffe9835bc39c","order_by":3,"name":"Hanya Lu","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hanya","middleName":"","lastName":"Lu","suffix":""},{"id":287170087,"identity":"a0c3c597-91a4-4d59-8d6d-c24a377fe983","order_by":4,"name":"Yanli Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Wang","suffix":""},{"id":287170088,"identity":"cd426128-3b3c-4222-9d7e-2266c7a07864","order_by":5,"name":"Shuya Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuya","middleName":"","lastName":"Wang","suffix":""},{"id":287170089,"identity":"7eb7bed4-2286-4966-923c-f8913901e81c","order_by":6,"name":"Zhiyi Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIie3QMWrDMBSAYQmBp9d6fcKG9AgqAYeCaa4iU0gWz50Fhk49gEt7jszPCNKl1AdIB3fJ7EApHkJplCFblIyF6gcJBO9DSIyFQn8x3O8ETAgiDTnEsTmbREXXpbNU1nQe2S0YX3e5zZXRfjF6rtafX9uPVL2yDHXZgmLE+015nPCX5WScwhqUZTPUbyuYCCPk0+I4EaizBNE6ssTicQU3hiJx4SERzr8TVI7wByx+3kGR9hPAMpO9dkQIpYFOE8TyPmFkQdqIdxruQNZN5X3LqJ4v5LC108u27ZsBbqdxXDX9xkP2XwC77YoOZ278825kcNednguFQqH/2i+JmlMwnyLoaAAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhiyi","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-26 04:59:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4167008/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4167008/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54160723,"identity":"b814be37-dc85-4f31-8103-07dd59831752","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9728086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHBS-3 expression is elevated in OA cartilage, causing a reduction in synthetic\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Proteomic analysis of knee joint tissues in normal mice and collagen-induced osteoarthritis (CIOA) model mice. (B) Western blot and quantitative analysis showed expressions of THBS-3 is up-regulated in human OA cartilages compared with healthy ones (n = 10). (C) RT-PCR and quantitative analysis showed expressions of THBS-3 is up-regulated in human OA cartilages compared with healthy ones (n = 10). (D) Utilizing the WB method, we investigated the protein expression levels of Aggrecan, MMP-13, and ADAMTs-5 in chondrocytes across various concentrations of THBS-3 (n=6). (E) Western blot and quantitative analysis showed expressions of siTHBS-3 in chondrocytes (n=6)\u003cstrong\u003e.\u003c/strong\u003e (F) RT-PCR and quantitative analysis showedexpressions of siTHBS-3 in chondrocytes (n=4). (G) Silencing of the THBS-3 gene modulates the expression of proteins associated with the synthesis and degradation metabolism in chondrocytes (n=4). Data are represented as the mean ± standard error of the mean (SEM). * denotes p\u0026lt;0.05 compared to the NC group, ** denotes p\u0026lt;0.01, *** denotes p\u0026lt;0.001. # denotes p\u0026lt;0.05 compared to the NC+IL-1β group, ## denotes p\u0026lt;0.01, ### denotes p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/25807eef4c6b6866a896d64a.jpg"},{"id":54160725,"identity":"761c29b4-db66-482b-a8d2-74a5cefd365b","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17864693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHBS-3 promotes the vascularization and osteogenesis coupling of chondrocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)WB was employed to assess the expression of ANG-2 (n=5), VEGF-A (n=5), and PDGF-B (n=4) in chondrocytes at different concentrations of THBS-3. (B) WB analysis was conducted to examine the expression of ANG-2 (n=5), VEGF-A (n=5), and PDGF-B (n=4) in the NC group, NC+IL-1β group, siNC group, and siTHBS-3 group. (C) Migration assays were conducted to assess the migratory response of HUVECs to different concentrations of THBS-3 (n=4). (D) Tube formation assays were conducted to examine the tube-forming capacity of HUVECs in response to various concentrations of THBS-3 (n=5). (E) The WB method was utilized to assess the expression of BMP-2 (n=4) and FGF-2 (n=5) in chondrocytes treated with different concentrations of THBS-3. (F) WB analysis was performed to examine the expression levels of BMP-2 (n=7) and FGF-2 (n=5) in different experimental groups: NC group, NC+IL-1β group, siNC group, and siTHBS-3 group. Data are represented as the mean ± standard error of the mean (SEM). * denotes p\u0026lt;0.05 compared to the NC group, ** denotes p\u0026lt;0.01, *** denotes p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/f851fd7b95c492d705f1a79a.jpg"},{"id":54160728,"identity":"3eab2673-b08e-4a36-a29d-1896461c4d23","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3699467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHBS-3 siRNA application demonstrated effective inhibition of bone spur formation and mitigated pathological changes in cartilage tissue among CIOA mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Three-dimensional Micro-CT Reconstruction of Knee Joint Tissues in Respective Mouse Groups. (B) Histological Assessment of Knee Joint Tissues in Each Group via HE Staining. Scale bar = 100 µm. (C) Conduct Safranin O-Fast Green staining on the knee joint tissues from each group of mice. Scale bar = 100 µm. (D) Evaluate the knee joint tissues of each group of mice using the OARSI scoring system. (E) Immunohistochemistry and statistical analysis were performed on MMP-13, ADAMTs-5, and Aggrecan in mice from the Control group, CIOA model group and CIOA+THBS-3 siRNA group(n=6). Scale bar = 50 µm. (F) Immunohistochemistry and statistical analysis were performed on ANG-2, BMP-2, and FGF-2 in mice from the Control group, CIOA model group and CIOA+THBS-3 siRNA group(n=6). Scale bar = 50 µm. Data are represented as the mean ± standard error of the mean (SEM). * denotes p\u0026lt;0.05 compared to the Control group, ** denotes p\u0026lt;0.01, *** denotes p\u0026lt;0.001. # denotes p\u0026lt;0.05 compared to the NCgroup, ## denotes p\u0026lt;0.01, ### denotes p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/2fcf3a594b18765c429f8fc8.jpg"},{"id":54160735,"identity":"c0dea4fa-51b4-4600-9fa8-084dbe1640e3","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":905689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTHBS-3 promotes the vascularization and osteogenesis coupling of chondrocytes through the TGF-β/Smad2/3 signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Quantification of TGF-β1 expression at different time points using WB analysis for THBS-3 (n=6). (B) Evaluation of p-smad2/3 expression in NC group, NC+IL-1β group, THBS-3+TGF-β i group, and THBS-3 group using WB analysis (n=6). (C) Using WB analysis to assess the expression of BMP-2(n=4), FGF-2 (n=6), ANG-2 (n=5),VEGF-A (n=5) AND PDGF-B (n=7) in NC group, TGF-β1 inhibitor group, and THBS-3 group. Data are represented as the mean ± standard error of the mean (SEM). * denotes p\u0026lt;0.05 compared to the NC group, ** denotes p\u0026lt;0.01, *** denotes p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/c43cb0ef3f91da034b92e1c9.jpg"},{"id":56039480,"identity":"4ffed4fa-f443-46f9-bf26-9f2d87e4d18c","added_by":"auto","created_at":"2024-05-07 19:12:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":964469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/2a9fb6a4-336a-4836-8b5e-668e42b12a9c.pdf"},{"id":54160726,"identity":"b0b1e53a-9041-4eee-a189-1bad7aed816b","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":48093,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/cead5b0665cb812280abbbf0.jpg"},{"id":54160724,"identity":"b22cc414-8985-4462-9245-6101c4344472","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":65851,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/5c188b76986236350f8f10e3.jpg"},{"id":54162509,"identity":"905ba05e-1830-4448-b5bd-7b39d1b27b6e","added_by":"auto","created_at":"2024-04-05 13:10:00","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5719174,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1B.tif","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/0fa10becb50fa8cb62af46e6.tif"},{"id":54160732,"identity":"3e6e7bb7-2196-44a9-9166-a1b028c28f20","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2538347,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1D.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/4db36266389f18a853fc6fb1.png"},{"id":54160734,"identity":"d5bf252a-8bad-40ae-9e08-20d950205ea6","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1160118,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1E.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/9d24786de2d44289cba408c2.png"},{"id":54160736,"identity":"12b0b1aa-de19-4125-a612-a2c5835e1f14","added_by":"auto","created_at":"2024-04-05 13:02:00","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4193117,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1G.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/09ba30581787c60d2a83dcb3.png"},{"id":54160731,"identity":"d21ea00f-58d5-4552-89a6-60dd0a46f97e","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1151718,"visible":true,"origin":"","legend":"","description":"","filename":"figure2A2E.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/cc15965f500c487c0a91ad67.png"},{"id":54160730,"identity":"9311ca7e-3e80-4b0e-b8bd-d39a2c3fc210","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":486429,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2B.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/1d9bfa2ada1acdff6ee555d1.png"},{"id":54160729,"identity":"f6a80d6c-fa97-407c-9dfe-521ae9e248e8","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":192274,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2F.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/7a033f29ce70cf058da1a144.png"},{"id":54160739,"identity":"da6f4168-cab3-46e5-93e3-8a1e31ed9ea3","added_by":"auto","created_at":"2024-04-05 13:02:00","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":342493,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4A.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/06ce3c21d8bd26a478b18624.png"},{"id":54160733,"identity":"d4a43a44-1592-4116-916d-0527e0b803dd","added_by":"auto","created_at":"2024-04-05 13:01:59","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":412672,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4B.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/80d93b2dae387c830d0009bc.png"},{"id":54160737,"identity":"9c64837e-051c-4a78-b047-b1554a685ca6","added_by":"auto","created_at":"2024-04-05 13:02:00","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":389316,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4C.png","url":"https://assets-eu.researchsquare.com/files/rs-4167008/v1/71c2279474fb55eae3d39a26.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"The mechanism study of THBS3 in regulating cartilage vascularization/bone coupling via the TGF-β/Smad2/3 pathway in osteoarthritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOA is an extremely common and multiarticular degenerative disease that leads to chronic joint pain and physical disability, resulting in a primary financial burden and extensive impairments in quality of life[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Accumulating studies have revealed that cartilage degradation, osteophyte formation, bone remodeling, neoangiogenesis, and synovial inflammation that lead to the pathogenesis of OA[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].But so far, the underlying mechanisms of OA remain abstruse.\u003c/p\u003e \u003cp\u003eNormal articular cartilage is avascular and aneural, but accumulating studies have suggested that increased angiogenesis at the bone-cartilage junction and in non-calcified cartilage in OA.The invasion of blood vessels across the osteochondral junction could potentially lead to cartilage calcification or ossification[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Within this context, the process of ossification within the cartilage contributes to the formation of bone spurs, exacerbating the progression of osteoarthritis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the context of osteoarthritis, the formation of new blood vessels plays a role in sustaining persistent inflammation. This occurs through the continuous provision of oxygen and nutrients to endothelial cells. These neo-vessels facilitate the entry of pro-inflammatory cytokines into the surrounding local microenvironment, further exacerbating the diseases processes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Angiogenesis is regulated by the balance of proangiogenic and antiangiogenic factors. However, pathological neovascularization in OA represents a breaking of these normal homeostatic mechanisms. Additionally, the process of vascularization is accompanied by sensory nerve growth, which may also be a contributing factor to the pain experienced by OA patients[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOsteogenesis and angiogenesis are coupled in space-time and interconnected manner.\u003c/p\u003e \u003cp\u003eIn order to maintain bone homeostasis, osteoblasts secrete angiogenic factors to promote growth of vessels and regulate their functions, while endothelial cells release angiocrine signals to regulate bone remodeling and repair. The bidirectional cross-talk between osteogenesis and angiogenesis is complex and several factors such as VEGF, FGFs, PDGF-B, BMP-2 have been involved in this interplay[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, regulating cartilage vascularization/bone coupling may contribute to the structural damage and pain associated with osteoarthritis and offer potential targets for novel treatments.\u003c/p\u003e \u003cp\u003eTHBS-3 belongs to the thrombospondin (THBS) protein family, which is a part of the ECM protein family. THBS form a family of five secreted, multimeric, modular matricellular glycoproteins[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. They exhibit specific spatio-temporal expression patterns in both embryonic and adult tissues and are involved in a wide range of essential processes, including angiogenesis, wound healing, cell proliferation, migration, as well as the organization of connective tissues[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. THBS-3 can also regulate postnatal skeletal maturation, influence endochondral ossification within cartilage, and participate in bone formation[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the exact relationships between THBS-3 and OA are still to be clarified, and there have been no reported studies on this connection. Further research is needed to understand the role of THBS-3 in OA and its implications for the disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1.In vitro experiments\u003c/p\u003e\n\u003cp\u003e1.1. Isolation and culture of chondrocytes from OA patients\u003c/p\u003e\n\u003cp\u003eCartilage tissue was obtained from discarded knee joints of osteoarthritis patients (n=10) who underwent total knee replacement surgery at the First Affiliated Hospital of Harbin Medical University. Cartilage tissues were washed three times with phosphate-buffered saline (PBS), and then cut into 1 mm\u003csup\u003e2\u003c/sup\u003e, digested in 2mg/ml collagenase type II (Sigma, St. Louis, MO, USA) 37℃ overnight. After that, the cells were filtered through a cell strainer. Primary cells were used from the P0-P1 passage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1.1.2 Culture of human normal chondrocytes\u003c/p\u003e\n\u003cp\u003eHuman normal articular chondrocytes were obtained from the C28/I2 human normal chondrocytes line(Otwo Biotech, Inc, Cat#HTX2308;China)and cultured in DMEM medium with the addition of 10% FBS and 0.5% penicillin-streptomycin, at 37 °C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e1.1.3 Culture of HMVEC\u003c/p\u003e\n\u003cp\u003eHuman microvascular ECs (HUVECs) (OTWO, Inc, Cat#HTX1922;China), were grown in endothelial cell medium (ECM)(ScienCell, Cat#1001; USA) supplemented with 25ml fetal bovine serum (FBS)(ScienCell, Cat#0025; USA), 5 ml Endothelial Cell growth supplement (ECGS)(ScienCell, Cat#1052; USA), 5 ml\u0026nbsp;\u0026nbsp;penicillin/streptomycin solution(P/S)(ScienCell, Cat#0503; USA), and used for tube formation and migration experiments. HUVECs were cultured to confluence, followed by incubation in serum-reduced ECM (1% FBS) for 24 hours prior to stimulation with THBS-3 for an additional 24 hours.\u003c/p\u003e\n\u003cp\u003e1.2 Small interfering RNA and transfection experiment\u003c/p\u003e\n\u003cp\u003eThe small interfering RNAs (siRNAs) and transfection reagent were purchased from RIBOBIO Co., Ltd. (RiboBio, China)according to manufacturer’s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTransfection target sequence (Table 1).\u003c/p\u003e\n\u003cp\u003e1.3 Western blotting\u003c/p\u003e\n\u003cp\u003eFollowing a triple wash with PBS, cells underwent protein extraction. Proteins were separated on 10% or 12% SDS-PAGE gels. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes (Thermo-Fisher, Hampton, NH) and probed with primary antibodies\u0026nbsp;against THBS-3 (Proteintech, Cat#19727-1-AP;USA), Aggrecan (Abbkine, Cat#ABP54013;China), ADAMTs-5 (Abcam,\u0026nbsp;\u0026nbsp;Cat#AB41037;USA), MMP-13(Bioword Technologyl, Cat#BS1231;China),BMP-2(Abbkine, Cat#ABP0179;China), FGF-2 (Abbkine, Cat#ABP53280;China), ANG-2(Abcam, Cat#AB155106;USA), VEGF-A (Abcam, Cat#AB214424;USA), PDGF-B(Abcam, Cat#AB178409;USA), TGF-β1\u0026nbsp;(Abcam, Cat#AB179695;USA), p-smad2/3 (Abbkine, Cat#ABP0048;China)and GAPDH (ABclonal, Cat#A19056;China),overnight at 4°C. The blots were then subjected to incubation with a secondary antibody (LI-COR, Cat#926-32211;USA) at room temperature for 1 hour. Visualization was achieved using a LI-COR Imaging System (Biosciences, USA), and subsequent quantification was carried out with Image-Pro Plus\u0026nbsp;software.\u003c/p\u003e\n\u003cp\u003e1.4 Real-time quantitative PCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTotal RNA from chondrocytes were extracted with Trizol reagent (Invitrogen, Cat#R1100; USA) according to manufacturer’s instructions. cDNA was then synthesized from 1 µg of total RNA using the 4*DN Master Mix with gDNA Remover and 5*RT Master Mix II ( TOYOBO ,Cat#FSQ-301; Japan) of the resultant cDNA, 25 ng was used for qRT-PCR. The qRT-PCR mix contains 5 μl iTaq Universal (BIO-RAD, Cat#1725124; ,USA), 0.5 μl of each primer, cDNA 1ul and Nuclease-free H\u003csub\u003e2\u003c/sub\u003eO with a total volume of 10 μl. Samples were amplified for 40 cycles using the Applied Biosystems® 7500 Real-Time PCR System (ThermoFisher Scientific). The 2^\u003csup\u003e-ΔΔCT\u0026nbsp;\u003c/sup\u003emethod was employed for calculating relative gene expression levels, with normalization against the housekeeping gene GAPDH.\u003c/p\u003e\n\u003cp\u003ePrimer sequences(Table 2)\u003c/p\u003e\n\u003cp\u003e1.5 Tube Formation Assay\u003c/p\u003e\n\u003cp\u003eEndothelial cells were seeded at a density of 2 × 10*4 cells per well in μ-Slide Angiogenesis (ibidi, Cat#81506; GER) pre-coated with 50 μL of Matrigel in each well (Corning, Cat#354230; USA). The plates were subsequently incubated at 37°C under various treatment conditions. After 6 hours of incubation, the cells were imaged and analyzed using an inverted microscope (Leica) and Image-Pro Plus software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1.6 Migration assay\u003c/p\u003e\n\u003cp\u003eThe wound scratch assay was employed to assess the migration of BMECs under various concentrations of human recombinant THBS-3 proteins. In brief, 5.0 × 10*5 cells were seeded per well in a Six-well culture plate and incubated for 24 hours at 37°C until reaching confluence. Subsequently, a scratch was created using a sterile p200 pipette tip, and the cell debris was washed three times with PBS. Following this, the cells were incubated in DMEM supplemented with 5% fetal bovine serum (FBS; Gibco, USA) under the various concentrations of\u0026nbsp;human recombinant THBS-3 proteins. Images of the wounds were captured immediately after scratching and again 24 hours later. Image J software was used to measure the change in the scratched areas. The migration rate was calculated as follows: migration area (%) = (A0 – An)/A0 × 100, where An and A0 represent the remaining area and initial area of the wound, respectively.\u003c/p\u003e\n\u003cp\u003e2 In vivo experiments\u003c/p\u003e\n\u003cp\u003e2.1 VII type collagenase-induced OA mouse models\u003c/p\u003e\n\u003cp\u003eFor the collagenase-induced OA model, 12-week-old male C57BL/6 (Animal Center of Harbin Medical University) mice were anesthetized, and then injected the VII type collagenase on the left knee. 2’OMe+5’Chol modified THBS-3 siRNA labeled with 5FAM were purchased from Ribo Company (RiboBio, China). Mice(n=18) were randomly divided into the following groups: Control(n=6), CIOA(n=6), \u0026nbsp;CIOA+siTHBS-3(n=6). At 7 days post-injection, 5nM siTHBS-3 (OA + siTHBS-3 group) were injected intra-articularly once a week into the mice knee joints. Mice were killed at week 7 for bone and cartilage analysis. All the animals were kept in a controlled environment that ensured they were free from specific pathogens. They were subjected to a 12-hour light and 12-hour dark cycle and were provided with a standard chow diet. All animal experiments were approved by the Harbin Medical University Committee on the Use and Care of Animals.\u003c/p\u003e\n\u003cp\u003e2.2 Micro-CT\u003c/p\u003e\n\u003cp\u003eSubchondral bone explants from the mouse specimens were initially fixed overnight in a 10% formalin solution. Subsequently, these explants were examined using\u0026nbsp;Microcomputed Tomography (Micro-CT) imaging (Quantum GX, Perkin Elmer, Waltham, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3 Histology.\u003c/p\u003e\n\u003cp\u003eKnee joints were fixed in 4% paraformaldehyde for 24 hours and then decalcified over an 8-week period using a 10% EDTA solution at pH 7.4. The samples were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek) or paraffin. The medial portion of the samples was longitudinally oriented and sectioned into 4 μm slices. The knee joint's medial compartment was subjected to staining procedures, including hematoxylin-eosin (H\u0026amp;E staining), Safranin O and fast green staining as well as immunohistochemistry. Images were captured using a microscope (Olympus).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.Statistical Analysis\u003c/p\u003e\n\u003cp\u003eThe data is presented as the mean ± standard error of the mean (SEM). For comparisons between two groups, a two-tailed Student's t-test was employed. In the case of multiple group comparisons, a one-way analysis of variance (ANOVA) was utilized. Initially, the homogeneity of variance was assessed, followed by post hoc multiple comparisons to evaluate differences between groups. Statistical significance was defined as p \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.THBS-3 expression is elevated in OA cartilage, causing a reduction in synthetic metabolism and an increase in catabolic metabolism.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomic analysis revealed a significant difference in THBS-3 levels between OA-induced mice and normal mice cartilage\u0026nbsp;(Fig1A). To investigate the involvement of THBS-3 in OA, we first analyzed the THBS-3 expression in human cartilage. In OA samples(n=10) from patients undergoing total knee arthroplasty (TKA), expressions of THBS-3 is significantly up-regulated (p=0.0236)(Fig1B)\u0026nbsp;and RT-qPCR(p=0.0002) (Fig1C) compared with healthy group(n=10). To explore a deeper understanding of the influence of THBS-3 on the catabolism and synthesis metabolism within chondrocytes, we exposed normal chondrocytes to 50, 100, 200nM concentrations of human recombinant THBS-3 protein for a 24-hour period. We assessed the expression of MMP-13, ADAMTs-5 and Aggrecan. Our findings indicated that THBS-3 actively promoted expression of MMP-13 and ADAMTs-5 while simultaneously suppressing expression of Aggrecan (Fig1D).\u003c/p\u003e\n\u003cp\u003eTo investigate whether IL-1\u0026beta; induces an increase in THBS-3 expression in normal chondrocytes, we examined THBS-3 expression in human normal chondrocytes after 24 hours of IL-1\u0026beta; (10 ng/ml) treatment. We designed three THBS-3 siRNAs (S1-S3)\u0026nbsp;and selected the most efficient THBS-3 siRNA\u0026nbsp;based on WB and RT-qPCR analysis for subsequent experiments. The WB analysis indicated a significant increase in THBS-3 expression in response to IL-1\u0026beta; (p=0.0138). In WB (p\u0026lt;0.0001) and RT-PCR (p\u0026lt;0.0001), the expression of THBS-3 is lowest in the S3 group (Fig1E).\u003c/p\u003e\n\u003cp\u003eFor a more in-depth examination of the influence of THBS-3 on IL-1\u0026beta;-induced chondrocyte extracellular matrix (ECM) degradation, chondrocytes were subjected to transfection with THBS-3 siRNA as well as a scrambled siRNA control. The THBS-3 siRNA resulted in a substantial reduction in the IL-1\u0026beta;-induced upregulation of MMP13 and ADAMTs 5 in chondrocytes. On the contrary, the THBS-3 siRNA led to an elevated expression of Aggrecan compared to normal chondrocytes treated by IL-1\u0026beta; alone (Fig1F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. THBS-3 promotes the vascularization and osteogenesis coupling of chondrocytes.\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eTo further investigate the role of THBS-3 in chondrocytes, we stimulated NC chondrocytes with\u0026nbsp;50, 100, 200nM\u0026nbsp;concentrations of recombinant human THBS-3 protein.\u0026nbsp;Subsequently, we conducted WB analysis to examine markers associated with angiogenesis and osteogenesis. Our research findings indicate that THBS-3 promotes the crosstalk between angiogenesis and osteogenesis processes (Fig2A,\u0026nbsp;E). On the contrary, the THBS-3 siRNA led to a decline expression of angiogenesis and osteogenesis (FigB, E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pivotal phase in angiogenesis is the migration of endothelial cells, which facilitates the extension of blood vessels. We prepared HUVEC cells that were either treated with THBS-3 (50,100,200nM) or left untreated, and we assessed the impact on angiogenesis through analysis of endothelial cell migration and tube formation. It was observed that THBS-3 significantly enhanced the migration of HUVEC cells compared to the control group. With the elevation of THBS-3 concentration, the migration area expands proportionally, reaching its most pronounced extent at a concentration of 100nM (p=0.0040) (Fig2C). The results of tube formation indicated that THBS-3 at concentrations of 50 nM (p=0.0036)\u0026nbsp;and 100 nM (p\u0026lt;0.0001)\u0026nbsp;significantly augmented tube formation compared to the NC group (Fig2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. THBS-3 siRNA application demonstrated effective inhibition of bone spur formation and mitigated pathological changes in cartilage tissue among CIOA mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe in vitro data encouraged us to further investigate the therapeutic effectiveness of THBS-3 inhibition in an experimental OA mice model induced by Type VII collagenase. Intra-articular injections of THBS-3 siRNA (5nM) were administered once a week for a duration of 7 weeks. Upon analyzing the results from each group, we observed that THBS-3 siRNA treatment led to a reduction in cartilage damage in OA mice through Micro-CT (Fig3A). Safranin O-Fast Green staining illustrated a decrease in cartilage damage in the knees of OA mice treated with THBS-3 siRNA when compared to control group (Fig3C). Furthermore, HE staining revealed that the tidemark, which had moved upwards in OA mice, was reversed by THBS-3 siRNA treatment (Fig3B). Evaluate the knee joint tissues of each group of mice using the OARSI scoring system (Fig3D). Compared to the Control group, the CIOA group showed a significant increase in scores. In comparison to the CIOA group, the CIOA+siTHBS-3 group exhibited a decrease in scores.\u0026nbsp;In other words, the inhibition of THBS-3 effectively counteracted the thickening of the calcified cartilage zone during the progression of OA. The inhibition of THBS-3 resulted in the restoration of Aggrecan expression, suppressed MMP-13 and ADAMTs-5 expression, as evaluated through immunohistochemistry in THBS-3 siRNA treated OA mice THBS-3 siRNA has inhibited vascularization /osteogenesis coupling in the articular cartilage tissue of CIOA mice(Fig3E-F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.THBS-3 promotes the vascularization and osteogenesis coupling of chondrocytes through the TGF-\u0026beta;/Smad2/3 signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether THBS-3 plays a role in OA through the TGF-\u0026beta; pathway, we made an intriguing discovery. After introducing 100nM of THBS into normal chondrocytes, we observed a significant upregulation of TGF-\u0026beta; expression within the 1-6 hour timeframe, peaking at 6 hours (p=0.0353) (Fig4A). Our current study revealed that the phosphorylation levels of SMAD2/3(p-SMAD2/3), which play a critical role in regulating the canonical TGF-\u0026beta; signaling pathway, were significantly lower in both the NC+IL-1\u0026beta;\u0026nbsp;group (p\u0026lt;0.0001) and the NC+THBS-3 group (p=0.0004) when compared to the NC group. Furthermore, in the NC + THBS-3 +TGF-\u0026beta; inhibitor group, the expression level of p-SMAD2/3 was significantly reduced compared to the NC+THBS-3 group (p=0.0070)(Fig4B).\u003c/p\u003e\n\u003cp\u003ePrevious studies have already pointed to the involvement of THBS-3 in vascularization and osteogenesis coupling within the context of OA. What\u0026apos;s particularly interesting is that when we pre-treated normal chondrocytes with a TGF-\u0026beta; inhibitor 10uM(SB-431542, MCE, China) and then introduced THBS-3, we observed a marked reduction in the expression of BMP-2 (p=0.0276), FGF-2 (p=0.0052), ANG-2 (p=0.0379), VEGF-A (p=0.0093) and PDGF-B (p=0.0065) associated with vascularization and osteogenesis coupling (Fig4C).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOA is a prevalent form of arthritis and a leading cause of chronic, non-traumatic disability among the elderly population. The results of this study can be succinctly summarized as follows:\u003c/p\u003e \u003cp\u003eFirst, we confirmed that OA resulted in up-regulated expressions of THBS-3. THBS-3 actively promoted degradation metabolism while simultaneously suppressing synthesis metabolism. Second, THBS-3 promotes the crosstalk between angiogenesis and osteogenesis processes in chondrocytes. Third, we demonstrated that THBS-3 in chondrocytes through activating TGF-β /smad2/3 signaling pathways and promoted angiogenesis and osteogenesis coupling in vitro, which may lead to vascular up-growth in OA.\u003c/p\u003e \u003cp\u003ePrevious studies have found that in a meta-analysis of gene expression in OA and non-OA chondrocytes, there was a significant increase in the expression of THBS-3 and COMP in OA[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our findings also highlighted a notable increase in the expression of THBS-3 in the OA. Posey's research discovered that mice lacking extracellular matrix proteins, THBS-1, THBS-3, THBS-5, and type IX collagen, exhibited skeletal abnormalities. THBS-3, THBS-5, and type IX collagen all play a direct role in regulating linear growth in growth plate tissue [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, Hankenson's study revealed that THBS-3 regulates postnatal bone maturation, influencing endochondral ossification. In cases of THBS3 deficiency, there was an accelerated ossification of the calcified cartilage in the femoral head[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These findings provide compelling evidence for the involvement of THBS-3 in the regulation of bone development, especially in the context of endochondral ossification during postnatal skeletal maturation. Nevertheless, the specific mechanism behind this phenomenon remains elusive at present.\u003c/p\u003e \u003cp\u003eThe exact pathogenic mechanism of OA remains elusive, but it is generally acknowledged that an imbalance in the synthesis and degradation metabolism of chondrocytes is a primary factor contributing to the development of OA. We observed that THBS-3 promotes chondrocyte degradation metabolism while inhibiting synthesis metabolism. Furthermore, research indicates that angiogenesis/osteogenesis coupling is involved in the progression of OA[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This coupling phenomenon is observed in the synovium, cartilage, and subchondral bone within the context of OA. In a study exploring the influence of THBS-3 on the biological behavior and survival of osteosarcoma patients, THBS-3 gene exhibited significant differential expression in osteosarcoma. Functioning as a potent stimulant for tumor progression, heightened levels of THBS-3 were found to actively stimulate angiogenesis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our research indicates that THBS-3 promotes the expression of VEGF, ANG-2, FGF-2, BMP-2 and PDGF-BB in osteoarthritis chondrocytes, thereby enhancing vascularization/bone coupling generation within the cartilage cells. In vivo experiments, immunohistochemical analysis yielded results consistent with the cellular experiments. Compared to the CIOA group, the CIOA\u0026thinsp;+\u0026thinsp;siTHBS-3 group showed a reduction in indicators related to vascularization/osteogenesis coupling in cartilage tissue.\u003c/p\u003e \u003cp\u003eTGFβ1 has been a subject of extensive investigation in the context of osteoarthritis and chondrocytes for many years. TGF-β1 is a crucial growth factor for the development, maintenance, and repair of articular cartilage[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have found that in in vitro culture of endothelial cells and in vivo vascular generation model experiments, THBS-4 promotes angiogenesis through TGF-β1 mediation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Suzuki Takahisa's research discovered an elevated expression of THBS-1 in synovial tissues of rheumatoid arthritis, and TGF-β1 significantly increased the expression of THBS-1 at both mRNA and protein levels in the synovium[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. THBS-1 may be involved in the pathogenesis of rheumatoid arthritis through the TGF-β1/THBS-1 pathway. Kimberly BD's study revealed that THBS-1 inhibits the osteogenic differentiation of human mesenchymal stem cells by activating TGF-β[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, it is evident that THBS-1 and 4 can promote osteogenesis and angiogenesis through TGF-β, although there is currently no research on THBS-3. Further, our findings indicate that the expression of TGF-β1 in chondrocytes stimulated with THBS-3 (100nM) progressively increased over time, with the peak expression observed at the 6-hour.These observations suggest that proper endogenous production of TGF-β1 is essential for maintaining chondrocyte homeostasis, whereas excessive or inadequate activation of TGF-β1 can be detrimental [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] The elevated TGF-β1 levels prompted us to further explore the regulatory mechanisms. Given the observed alterations in the expression levels of TGF-β signaling receptors in the context of OA, it is reasonable to expect changes in the activity of their signal mediators, Smad2/3.\u003c/p\u003e \u003cp\u003eResearch has provided compelling evidence of reduced Smad2 phosphorylation levels in cartilage during OA progression, evident in both spontaneous (STR/Ort) and collagenase-induced mouse models of OA [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, it has been observed that Smad2 phosphorylation diminishes in cartilage in older mice when compared to their younger counterparts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While Smad3 phosphorylation was not specifically examined in these models, a recent study has reported decreased Smad3 phosphorylation levels in Smurf-2 transgenic mice that spontaneously develop an OA-like phenotype[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These collective findings strongly suggest that OA is associated with a compromised TGF-β/Smad2/3 signaling pathway. Our findings are consistent with previous research. In the chondrocyte osteoarthritis (OA) model induced by IL-1 stimulation, a reduction in p-Smad2/3 expression was observed.\u003c/p\u003e \u003cp\u003eTo delve deeper into the pathways associated with THBS-3, stimulation of chondrocytes with THBS-3 revealed an inhibitory effect on p-Smad2/3 expression. Notably, the addition of a TGF inhibitor reversed the expression of p-Smad2/3, underscoring the intricate regulatory role of THBS-3 in this context.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur conclusion can be summarized as follows: THBS3 in regulating cartilage vascularization/bone coupling via the TGFB1/Smad2/3 pathway in OA. Furthermore, this presents novel therapeutic targets for addressing OA.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eECGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eEndothelial Cell growth supplement\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eECM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eextracellular matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eFBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003efetal bovine serum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eH\u0026amp;E staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003ehematoxylin-eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eHUVECs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eHuman microvascular ECs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eMicro-CT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eMicrocomputed Tomography\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eOA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eosteoarthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eOCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eoptimal cutting temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eP/S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003epenicillin/streptomycin solution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003ephosphate-buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eTHBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003ethrombospondin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.63636363636363%\" valign=\"top\"\u003e\n \u003cp\u003eWB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"61.36363636363637%\" valign=\"top\"\u003e\n \u003cp\u003eWestern blot\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedures of this study were approved by the Institutional Ethics Committee of the First Affiliated Hospital of Harbin Medical University. (NO:2023JS57)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eNot applicable\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eNot applicable\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China to Zhiyi Zhang (NSFC 82271826) and Shuya Wang (NSFC 82202020), and partially by the First Afliated Hospital of HMU Merit Review Frontiers grant to Shuya Wang (HYD2020YQ0008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors` contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJY.Y and SY.W contributed to the conception and design of the study. HY.L and XY.Z performed the experiments and contributed to the analysis and interpretation of data. YP. Z and ZY.Z contributed to draft manuscript. All approved the submitted manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicts of interest were declared.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbramoff B, Caldera FE. Osteoarthritis: Pathology, Diagnosis, and Treatment Options. Med Clin North Am. 2020,104(2):293-311. \u003c/li\u003e\n\u003cli\u003eQuicke JG, Conaghan PG, Corp N, Peat G. Osteoarthritis year in review 2021: epidemiology \u0026amp; therapy. Osteoarthritis Cartilage. 2022, 30(2):196-206.\u003c/li\u003e\n\u003cli\u003eBonnet CS, Walsh DA. Osteoarthritis, angiogenesis and inflammation. Rheumatology (Oxford). 2005, 44(1):7-16. \u003c/li\u003e\n\u003cli\u003eGrosso A, Burger MG, Lunger A, Schaefer DJ, Banfi A, Di Maggio N. It Takes Two to Tango: Coupling of Angiogenesis and Osteogenesis for Bone Regeneration. Front Bioeng Biotechnol. 2017, 5:68. \u003c/li\u003e\n\u003cli\u003eZoricic S, Maric I, Bobinac D, Vukicevic S. Expression of bone morphogenetic proteins and cartilage-derived morphogenetic proteins during osteophyte formation in humans. J Anat. 2003;202(Pt 3):269-277.\u003c/li\u003e\n\u003cli\u003eCosta C, Incio J, Soares R. Angiogenesis and chronic inflammation: cause or consequence?. Angiogenesis. 2007;10(3):149-166. \u003c/li\u003e\n\u003cli\u003eMapp PI, Walsh DA. Mechanisms and targets of angiogenesis and nerve growth in osteoarthritis. Nat Rev Rheumatol. 2012;8(7):390-398. \u003c/li\u003e\n\u003cli\u003eAdams JC, Lawler J. The thrombospondins. Cold Spring Harb Perspect Biol. 2011, 3(10):a009712\u003c/li\u003e\n\u003cli\u003eCarminati L, Taraboletti G. Thrombospondins in bone remodeling and metastatic bone disease. Am J Physiol Cell Physiol. 2020, 319(6):C980-C990.\u003c/li\u003e\n\u003cli\u003eHankenson KD, Hormuzdi SG, Meganck JA, Bornstein P. Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head. Mol Cell Biol. 2005, 25(13):5599-5606. \u003c/li\u003e\n\u003cli\u003eTran TM, Sosa B, O\u0026apos;Connell A, Chu T, Cottrell JA, Chang SL. A Meta-Analysis of Non-Osteoarthritis and Osteoarthritis Chondrocyte Gene Expression to Determine the Efficacy of Autologous Chondrocyte Transplantation as a Viable Treatment Option. Med Case Rep Short Rev. 2019, 2(1):264.\u003c/li\u003e\n\u003cli\u003ePosey KL, Hankenson K, Veerisetty AC, Bornstein P, Lawler J, Hecht JT. Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen. Am J Pathol. 2008;172(6):1664-1674. \u003c/li\u003e\n\u003cli\u003eHankenson KD, Hormuzdi SG, Meganck JA, Bornstein P. Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head. Mol Cell Biol. 2005;25(13):5599-5606. \u003c/li\u003e\n\u003cli\u003eDalla-Torre CA, Yoshimoto M, Lee CH, et al. Effects of THBS3, SPARC and SPP1 expression on biological behavior and survival in patients with osteosarcoma. BMC Cancer. 2006, 6:237.\u003c/li\u003e\n\u003cli\u003eBlaney Davidson EN, van der Kraan PM, van den Berg WB. TGF-beta and osteoarthritis. Osteoarthritis Cartilage. 2007, 15(6):597-604. \u003c/li\u003e\n\u003cli\u003eMuppala S, Xiao R, Krukovets I. Thrombospondin-4 mediates TGF-\u0026beta;-induced angiogenesis. Oncogene. 2017, 36(36):5189-5198. \u003c/li\u003e\n\u003cli\u003eSuzuki T, Iwamoto N, Yamasaki S. Upregulation of Thrombospondin 1 Expression in Synovial Tissues and Plasma of Rheumatoid Arthritis: Role of Transforming Growth Factor-\u0026beta;1 toward Fibroblast-like Synovial Cells [published correction appears in J Rheumatol. 2017 Jan;44(1):131]. J Rheumatol. 2015, 42(6):943-947. \u003c/li\u003e\n\u003cli\u003eHunter DJ, Bierma-Zeinstra S. Osteoarthritis. Lancet. 2019, 393(10182):1745-1759. \u003c/li\u003e\n\u003cli\u003eZhen G, Guo Q, Li Y, et al. Mechanical stress determines the configuration of TGF\u0026beta; activation in articular cartilage. Nat Commun. 2021;12(1):1706.\u003c/li\u003e\n\u003cli\u003eBlaney Davidson EN, Vitters EL, van der Kraan PM, van den Berg WB. Expression of transforming growth factor-beta (TGFbeta) and the TGFbeta signalling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: role in cartilage degradation, chondrogenesis and osteophyte formation. Ann Rheum Dis. 2006;65(11):1414-1421. \u003c/li\u003e\n\u003cli\u003eBlaney Davidson EN, Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB. Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity. Arthritis Res Ther. 2005;7(6):R1338-R1347.\u003c/li\u003e\n\u003cli\u003eWu Q, Huang JH, Sampson ER, et al. Smurf2 induces degradation of GSK-3beta and upregulates beta-catenin in chondrocytes: a potential mechanism for Smurf2-induced degeneration of articular cartilage. Exp Cell Res. 2009;315(14):2386-2398. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, THBS-3, vascularization, ossification","lastPublishedDoi":"10.21203/rs.3.rs-4167008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4167008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eVascularization and osteogenesis coupling is observed in osteoarthritis (OA) cartilage. THBS-3 belongs to the extracellular matrix (ECM) proteins and is highly expressed in cartilage tissue. The effect of THBS-3 on OA is unclear. This study aims to explore the mechanistic role of THBS-3 in OA.\u003c/p\u003e\u003ch2\u003eDesign:\u003c/h2\u003e \u003cp\u003eExpressions of THBS-3 was detected by Western blot (WB) and RT-qPCR. WB was employed to measure the expression levels of synthesis and degradation metabolism, as well as vascularization/ossification coupling. Migration and tube formation experiments were conducted to assess the migratory and tube-forming abilities of HUVECs influenced by THBS-3. Micro-CT was utilized for 3D imaging in mice. Immunohistochemistry was employed to detect the expression of synthesis, degradation metabolism, and vascularization/ossification coupling-related markers. Additionally, WB was utilized to assess the transforming growth factor-beta (TGF-β) signaling pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eProteinomics sequencing has revealed a higher expression level of THBS-3 in OA cartilage. Chondrocytes from OA joints exhibited significantly higher expression of THBS-3 relative to healthy individuals. In experiments conducted both in vivo and in vitro, THBS-3 exhibited a dual impact by enhancing catabolic metabolism, suppressing synthetic metabolism, and fostering the coupling of vascularization and osteogenesis within the cartilage. THBS-3 activated the TGF-β signaling pathway, and blockade of the TGF-β signaling pathway resulted in increased p-Smad2/3 expression in OA cartilage cells and decreased expression of vascularization /ossification coupling.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTHBS-3 can promote the vascularization/ossification coupling of cartilage cells by activating the TGF-β/Smad2/3 signaling pathway, providing new insights and targets for the treatment of OA.\u003c/p\u003e","manuscriptTitle":"The mechanism study of THBS3 in regulating cartilage vascularization/bone coupling via the TGF-β/Smad2/3 pathway in osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-05 13:01:54","doi":"10.21203/rs.3.rs-4167008/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d6e222c-24c8-432e-bd9e-a1a5472cf98a","owner":[],"postedDate":"April 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-07T19:03:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-05 13:01:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4167008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4167008","identity":"rs-4167008","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}