Pyroptosis: A spoiler of peaceful coexistence between cells in degenerative bone and joint diseases.

BACKGROUND: As people age, degenerative bone and joint diseases (DBJDs) become more prevalent. When middle-aged and elderly people are diagnosed with one or more disorders such as osteoporosis (OP), osteoarthritis (OA), and intervertebral disc degeneration (IVDD), it often signals the onset of prolonged pain and reduced functionality. Chronic inflammation has been identified as the underlying cause of various degenerative diseases, including DBJDs. Recently, excessive activation of pyroptosis, a form of programed cell death (PCD) mediated by inflammasomes, has emerged as a primary driver of harmful chronic inflammation. Consequently, pyroptosis has become a potential target for preventing and treating DBJDs. AIM OF REVIEW: This review explored the physiological and pathological roles of the pyroptosis pathway in bone and joint development and its relation to DBJDs. Meanwhile, it elaborated the molecular mechanisms of pyroptosis within individual cell types in the bone marrow and joints, as well as the interplay among different cell types in the context of DBJDs. Furthermore, this review presented the latest compelling evidence supporting the idea of regulating the pyroptosis pathway for DBJDs treatment, and discussed the potential, limitations, and challenges of various therapeutic strategies involving pyroptosis regulation. KEY SCIENTIFIC CONCEPTS OF REVIEW: In summary, an interesting identity for the unregulated pyroptosis pathway in the context of DBJDs was proposed in this review, which was undertaken as a spoiler of peaceful coexistence between cells in a degenerative environment. Over the extended course of DBJDs, pyroptosis pathway perpetuated its activity through crosstalk among pyroptosis cascades in different cell types, thus exacerbating the inflammatory environment throughout the entire bone marrow and joint degeneration environment. Correspondingly, pyroptosis regulation therapy emerged as a promising option for clinical treatment of DBJDs.

Genetically Engineered Biomimetic Nanoparticles for Targeted Delivery of mRNA to Treat Rheumatoid Arthritis.

In addition to inhibiting persistent inflammation, phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is known as an important therapeutic target for alleviating rheumatoid arthritis (RA) symptoms. Modulation of PTEN gene expression in synovial tissue using messenger RNA (mRNA) is a promising approach to combat RA. However, mRNA therapeutics are often hampered by unsatisfactory stability and inefficient localization in synovial tissue. In this study, a genetically engineered biomimetic membrane-coated mRNA (MR@P-mPTEN) carrier that effectively delivers mRNA-PTEN (mPTEN) directly to the RA joint is presented. By overexpressing tumor necrosis factor (TNF-α) receptors on macrophage biomimetic membranes via plasmid transfection, decoys that reduce inflammatory pathway activation are prepared for TNF-α. The resulting construct, MR@P-mPTEN, shows good stability and RA targeting based on in vivo fluorescence imaging. It is also found that MR@P-mPTEN competitively binds TNF-α and activates the PTEN pathway in vitro and in vivo, thereby inhibiting synovitis and joint damage. Clinical micro-computed tomography and histological analyses confirm the treatment effects. These results suggest that the genetically engineered biomimetic therapeutic platform MR@P-mPTEN both inhibits pro-inflammatory cytokines and upregulates PTEN protein expression to alleviate RA damage, providing a new a new combination strategy for RA treatment.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

Objective: The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Methods: The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. Results: The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. Conclusion: The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. The translational potential of this article: Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

A Three-Dimensional Co-Culture Model for Rheumatoid Arthritis Pannus Tissue.

Three-dimensional (3D) co-culture models have closer physiological cell composition and behavior than traditional 2D culture. They exhibit pharmacological effects like in vivo responses, and therefore serve as a high-throughput drug screening model to evaluate drug efficacy and safety in vitro. In this study, we created a 3D co-culture environment to mimic pathological characteristics of rheumatoid arthritis (RA) pannus tissue. 3D scaffold was constructed by bioprinting technology with synovial fibroblasts (MH7A), vascular endothelial cells (EA.hy 926) and gelatin/alginate hydrogels. Cell viability was observed during 7-day culture and the proliferation rate of co-culture cells showed a stable increase stage. Cell-cell interactions were evaluated in the 3D printed scaffold and we found that spheroid size increased with time. TNF-α stimulated MH7A and EA.hy 926 in 3D pannus model showed higher vascular endothelial growth factor (VEGF) and angiopoietin (ANG) protein expression over time. For drug validation, methotrexate (MTX) was used to examine inhibition effects of angiogenesis in 3D pannus co-culture model. In conclusion, this 3D co-culture pannus model with biological characteristics may help the development of anti-RA drug research.

PLGA/ß-TCP composite scaffold incorporating cucurbitacin B promotes bone regeneration by inducing angiogenesis.

OBJECTIVES: Vascularization is an essential step in successful bone tissue engineering. The induction of angiogenesis in bone tissue engineering can be enhanced through the delivery of therapeutic agents that stimulate vessel and bone formation. In this study, we show that cucurbitacin B (CuB), a tetracyclic terpene derived from Cucurbitaceae family plants, facilitates the induction of angiogenesis in vitro. METHODS: We incorporated CuB into a biodegradable poly (lactide-co-glycolide) (PLGA) and ß-tricalcium phosphate (ß-TCP) biomaterial scaffold (PT/CuB) Using 3D low-temperature rapid prototyping (LT-RP) technology. A rat skull defect model was used to verify whether the drug-incorporated scaffold has the effects of angiogenesis and osteogenesis in vivo for the regeneration of bone defect. Cytotoxicity assay was performed to determine the safe dose range of the CuB. Tube formation assay and western blot assay were used to analyze the angiogenesis effect of CuB. RESULTS: PT/CuB scaffold possessed well-designed bio-mimic structure and improved mechanical properties. CuB was linear release from the composite scaffold without affecting pH value. The results demonstrated that the PT/CuB scaffold significantly enhanced neovascularization and bone regeneration in a rat critical size calvarial defect model compared to the scaffold implants without CuB. Furthermore, CuB stimulated angiogenic signaling via up-regulating VEGFR2 and VEGFR-related signaling pathways. CONCLUSION: CuB can serve as promising candidate compound for promoting neovascularization and osteogenesis, especially in tissue engineering for repair of bone defects. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study highlights the potential use of CuB as a therapeutic agent and strongly support its adoption as a component of composite scaffolds for tissue-engineering of bone repair.

Treatment of collagen-induced arthritis rat model by using Notch signalling inhibitor.

BACKGROUND: The Notch signalling pathway has been reported to play a key role in rheumatoid arthritis (RA) development. Thus, inhibition of the activation of this signalling pathway may be a promising approach to the treatment of RA. In this study, the Notch signalling inhibitor LY411575, which can inhibit both Notch1 and Notch3, was used for the treatment of collagen-induced arthritis (CIA) rats. METHODS: Wistar rats were immunised with bovine type II collagen (CII) to establish rats CIA model. The inhibitory effects of LY411575 on Notch1 intracellular domain (N1ICD) and Notch3 intracellular domain (N3ICD) protein was verified by western blot (WB) in vitro. CIA rats were treated with different doses of LY411575 for 15 and 28 days, respectively. Methotrexate and sodium carboxymethyl cellulose (CMC-Na) were used as positive and negative (vehicle) control respectively. Destruction of the rat ankle joint and the bone loss on the periarticular side were evaluated by micro-computed tomography (Micro-CT). In addition, destruction of the ankle articular cartilage and the osteoclast numbers were determined by histology. Expression of N1ICD and N3ICD in the ankle joint was detected by immunohistochemistry. RESULTS: LY411575 could significantly inhibit the expression of N1ICD and N3ICD in vitro. Micro-CT test showed that the ankle joint destruction significantly improved after treatment with LY411575 (5 â€‹mg/kg and 10 â€‹mg/kg, respectively). The bone quality in the LY411575 (5 â€‹mg/kg and 10 â€‹mg/kg, respectively) groups were improved compared with the vehicle group. Histological analysis showed that LY411575 (5 â€‹mg/kg and 10 â€‹mg/kg, respectively) treatment reduced the severity of ankle joint inflammation in CIA rats (including ankle joint destruction, pannus formation, and cartilage damage) and reduced the expression of N1ICD and N3ICD in CIA rats ankle joints significantly. CONCLUSION: The inhibitor of Notch signalling LY411575 is an effective treatment for CIA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our study provides new evidence to support the potential clinical application of Notch signalling pathway inhibitor LY411575 as a drug candidate for the treatment of RA.

Notch-1 and Notch-3 Mediate Hypoxia-Induced Activation of Synovial Fibroblasts in Rheumatoid Arthritis.

OBJECTIVE: To investigate the molecular mechanism of hypoxia-induced rheumatoid arthritis synovial fibroblast (RASF) activation via Notch-1 and Notch-3 signaling, and to evaluate its potential as a therapeutic target. METHODS: Expression of Notch-1 intracellular domain (N1ICD), N3ICD, and hypoxia-inducible factor 1α (HIF-1α) was assessed by immunhistology in synovial tissue from patients with RA. RASFs were cultured under hypoxic conditions and normoxic conditions with or without small interfering RNAs (siRNAs), and N1ICD and N3ICD were overexpressed under normoxic conditions. Rats with collagen-induced arthritis (CIA) were administered LY411575 (inhibitor of N1ICD and N3ICD) for 15 days and 28 days, and its therapeutic efficacy was assessed by histologic and radiologic evaluation of the rat synovial tissue, and by analysis of inflammatory cytokine production in the serum of rats. RESULTS: N1ICD, N3ICD, and HIF-1α were expressed abundantly in the synovial tissue of RA patients. HIF-1α was shown to directly regulate the expression of Notch-1 and Notch-3 genes under hypoxic conditions. Moreover, hypoxia-induced N1ICD and N3ICD expression in RASFs was blocked by HIF-1α siRNA. Notch-1 siRNA and Notch-3 siRNA inhibited hypoxia-induced RASF invasion and angiogenesis in vitro, whereas overexpression of N1ICD and N3ICD promoted these processes. In addition, Notch-1 was shown to regulate RASF migration and epithelial-mesenchymal transition under hypoxic conditions, whereas Notch-3 was shown to regulate the processes of anti-apoptosis and autophagy. Furthermore, in vivo studies in rats with CIA showed that the N1ICD and N3ICD inhibitor LY411575 had a therapeutic effect in terms of ameliorating the symptoms and severity of the disease. CONCLUSION: This study identified a functional link between HIF-1α, Notch-1, and Notch-3 signaling in regulating activation of RASFs and the processes involved in the pathogenesis of RA.

Repurposing of Pirfenidone (Anti-Pulmonary Fibrosis Drug) for Treatment of Rheumatoid Arthritis.

Clinical studies have shown that pirfenidone (PFD) effectively relieves joint pain in rheumatoid arthritis (RA) patients. However, the detailed mechanisms underlying the anti-RA effects of PFD have not been investigated. This study was undertaken to investigate the repurposing of PFD for the treatment of RA, and explore its anti-rheumatic mechanisms. A collagen-induced arthritis (CIA) rat model was used to observe joint pathological changes following PFD treatment. Based on bioinformatics to predict the mechanism of PFD anti-RA, using EA. hy926 and TNF-α-induced MH7A cells to establish in vitro model to explore its biological mechanism from the perspectives of synovial inflammation and angiogenesis. PFD significantly relieved pathological changes, including joint swelling, synovial hyperplasia, inflammatory cell infiltration and joint destruction. PFD was also associated with reduced expression of MMP-3 and VEGF in articular chondrocytes and synovial cells of CIA rats (p < 0.05). Using bioinformatic methods, we predicted that PFD inhibits cell inflammation and migration by interfering with the JAK2/STAT3 and Akt pathways. These results were verified using in vitro models. In particular, PFD effectively reduced the expression of pro-inflammatory, chondrogenic, and angiogenic cytokines, such as IL-1ß, IL-6, IL-8, MMP-1/3/2/9 and VEGF (p < 0.05), in TNF-α-induced MH7A cells. In addition, PFD significantly reduced the production of MMP-2/9 and VEGF in EA. hy926 cells, thereby weakening migration and inhibiting angiogenesis (p < 0.05). These findings suggest that PFD may alleviate the pathological process in CIA rats, by inhibiting inflammation and angiogenesis through multiple pathways, and serve as a potential therapeutic drug for RA.

Biphasic Effect of Pirfenidone on Angiogenesis.

Pirfenidone (PFD), a synthetic arsenic compound, has been found to inhibit angiogenesis at high concentrations. However, the biphasic effects of different PFD concentrations on angiogenesis have not yet been elucidated, and the present study used an in vitro model to explore the mechanisms underlying this biphasic response. The effect of PFD on the initial angiogenesis of vascular endothelial cells was investigated through a Matrigel tube formation assay, and the impact of PFD on endothelial cell migration was evaluated through scratch and transwell migration experiments. Moreover, the expression of key migration cytokines, matrix metalloproteinase (MMP)-2 and MMP-9, was examined. Finally, the biphasic mechanism of PFD on angiogenesis was explored through cell signaling and apoptosis analyses. The results showed that 10-100 µM PFD has a significant and dose-dependent inhibitory effect on tube formation and migration, while 10 nM-1 µM PFD significantly promoted tube formation and migration, with 100 nM PFD having the strongest effect. Additionally, we found that a high concentration of PFD could significantly inhibit MMP-2 and MMP-9 expression, while low concentrations of PFD significantly promoted their expression. Finally, we found that high concentrations of PFD inhibited EA.hy926 cell tube formation by promoting apoptosis, while low concentrations of PFD promoted tube formation by increasing MMP-2 and MMP-9 protein expression predominantly via the EGFR/p-p38 pathway. Overall, PFD elicits a biphasic effect on angiogenesis through different mechanisms, could be used as a new potential drug for the treatment of vascular diseases.