Ti3C2 nanosheet-induced autophagy derails ovarian functions.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have gained significant attention in various biomedical applications. Although previous studies have described the accumulation and associated damage of Ti3C2 nanosheets in the testes and placenta. However, it is currently unclear whether Ti3C2 nanosheets can be translocated to the ovaries and cause ovarian damage, thereby impairing ovarian functions. RESULTS: We established a mouse model with different doses (1.25, 2.5, and 5 mg/kg bw/d) of Ti3C2 nanosheets injected intravenously for three days. We demonstrated that Ti3C2 nanosheets can enter the ovaries and were internalized by granulosa cells, leading to a decrease in the number of primary, secondary and antral follicles. Furthermore, the decrease in follicles is closely associated with higher levels of FSH and LH, as well as increased level of E2 and P4, and decreased level of T in mouse ovary. In further studies, we found that exposure toTi3C2 nanosheets increased the levels of Beclin1, ATG5, and the ratio of LC3II/Ι, leading to autophagy activation. Additionally, the level of P62 increased, resulting in autophagic flux blockade. Ti3C2 nanosheets can activate autophagy through the PI3K/AKT/mTOR signaling pathway, with oxidative stress playing an important role in this process. Therefore, we chose the ovarian granulosa cell line (KGN cells) for in vitro validation of the impact of autophagy on the hormone secretion capability. The inhibition of autophagy initiation by 3-Methyladenine (3-MA) promoted smooth autophagic flow, thereby partially reduced the secretion of estradiol and progesterone by KGN cells; Whereas blocking autophagic flux by Rapamycin (RAPA) further exacerbated the secretion of estradiol and progesterone in cells. CONCLUSION: Ti3C2 nanosheet-induced increased secretion of hormones in the ovary is mediated through the activation of autophagy and impairment of autophagic flux, which disrupts normal follicular development. These results imply that autophagy dysfunction may be one of the underlying mechanisms of Ti3C2-induced damage to ovarian granulosa cells. Our findings further reveal the mechanism of female reproductive toxicity induced by Ti3C2 nanosheets.

An Hsp70 promoter-based mouse for heat shock-induced gene modulation.

Physical therapy is extensively employed in clinical settings. Nevertheless, the absence of suitable animal models has resulted in an incomplete understanding of the in vivo mechanisms and cellular distribution that respond to physical stimuli. The objective of this research was to create a mouse model capable of indicating the cells affected by physical stimuli. In this study, we successfully established a mouse line based on the heat shock protein 70 (Hsp70) promoter, wherein the expression of CreERT2 can be induced by physical stimuli. Following stimulation of the mouse tail, ear, or cultured calvarias with heat shock (generated by heating, ultrasound, or laser), a distinct Cre-mediated excision was observed in cells stimulated by these physical factors with minimal occurrence of leaky reporter expression. The application of heat shock to Hsp70-CreERT2; FGFR2-P253R double transgenic mice or Hsp70-CreERT2 mice infected with AAV-BMP4 at calvarias induced the activation of Cre-dependent mutant FGFR2-P253R or BMP4 respectively, thereby facilitating the premature closure of cranial sutures or the repair of calvarial defects. This novel mouse line holds significant potential for investigating the underlying mechanisms of physical therapy, tissue repair and regeneration, lineage tracing, and targeted modulation of gene expression of cells in local tissue stimulated by physical factor at the interested time points. KEY MESSAGES: In the study, an Hsp70-CreERT2 transgenic mouse was generated for heat shock-induced gene modulation. Heat shock, ultrasound, and laser stimulation effectively activated Cre expression in Hsp70-CreERT2; reporter mice, which leads to deletion of floxed DNA sequence in the tail, ear, and cultured calvaria tissues of mice. Local laser stimuli on cultured calvarias effectively induce Fgfr2-P253R expression in Hsp70-mTmG-Fgfr2-P253R mice and result in accelerated premature closure of cranial suture. Heat shock activated AAV9-FLEX-BMP4 expression and subsequently promoted the repair of calvarial defect of Hsp70-CreERT2; Rosa26-mTmG mice.

A Novel Cargo Delivery System-AnCar-ExoLaIMTS Ameliorates Arthritis via Specifically Targeting Pro-Inflammatory Macrophages.

Macrophages are heterogenic phagocytic cells that play distinct roles in physiological and pathological processes. Targeting different types of macrophages has shown potent therapeutic effects in many diseases. Although many approaches are developed to target anti-inflammatory macrophages, there are few researches on targeting pro-inflammatory macrophages, which is partially attributed to their non-s pecificity phagocytosis of extracellular substances. In this study, a novel recombinant protein is constructed that can be anchored on an exosome membrane with the purpose of targeting pro-inflammatory macrophages via antigen recognition, which is named AnCar-ExoLaIMTS . The data indicate that the phagocytosis efficiencies of pro-inflammatory macrophages for different AnCar-ExoLaIMTS show obvious differences. The AnCar-ExoLaIMTS3 has the best targeting ability for pro-inflammatory macrophages in vitro and in vivo. Mechanically, AnCar-ExoLaIMTS3 can specifically recognize the leucine-rich repeat domain of the TLR4 receptor, and then enter into pro-inflammatory macrophages via the TLR4-mediated receptor endocytosis pathway. Moreover, AnCar-ExoLaIMTS3 can efficiently deliver therapeutic cargo to pro-inflammatory macrophages and inhibit the synovial inflammatory response via downregulation of HIF-1α level, thus ameliorating the severity of arthritis in vivo. Collectively, the work established a novel gene/drug delivery system that can specifically target pro-inflammatory macrophages, which may be beneficial for the treatments of arthritis and other inflammatory diseases.

IRE1α protects against osteoarthritis by regulating progranulin-dependent XBP1 splicing and collagen homeostasis.

Osteoarthritis (OA) is a full-joint, multifactorial, degenerative and inflammatory disease that seriously affects the quality of life of patients due to its disabling and pain-causing properties. ER stress has been reported to be closely related to the progression of OA. The inositol-requiring enzyme 1α/X-box-binding protein-1 spliced (IRE1α/XBP1s) pathway, which is highly expressed in the chondrocytes of OA patients, promotes the degradation and refolding of abnormal proteins during ER stress and maintains the stability of the ER environment of chondrocytes, but its function and the underlying mechanisms of how it contributes to the progression of OA remain unclear. This study investigates the role of IRE1α/ERN1 in OA. Specific deficiency of ERN1 in chondrocytes spontaneously resulted in OA-like cartilage destruction and accelerated OA progression in a surgically induced arthritis model. Local delivery of AdERN1 relieved degradation of the cartilage matrix and prevented OA development in an ACLT-mediated model. Mechanistically, progranulin (PGRN), an intracellular chaperone, binds to IRE1α, promoting its phosphorylation and splicing of XBP1u to generate XBP1s. XBP1s protects articular cartilage through TNF-α/ERK1/2 signaling and further maintains collagen homeostasis by regulating type II collagen expression. The chondroprotective effect of IRE1α/ERN1 is dependent on PGRN and XBP1s splicing. ERN1 deficiency accelerated cartilage degeneration in OA by reducing PGRN expression and XBP1s splicing, subsequently decreasing collagen II expression and triggering collagen structural abnormalities and an imbalance in collagen homeostasis. This study provides new insights into OA pathogenesis and the UPR and suggests that IRE1α/ERN1 may serve as a potential target for the treatment of joint degenerative diseases, including OA.

Expansion of FGFR3-positive nucleus pulposus cells plays important roles in postnatal nucleus pulposus growth and regeneration.

BACKGROUND: Intervertebral disc degeneration (IVDD) can cause low back pain, a major public health concern. IVDD is characterized with loss of cells especially those in nucleus pulposus (NP), due to the limited proliferative potential and regenerative ability. Few studies, however, have been carried out to investigate the in vivo proliferation events of NP cells and the cellular contribution of a specific subpopulation of NP during postnatal growth or regeneration. METHODS: We generated FGFR3-3*Flag-IRES-GFP mice and crossed FGFR3-CreERT2 mice with Rosa26-mTmG, Rosa26-DTA and Rosa26-Confetti mice, respectively, to perform inducible genetic tracing studies. RESULTS: Expression of FGFR3 was found in the outer region of NP with co-localized expressions of proliferating markers. By fate mapping studies, FGFR3-positive (FGFR3+) NP cells were found proliferate from outer region to inner region of NP during postnatal growth. Clonal lineage tracing by Confetti mice and ablation of FGFR3·+ NP cells by DTA mice further revealed that the expansion of the FGFR3+ cells was required for the morphogenesis and homeostasis of postnatal NP. Moreover, in degeneration and regeneration model of mouse intervertebral disc, FGFR3+ NP cells underwent extensive expansion during the recovery stage. CONCLUSION: Our present work demonstrates that FGFR3+ NP cells are novel subpopulation of postnatal NP with long-existing proliferative capacity shaping the adult NP structure and participating in the homeostasis maintenance and intrinsic repair of NP. These findings may facilitate the development of new therapeutic approaches for IVD regeneration.

Inhibition of aberrant Hif1α activation delays intervertebral disc degeneration in adult mice.

The intervertebral disc (IVD) is the largest avascular tissue. Hypoxia-inducible factors (HIFs) play essential roles in regulating cellular adaptation in the IVD under physiological conditions. Disc degeneration disease (DDD) is one of the leading causes of disability, and current therapies are ineffective. This study sought to explore the role of HIFs in DDD pathogenesis in mice. The findings of this study showed that among HIF family members, Hif1α was significantly upregulated in cartilaginous endplate (EP) and annulus fibrosus (AF) tissues from human DDD patients and two mouse models of DDD compared with controls. Conditional deletion of the E3 ubiquitin ligase Vhl in EP and AF tissues of adult mice resulted in upregulated Hif1α expression and age-dependent IVD degeneration. Aberrant Hif1α activation enhanced glycolytic metabolism and suppressed mitochondrial function. On the other hand, genetic ablation of the Hif1α gene delayed DDD pathogenesis in Vhl-deficient mice. Administration of 2-methoxyestradiol (2ME2), a selective Hif1α inhibitor, attenuated experimental IVD degeneration in mice. The findings of this study show that aberrant Hif1α activation in EP and AF tissues induces pathological changes in DDD, implying that inhibition of aberrant Hif1α activity is a potential therapeutic strategy for DDD.

Effect Of XBP1 Deficiency In Cartilage On The Regulatory Network Of LncRNA/circRNA-miRNA-mRNA.

X-box binding protein 1(XBP1) is a critical component for unfolded protein response (UPR) in ER stress. According to previous studies performed with different XBP1-deficient mice, the XBP1 gene affects mouse cartilage development and causes other related diseases. However, how the complete transcriptome, including mRNA and ncRNAs, affects the function of cartilage and other tissues when XBP1 is deficient in chondrocytes is unclear. In this study, we aimed to screen the differentially expressed (DE) mRNAs, circRNAs, lncRNAs and miRNAs in XBP1 cartilage-specific knockout (CKO) mice using high throughput sequencing and construct the circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA regulatory networks. DE LncRNAs (DE-LncRNAs), circRNAs (DE-circRNAs), miRNAs (DE-miRNAs), and mRNAs [differentially expressed genes (DEGs)] between the cartilage tissue of XBP1 CKO mice and controls were identified, including 441 DE-LncRNAs, 15 DE-circRNAs, 6 DE-miRNAs, and 477 DEGs. Further, 253,235 lncRNA-miRNA-mRNA networks and 1,822 circRNA-miRNA-mRNA networks were constructed based on the correlation between lncRNAs/circRNAs, miRNAs, mRNAs. The whole transcriptome analysis revealed that XBP1 deficiency in cartilage affects the function of cartilage and other different tissues, as well as associated diseases. Overall, our findings may provide potential biomarkers and mechanisms for the diagnosis and treatment of cartilage and other related diseases.

Long term usage of dexamethasone accelerating accelerates the initiation of osteoarthritis via enhancing chondrocyte apoptosis and the extracellular matrix calcification and apoptosis of chondrocytes.

Systemic application of glucocorticoids is an essential anti-inflammatory and immune-modulating therapy for severe inflammatory or autoimmunity conditions. However, its long-term effects on articular cartilage of patients' health need to be further investigated. In this study, we studied the effects of dexamethasone (Dex) on the homeostasis of articular cartilage and the progress of destabilization of medial meniscus (DMM)-induced osteoarthritis (OA) in adult mice. Long-term administration of Dex aggravates the proteoglycan loss of articular cartilage and drastically accelerates cartilage degeneration under surgically induced OA conditions. In addition, Dex increases calcium content in calcified cartilage layer of mice and the samples from OA patients with a history of long-term Dex treatment. Moreover, long term usage of Dex results in decrease subchondral bone mass and bone density. Further studies showed that Dex leads to calcification of extracellular matrix of chondrocytes partially through activation of AKT, as well as promotes apoptosis of chondrocytes in calcified cartilage layer. Besides, Dex weakens the stress-response autophagy with the passage of time. Taken together, our data indicate that long-term application of Dex may predispose patients to OA and or even accelerate the OA disease progression development of OA patients.

Low-intensity pulsed ultrasound inhibits fibroblast-like synoviocyte proliferation and reduces synovial fibrosis by regulating Wnt/ß-catenin signaling.

OBJECTIVE: Synovial fibrosis is a characteristic symptom of osteoarthritis (OA), which is closely associated with joint pain and stiffness. Previous studies have reported that low-intensity pulsed ultrasound (LIPUS) can alleviate cartilage degradation in OA. However, the functions and mechanisms of LIPUS in OA synovial fibrosis are still unknown. METHODS: The destabilization of the medial meniscus (DMM) mouse model of OA was established in C57 male mice and fibroblast-like synoviocytes (FLS) were isolated from synovial tissue of OA patients. The knee joint diameter, Masson's trichrome (MT) and Hematoxylin-eosin (HE) staining were used to evaluate synovial fibrosis and hyperplasia. The Immunohistochemistry (IHC) staining was performed to detected the expression of synovial fibrosis makers and the activation of Wnt/ß-catenin signaling in vivo. FLS were treated with TGF-ß1 to serve as an in vitro model of synovial fibrosis, Wnt3a was used to activate the Wnt/ß-catenin signaling in cells. Cell proliferation was detected by using EdU assay, cell viability was performed by CCK8 assay. The protein levels of α-SMA, CTGF, Col Ⅰ, ß-catenin, active ß-catenin, c-Myc and cyclin D1 were examined by western blot and immunofluorescence staining. RESULTS: Two weeks after the LIPUS treatment, the synovial fibrosis, synovial hyperplasia and synoviocyte proliferation in the DMM model were significantly decreased. In vitro, LIPUS directly inhibited the TGF-ß1-induced fibrotic response and proliferation of FLS. Meanwhile, LIPUS suppressed Wnt/ß-catenin signaling in the synovium of DMM mice and cultured FLS. More importantly, we found that the synovial fibrosis makers, Wnt/ß-catenin pathway downstream proteins and FLS proliferation were significantly decreased in Wnt3a-stimulated FLS following LIPUS treatment. CONCLUSIONS: Our results present a novel role of LIPUS in OA-related synovial fibrosis, which is associated with its ability to repress Wnt/ß-catenin signaling in FLS. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study provides new insight into the clinical application of LIPUS as a therapeutic option to manage synovial fibrosis in OA.

Treating Autoimmune Inflammatory Diseases with an siERN1-Nanoprodrug That Mediates Macrophage Polarization and Blocks Toll-like Receptor Signaling.

The clinical application of small interfering RNA (siRNA) drugs provides promising opportunities to develop treatment strategies for autoimmune inflammatory diseases. In this study, siRNAs targeting the endoplasmic reticulum to nucleus signaling 1 (ERN1) gene (siERN1) were screened. Two cationic polymers, polyethylenimine (PEI) and poly(ß-amino amine) (PBAA), which can improve the efficiency of the siRNA transfection, were used as siERN1 delivery carriers. They were implemented to construct a nanodrug delivery system with macrophage-targeting ability and dual responsiveness for the treatment of autoimmune inflammatory diseases. In terms of the mechanism, siERN1 can regulate the intracellular calcium ion concentration by interfering with the function of inositol 1,4,5-trisphosphate receptor 1/3 (IP3R1/3) and thus inducing M2 polarization of macrophages. Furthermore, siERN1-nanoprodrug [FA (folic acid)-PEG-R(RKKRRQRRR)-NPs(ss-PBAA-PEI)@siERN1] acts as a conductor of macrophage polarization by controlling the calcium ion concentration and is an inhibitor of MyD88-dependent Toll-like receptor signaling. The results revealed that the FA-PEG-R-NPs@siERN1 has universal biocompatibility, long-term drug release responsiveness, superior targeting properties, and therapeutic effects in mouse collagen-induced arthritis and inflammatory bowel disease models. In conclusion, this study reveals a potential strategy to treat autoimmune inflammatory disorders.

Targeting local lymphatics to ameliorate heterotopic ossification via FGFR3-BMPR1a pathway.

Acquired heterotopic ossification (HO) is the extraskeletal bone formation after trauma. Various mesenchymal progenitors are reported to participate in ectopic bone formation. Here we induce acquired HO in mice by Achilles tenotomy and observe that conditional knockout (cKO) of fibroblast growth factor receptor 3 (FGFR3) in Col2+ cells promote acquired HO development. Lineage tracing studies reveal that Col2+ cells adopt fate of lymphatic endothelial cells (LECs) instead of chondrocytes or osteoblasts during HO development. FGFR3 cKO in Prox1+ LECs causes even more aggravated HO formation. We further demonstrate that FGFR3 deficiency in LECs leads to decreased local lymphatic formation in a BMPR1a-pSmad1/5-dependent manner, which exacerbates inflammatory levels in the repaired tendon. Local administration of FGF9 in Matrigel inhibits heterotopic bone formation, which is dependent on FGFR3 expression in LECs. Here we uncover Col2+ lineage cells as an origin of lymphatic endothelium, which regulates local inflammatory microenvironment after trauma and thus influences HO development via FGFR3-BMPR1a pathway. Activation of FGFR3 in LECs may be a therapeutic strategy to inhibit acquired HO formation via increasing local lymphangiogenesis.

TGF-ß/Alk5 signaling prevents osteoarthritis initiation via regulating the senescence of articular cartilage stem cells.

Osteoarthritis (OA) is the most common joint disease. The surface of joint cartilage is a defensive and first affected structure of articular cartilage (AC) during the pathogenesis of OA. Alk5 signaling is critical for maintaining AC homeostasis, however, the role and underlying mechanism for the involvement of Alk5 signaling in the phenotypes of articular cartilage stem cells (ACSCs) at the surface of AC is still unclear. The role of Alk5 in OA development was explored using an ACSCs-specific Alk5-deficient (cKO) mouse model. Alterations in cartilage structure were evaluated histologically. Senescence was detected by SA-ß-gal, while reactive oxygen species (ROS), MitoTracker, and LysoTracker staining were used to detect changes related to senescence. In addition, mice were injected intra-articularly with ganciclovir to limit the detrimental roles of senescent cells (SnCs). Alk5 cKO mice showed a decreased number of the slow-cell cycle cells and less lubricant secretion at the surface accompanied with drastically accelerated cartilage degeneration under ageing and surgically induced OA conditions. Further studies showed that Alk5 deficient ACSCs exhibited senescence-like manifestations including decreased proliferation and differentiation, more SA-ß-gal-positive cells and ROS production, as well as significantly swollen mitochondria and lysosome breakdown. We further found that local limitation of the detrimental roles of SnCs can attenuate the development of posttraumatic OA. Taken together, our findings suggest that Alk5 signaling acts as an important regulator of the SnCs in the superficial layer during AC maintenance and OA initiation.

FGF/FGFR signaling in health and disease.

Growing evidences suggest that the fibroblast growth factor/FGF receptor (FGF/FGFR) signaling has crucial roles in a multitude of processes during embryonic development and adult homeostasis by regulating cellular lineage commitment, differentiation, proliferation, and apoptosis of various types of cells. In this review, we provide a comprehensive overview of the current understanding of FGF signaling and its roles in organ development, injury repair, and the pathophysiology of spectrum of diseases, which is a consequence of FGF signaling dysregulation, including cancers and chronic kidney disease (CKD). In this context, the agonists and antagonists for FGF-FGFRs might have therapeutic benefits in multiple systems.

Fgfr3 mutation disrupts chondrogenesis and bone ossification in zebrafish model mimicking CATSHL syndrome partially via enhanced Wnt/ß-catenin signaling.

CATSHL syndrome, characterized by camptodactyly, tall stature and hearing loss, is caused by loss-of-function mutations of fibroblast growth factor receptors 3 (FGFR3) gene. Most manifestations of patients with CATSHL syndrome start to develop in the embryonic stage, such as skeletal overgrowth, craniofacial abnormalities, however, the pathogenesis of these phenotypes especially the early maldevelopment remains incompletely understood. Furthermore, there are no effective therapeutic targets for this skeleton dysplasia. Methods: We generated fgfr3 knockout zebrafish by CRISPR/Cas9 technology to study the developmental mechanisms and therapeutic targets of CATSHL syndrome. Several zebrafish transgenic lines labeling osteoblasts and chondrocytes, and live Alizarin red staining were used to analyze the dynamical skeleton development in fgfr3 mutants. Western blotting, whole mount in situ hybridization, Edu labeling based cell proliferation assay and Wnt/ß-catenin signaling antagonist were used to explore the potential mechanisms and therapeutic targets. Results: We found that fgfr3 mutant zebrafish, staring from early development stage, showed craniofacial bone malformation with microcephaly and delayed closure of cranial sutures, chondroma-like lesion and abnormal development of auditory sensory organs, partially resembling the clinical manifestations of patients with CATSHL syndrome. Further studies showed that fgfr3 regulates the patterning and shaping of pharyngeal arches and the timely ossification of craniofacial skeleton. The abnormal development of pharyngeal arch cartilage is related to the augmented hypertrophy and disordered arrangement of chondrocytes, while decreased proliferation, differentiation and mineralization of osteoblasts may be involved in the delayed maturation of skull bones. Furthermore, we revealed that deficiency of fgfr3 leads to enhanced IHH signaling and up-regulated canonical Wnt/ß-catenin signaling, and pharmacological inhibition of Wnt/ß-catenin could partially alleviate the phenotypes of fgfr3 mutants. Conclusions: Our study further reveals some novel phenotypes and underlying developmental mechanism of CATSHL syndrome, which deepens our understanding of the pathogenesis of CATSHL and the role of fgfr3 in skeleton development. Our findings provide evidence that modulation of Wnt/ß-catenin activity could be a potential therapy for CATSHL syndrome and related skeleton diseases.

Pulsed Electromagnetic Field Inhibits Synovitis via Enhancing the Efferocytosis of Macrophages.

Synovitis plays an important role in the pathogenesis of arthritis, which is closely related to the joint swell and pain of patients. The purpose of this study was to investigate the anti-inflammatory effects of pulsed electromagnetic fields (PEMF) on synovitis and its underlying mechanisms. Destabilization of the medial meniscus (DMM) model and air pouch inflammation model were established to induce synovitis in C57BL/6 mice. The mice were then treated by PEMF (pulse waveform, 1.5 mT, 75 Hz, 10% duty cycle). The synovitis scores as well as the levels of IL-1ß and TNF-α suggested that PEMF reduced the severity of synovitis in vivo. Moreover, the proportion of neutrophils in the synovial-like layer was decreased, while the proportion of macrophages increased after PEMF treatment. In addition, the phagocytosis of apoptotic neutrophils by macrophages (efferocytosis) was enhanced by PEMF. Furthermore, the data from western blot assay showed that the phosphorylation of P38 was inhibited by PEMF. In conclusion, our current data show that PEMF noninvasively exhibits the anti-inflammatory effect on synovitis via upregulation of the efferocytosis in macrophages, which may be involved in the phosphorylation of P38.

Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis.

Low-intensity pulsed ultrasound (LIPUS), a noninvasive physical therapy, was recently demonstrated to be an effective treatment for osteoarthritis (OA). Vascular endothelium growth factor A (VEGFA) has been found to be upregulated in the articular cartilage, synovium and subchondral bone of OA patients, leading to cartilage degeneration, synovitis and osteophyte formation. However, the functions and mechanisms of LIPUS in regulating chondrocyte-derived VEGFA expression are still unclear. In this study, we investigated whether LIPUS attenuated OA progression by (a) decreasing the percentage of VEGFA-positive cells in mouse articular cartilage destabilised through medial meniscus surgery and (b) relieving interleukin-1ß-induced VEGFA expression in mouse primary chondrocytes. However, this function was negated by a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. In addition, we found that LIPUS ameliorated VEGFA-mediated disorders in cartilage extracellular matrix metabolism and chondrocyte hypertrophy during OA development. In conclusion, our data indicate a novel effect of LIPUS in regulating the expression of osteoarthritic chondrocyte-derived VEGFA through the suppression of p38 MAPK activity.

SQSTM1-dependent autophagic degradation of PKM2 inhibits the production of mature IL1B/IL-1ß and contributes to LIPUS-mediated anti-inflammatory effect.

Synovitis is implicated in the pathology of osteoarthritis (OA) and significantly contributes to the development of OA. As a noninvasive physical therapy, low-intensity pulsed ultrasound (LIPUS) has been reported to possess anti-inflammatory effect in recent years. However, the role of LIPUS on synovitis of OA and the underlying mechanisms are little known. The present study showed that LIPUS ameliorated synovial inflammation in destabilization of the medial meniscus (DMM) mouse model and air pouch model, and alleviated pain gait patterns of DMM mouse. LIPUS dramatically inhibited the production of mature IL1B/IL-1ß (interleukin 1 beta) in vitro and in vivo. In addition, LIPUS upregulated the macroautophagy/autophagy level as well as accelerated the formation of an SQSTM1 (sequestosome1)-PKM (pyruvate kinase, muscle) complex in the lipopolysaccharide (LPS)-adenosine triphosphate (ATP)-treated macrophages. Besides, LIPUS downregulated the level of PKM2 in LPS-ATP-treated macrophages, which could be reversed by SQSTM1 knockdown. In brief, the present study for the first time demonstrates that LIPUS inhibits the production of mature IL1B partially via SQSTM1-dependent autophagic degradation of PKM2 in LPS-ATP-treated macrophages, which may further ameliorate the synovial inflammation and gait patterns in animal models. Our data provide new clues for the treatments of synovitis and other inflammatory diseases using LIPUS. ABBREVIATIONS: 3-MA: 3-methyladenene; ATG7: autophagy-related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; BMDMs: bone marrow derived macrophages; CHX: cycloheximide; DMM: destabilization of the medial meniscus; ELISA: enzyme-linked immunosorbent assay; GFP: green fluorescent protein; IL1B/IL-1ß: interleukin 1 beta; LIPUS: low-intensity pulsed ultrasound; LIR: LC3-interacting region; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDP: muramyl dipeptide; NFKB/NF-κB: nuclear factor kappa B; NLRP3: NLR family, pyrin domain containing 3; OA: osteoarthritis; PKM/PKM2: pyruvate kinase M1/2; PMA: phorbol-12-myristate-13-acetate; PYCARD/ASC; PYD and CARD domain containing; RFP: red fluorescent protein; siRNAs: small interfering RNAs; SQSTM1: sequestosome 1; TEM: transmission electron microscopy.

FGFR3 deficiency enhances CXCL12-dependent chemotaxis of macrophages via upregulating CXCR7 and aggravates joint destruction in mice.

OBJECTIVES: This study aims to investigate the role and mechanism of FGFR3 in macrophages and their biological effects on the pathology of arthritis. METHODS: Mice with conditional knockout of FGFR3 in myeloid cells (R3cKO) were generated. Gait behaviours of the mice were monitored at different ages. Spontaneous synovial joint destruction was evaluated by digital radiographic imaging and µCT analysis; changes of articular cartilage and synovitis were determined by histological analysis. The recruitment of macrophages in the synovium was examined by immunostaining and monocyte trafficking assay. RNA-seq analysis, Western blotting and chemotaxis experiment were performed on control and FGFR3-deficient macrophages. The peripheral blood from non-osteoarthritis (OA) donors and patients with OA were analysed. Mice were treated with neutralising antibody against CXCR7 to investigate the role of CXCR7 in arthritis. RESULTS: R3cKO mice but not control mice developed spontaneous cartilage destruction in multiple synovial joints at the age of 13 months. Moreover, the synovitis and macrophage accumulation were observed in the joints of 9-month-old R3cKO mice when the articular cartilage was not grossly destructed. FGFR3 deficiency in myeloid cells also aggravated joint destruction in DMM mouse model. Mechanically, FGFR3 deficiency promoted macrophage chemotaxis partly through activation of NF-κB/CXCR7 pathway. Inhibition of CXCR7 could significantly reverse FGFR3-deficiency-enhanced macrophage chemotaxis and the arthritic phenotype in R3cKO mice. CONCLUSIONS: Our study identifies the role of FGFR3 in synovial macrophage recruitment and synovitis, which provides a new insight into the pathological mechanisms of inflammation-related arthritis.

The exosome-like vesicles from osteoarthritic chondrocyte enhanced mature IL-1ß production of macrophages and aggravated synovitis in osteoarthritis.

Synovitis, a common clinical symptom for osteoarthritis (OA) patients, is highly related to OA pathological progression and pain manifestation. The activated synovial macrophages have been demonstrated to play an important role in synovitis, but the mechanisms about macrophage activation are still not clear. In this study, we found that the exosome-like vesicles from osteoarthritic chondrocytes could be a new biological factor to stimulate inflammasome activation and increase mature IL-1ß production in macrophages. The degraded cartilage explants produced more exosome-like vesicles than the nondegraded ones, while the exosome-like vesicles from chondrocytes could enter into joint synovium tissue and macrophages. Moreover, the exosome-like vesicles from osteoarthritic chondrocytes enhanced the production of mature IL-1ß in macrophages. These vesicles could inhibit ATG4B expression via miR-449a-5p, leading to inhibition of autophagy in LPS-primed macrophages. The decreased autophagy promoted the production of mitoROS, which further enhanced the inflammasome activation and subsequent IL-1ß processing. Ultimately, the increase of mature IL-1ß may aggravate synovial inflammation and promote the progression of OA disease. Our study provides a new perspective to understand the activation of synovial macrophages and synovitis in OA patients, which may be beneficial for therapeutic intervention in synovitis-related OA patients.

Imbalanced development of anterior and posterior thorax is a causative factor triggering scoliosis.

OBJECTIVE: Scoliosis is a common disease characterized by spinal curvature with variable severities. There is no generally accepted theory about the physical origin of the spinal deformation of scoliosis. The aim of this study was to explore a new hypothesis suggesting that the curvatures in scoliosis may be associated with the imbalance growth between thoracic vertebral column and sternum. METHODS: We undertook a comparative computed tomography (CT) based morphology study of thoracic vertebrae and sternum of patients with adolescent idiopathic scoliosis (AIS) and age-gender matched normal subjects. We further measured the ratios between the lengths of the sternum and thoracic vertebra of mice with deficiency of fibroblast growth factor receptor 3 (FGFR3), which exhibit scoliosis. Three-week-old C57BL/6J mice were used to generate bipedal and sternal growth plate injury model. Radiographs and histological images were obtained to observe the presence of sternal and spinal deformity. RESULTS: There was a significant correlation between the severities of scoliosis and the ratios of the sternum to thoracic vertebral lengths. We also found that FGFR3 deficient mice showed smaller ratio of the sternum to thoracic vertebra lengths than that of the wild-type mice, which were similar with that of the AIS patients. Surgery-induced injuries of sternal growth plates can accelerate and aggravate the scoliosis in bipedal mice and imbalanced development of anterior and posterior thoracic occurred before the appearance of scoliosis. CONCLUSIONS: Our findings suggest that the imbalanced growth between the thoracic vertebral column and the sternum is an important causative factor for the pathogenesis of scoliosis including AIS. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Imbalanced growth between the thoracic vertebral column and the sternum is associated with scoliosis. Surgical or rehabilitation intervention for scoliosis should focus on all components involved in the pathogenesis of curvature to obtain better outcome.