mTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions.

BACKGROUND: The larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated. MATERIALS AND RESULTS: This study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration. CONCLUSION: In summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

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.

Cross-sectional studies of the causal link between asthma and osteoporosis: insights from Mendelian randomization and bioinformatics analysis.

The study, using data from Chongqing, China, and employing Mendelian randomization along with bioinformatics, establishes a causal link between asthma and osteoporosis, beyond glucocorticoid effects. Asthma may contribute to osteoporosis by accelerating bone turnover through inflammatory factors, disrupting the coupling between osteoblasts and osteoclasts, ultimately leading to osteoporosis. INTRODUCTION: Asthma and osteoporosis are prevalent health conditions with substantial public health implications. However, their potential interplay and the underlying mechanisms have not been fully elucidated. Previous research has primarily focused on the impact of glucocorticoids on osteoporosis, often overlooking the role of asthma itself. METHODS: We conducted a multi-stage stratified random sampling in Chongqing, China and excluded individuals with a history of glucocorticoid use. Participants underwent comprehensive health examinations, and their clinical data, including asthma status, were recorded. Logistic regression and Mendelian randomization were employed to investigate the causal link between asthma and osteoporosis. Furthermore, bioinformatics analyses and serum biomarker assessments were conducted to explore potential mechanistic pathways. RESULTS: We found a significant association between asthma and osteoporosis, suggesting a potential causal link. Mendelian Randomization analysis provided further support for this causal link. Bioinformatics analyses revealed that several molecular pathways might mediate the impact of asthma on bone health. Serum alkaline phosphatase levels were significantly elevated in the asthma group, suggesting potential involvement in bone turnover. CONCLUSION: Our study confirms a causal link between asthma and osteoporosis and highlights the importance of considering asthma in osteoporosis prediction models. It also suggests that asthma may accelerate osteoporosis by increasing bone turnover through inflammatory factors, disrupting the coupling between osteoblasts and osteoclasts, ultimately leading to bone loss.

Lymphatic vessels: roles and potential therapeutic intervention in rheumatoid arthritis and osteoarthritis.

Lymphatic vessel networks are a main part of the vertebrate cardiovascular system, which participate in various physiological and pathological processes via regulation of fluid transport and immunosurveillance. Targeting lymphatic vessels has become a potent strategy for treating various human diseases. The presence of varying degrees of inflammation in joints of rheumatoid arthritis (RA) and osteoarthritis (OA), characterized by heightened infiltration of inflammatory cells, increased levels of inflammatory factors, and activation of inflammatory signaling pathways, significantly contributes to the disruption of cartilage and bone homeostasis in arthritic conditions. Increasing evidence has demonstrated the pivotal role of lymphatic vessels in maintaining joint homeostasis, with their pathological alterations closely associated with the initiation and progression of inflammatory joint diseases. In this review, we provide a comprehensive overview of the evolving knowledge regarding the structural and functional aspects of lymphatic vessels in the pathogenesis of RA and OA. In addition, we summarized the potential regulatory mechanisms underlying the modulation of lymphatic function in maintaining joint homeostasis during inflammatory conditions, and further discuss the distinctions between RA and OA. Moreover, we describe therapeutic strategies for inflammatory arthritis based on lymphatic vessels, including the promotion of lymphangiogenesis, restoration of proper lymphatic vessel function through anti-inflammatory approaches, enhancement of lymphatic contractility and drainage, and alleviation of congestion within the lymphatic system through the elimination of inflammatory cells. At last, we envisage potential research perspectives and strategies to target lymphatic vessels in treating these inflammatory joint diseases.

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.

Effects of different over-fired drying methods on the aroma of Lu'an Guapian tea.

Over-fired drying, a crucial process in the production of Lu'an Guapian (LAGP) tea, greatly enriches the tea's aroma. In this study, the aroma compounds of LAGP tea processed through pulley charcoal drying (PCD), roller drying (RD), roller-conveyor drying (RCD), and hot air drying (HD) were analyzed using gas chromatography-mass spectrometry. A subsequent analysis of aroma extraction dilution analysis and odor activity values revealed that (E)-ß-ionone, dimethyl sulfide, (E,E)-2,4-heptadienal, geraniol, linalool, benzeneacetaldehyde, coumarin, 2-ethyl-3,5-dimethyl-pyrazine, indole, hexanal, (Z)-jasmone, and (Z)-3-hexen-1-ol were the key contributors to the samples' aroma variation. Moreover, a quantitative descriptive analysis and aroma recombination and omission experiments analysis revealed that (E)-ß-ionone is the most critical contributor to the formation of floral aroma in tea processed using PCD, whereas (E,E)-2,4-heptadienal is responsible for the more pronounced fresh aroma in tea processed using HD. In addition, 2-ethyl-3,5-dimethyl-pyrazine contributes to the formation of a roasted aroma in tea processed using RD and RCD. The study results provide a theoretical basis for choosing the processing method, especially for drying, to obtain high-quality LAGP tea.

Dexmedetomidine Suppresses Glutamate-Stimulated Neuronal Injury Via MicroRNA-433/Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway.

Background: Cerebral ischemia has been a hotpot in the prevention and treatment of cerebral ischemia. Dexmedetomidine (Dex) is a new type of highly selective α2 adrenergic receptor agonist with pharmacological properties. Objective: Quantitative studies have shown that Dex has a protective effect on glutamate (Glu)-induced neuronal damage. however, its mechanism has not been fully elucidated. The purpose of this study was to explore the underlying molecular mechanism by which Dex ameliorates Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 axis. Materials and Methods: A model of neuronal injury was constructed by Glu treatment and intervened with Dex. miRNA expression profiling assay was conducted to screen potential miRNAs affected by Dex. Cell viability, lactate dehydrogenase (LDH) release and apoptosis were detected by MTT assay, LDH kit, and TUNEL staining, respectively. Oxidative stress indicators were assessed by ELISA whereas mitochondrial membrane potential (MMP) was assessed by C11-BODIPY581/591 staining. The targeting relationship between the miR-433 and JAK2 was verified by dual-luciferase reporter assay and gene expression was analyzed by quantitative PCR and Western blot. Results: Glu treatment decreased cell viability and MMP and promoted LDH release, apoptosis and oxidative damage. Glu-induced changes in neurons were reversed after Dex treatment through upregulating the miR-433 expression to block the activation of JAK2/STAT3 pathway. Conclusions: Dex protects against Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 pathway, which provides new insights into the treatment of neuronal injury.

Exogenous Indian hedgehog antagonist damages intervertebral discs homeostasis in adult mice.

Background: Vismodegib, as an exogenous Indian hedgehog (Ihh) antagonist, has been approved by the Food and Drug Administration (FDA) for the clinical treatment of patients with basal cell carcinoma, and previous observations implicate the potential therapeutic of vismodegib in osteoarthritis treatment. However, there is no direct evidence for the role of Ihh signaling in intervertebral discs (IVDs) homeostasis of adult mice. The aim of the present study is to assess the effect of systemic administration of Smoothened inhibitor (SMOi) - vismodegib on IVDs homeostasis during the adult stage. Methods: The expression of glioma-associated oncogene homolog 1 (Gli1), the downstream targeting gene of Ihh signaling, in IVDs of adult mice after receiving systemic administration of SMOi was examined by immunohistochemistry. The pathological changes of vertebral bodies after SMOi treatment were evaluated by X-ray and micro-CT. The effects of SMOi on homeostasis of IVDs including cartilaginous endplates (CEP), growth plates (GP) and annulus fibrous (AF) were evaluated by histological analysis. The expressions of Aggrecan, Matrix metalloproteinase 13 (MMP13) and Runt-related transcription factor 2 (Runx2), in IVDs were also investigated by immunohistochemistry. Changes in chondrocyte apoptosis and proliferation in IVDs were evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and analyzing the expression of the cell proliferation antigen Ki-67. Results: Systemic administration of SMOi significantly decreased the expression of Gli1 in IVDs that indicating effective inhibition of Ihh signaling. Bone mass of vertebral bodies was diminished after SMOi treatment. Moreover, IVDs degeneration (IDD) like defects including CEP sclerosis, degenerative nucleus pulposus (NP) and fissure within AF, as well as narrowed or fused GP and loss bone mass of vertebral bodies was observed in SMOi-treated mice. The severity of IDD was time-dependent with the administration of SMOi treatment after 2-8 weeks. The expressions of Aggrecan, MMP13 and Runx2 in IVDs of mice receiving SMOi treatment were significantly decreased. In addition, chondrocyte apoptosis was significantly enhanced, while chondrocyte proliferation was significantly inhibited. Conclusions: Our study propose that systemic administration of vismodegib damages IVDs homeostasis via inhibition of Ihh signaling in adult mice. The clinical application of Ihh signaling antagonists such as vismodegib should be careful considering these side adverse. The Translational Potential of this Article: Vismodegib as an exogenous antagonist of Ihh signaling has been approved by the FDA for the clinical treatment of patients with basal cell carcinoma. However, it is still unknown whether vismodegib will has adverse effects on the patient or animal model of IVDs cartilage homeostasis. Based on our study, systemic administration of vismodegib damages IVDs homeostasis via inhibition of Ihh signaling in adult mice and special attention should be paid to the clinical application of vismodegib.

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.

Improving the flavor of summer green tea (Camellia sinensis L.) using the yellowing process.

Summer green tea (SGT) has poor flavor due to its high levels of bitterness and astringency. The present study aimed to improve the flavor of SGT using the yellowing process. The results showed that after the yellowing process, the sweetness and overall acceptability increased, and the content of gallated catechins and flavonol glycosides decreased by 30.2% and 27.4%, respectively, as did the bitterness and astringency of SGT. Yellowing caused a decrease in the concentration of some aroma compounds, such as (z)-3-hexen-1-ol, 1-hexanol, pentanal, heptanal and 1-octanol, which caused grassy, floral and fruity aromas. In contrast, the concentrations of 1-octen-3-ol, benzene acetaldehyde and ß-ionone increased, which have mushroom and sweet aromas. Meanwhile, the sweetness and umami of SGT were enhanced by the addition of selected aroma compounds (1-octen-3-ol, benzene acetaldehyde and ß-ionone), demonstrating that the yellowing process improves the flavor of SGT through odor-taste interactions.

Sevoflurane Suppresses the Growth, Metastasis, and Invasion of Endometrial Carcinoma Cells via miR-195-5p/JAK2 Axis.

BACKGROUND: Highly invasive and destructive endometrioma is one of the most familiar primary malignant tumors among women. It has been studied that sevoflurane can influence the development of various malignancies. But whatever sevoflurane could influence endometrial tumors is unknown. MATERIALS AND METHODS: Through CCK8 and transwell analysis, we investigated the influence of sevoflurane on the development of endometrial tumors in vitro. Then, we studied the function of miRNA-195-5p to promote sevoflurane to inhibit the development of endometrial tumors. Then, we predicted the target genes of miRNA-195-5p by online software and focused on JAK2. Through luciferase assay, we proved the direct binding and regulation of miRNA-195-5p to JAK2. RESULTS: We showed that sevoflurane could inhibit the growth, metastasis, and invasion of endometrial tumors via miRNA-195-5p/JAK2 axis. CONCLUSIONS: Our research shows the function of sevoflurane in inhibiting the development of endometrial tumors via miRNA-195-5p/JAK2 axis. Our findings proved that sevoflurane is potentially beneficial for endometrial carcinoma patients with surgery and may be helpful for the choice of anesthetics in endometrial carcinoma operations.

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.

Rapid identification of the green tea geographical origin and processing month based on near-infrared hyperspectral imaging combined with chemometrics.

The geographical origin and processing month of green tea greatly affect its economic value and consumer acceptance. This study investigated the feasibility of combining near-infrared hyperspectral imaging (NIR-HSI) with chemometrics for the identification of green tea. Tea samples produced in three regions of Chongqing (southeastern Chongqing, northeastern Chongqing, and western Chongqing) for four months (from May to August 2020) were collected. Principal component analysis (PCA) was used to reduce data dimensionality and visualize the clustering of samples in different categories. Linear partial least squares-discriminant analysis (PLS-DA) and nonlinear support vector machine (SVM) algorithms were used to develop discriminant models. The PCA-SVM models based on the first four and first five principal components (PCs) achieved the best accuracies of 97.5% and 95% in the prediction set for geographical origin and processing month of green tea, respectively. This study demonstrated the feasibility of HSI in the identification of green tea species, providing a rapid and nondestructive method for the evaluation and control of green tea quality.

Association rules analysis on patterns of multimorbidity in adults: based on the National Health and Nutrition Examination Surveys database.

OBJECTIVE: To explore the prevalence and patterns of multimorbidity in population with different genders and age ranges. DESIGN: A cross-sectional study. SETTING: National Health and Nutrition Examination Surveys database. PARTICIPANTS: 12 576 patients. PRIMARY AND SECONDARY OUTCOME MEASURES: The prevalence and patterns of multimorbidity. RESULTS: High cholesterol had the highest prevalence in all population (33.4 (95% CI: 32.0 to 34.9)) and males. In females <65 years, the most prevalent disease was sleep disorder (32.1 (95% CI: 29.6 to 34.5)) while in females ≥65 years, hypertension was the most prevalent disease (63.9 (95% CI: 59.9 to 67.9)). Hypertension and high cholesterol were associated with the highest support (occur together most frequently) in all population regardless of genders. Hypertension displayed the highest betweenness centrality (mediating role in the network) followed by high cholesterol and arthritis in all population. For males aged <65 years, hypertension and high cholesterol presented the highest betweenness centrality. In males ≥65 years, hypertension, high cholesterol and arthritis were the top three diseases of degree centrality (direct association with other conditions). As for females ≥65 years, hypertension showed the highest betweenness centrality followed by high cholesterol and arthritis. The associations of hypertension, arthritis and one other item with high cholesterol presented the highest support in all population. In males, the associations of depression, hypertension with sleep disorders had the highest lift (the chance of co-occurrence of the conditions and significant association). Among females, the associations of depression, arthritis with sleep disorders had the highest lift. CONCLUSION: Hypertension and high cholesterol were prevalent in all population, regardless of females and males. Hypertension and high cholesterol, arthritis and hypertension, and diabetes and hypertension were more likely to coexist. The findings of this study might help make plans for the management and primary care of people with one or more 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.

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.

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.

Dstyk mutation leads to congenital scoliosis-like vertebral malformations in zebrafish via dysregulated mTORC1/TFEB pathway.

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.

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.