CD_99 G1 neutrophils modulate osteogenic differentiation of mesenchymal stem cells in the pathological process of ankylosing spondylitis.

OBJECTIVES: This study aimed to identify the types and heterogeneity of cells within the spinal enthesis and investigate the underlying mechanisms of osteogenesis. METHODS: Single-cell RNA sequencing was used to identify cell populations and their gene signatures in the spinal enthesis of five patients with ankylosing spondylitis (AS) and three healthy individuals. The transcriptomes of 40 065 single cells were profiled and divided into 7 clusters: neutrophils, monocytic cells, granulomonocytic progenitor_erythroblasts, T cells, B cells, plasma cells and stromal cells. Real-time quantitative PCR, immunofluorescence, flow cytometry, osteogenesis induction, alizarin red staining, immunohistochemistry, short hairpin RNA and H&E staining were applied to validate the bioinformatics analysis. RESULTS: Pseudo-time analysis showed two differentiation directions of stromal cells from the mesenchymal stem cell subpopulation MSC-C2 to two Cxcl12-abundant-reticular (CAR) cell subsets, Osteo-CAR and Adipo-CAR, within which three transcription factors, C-JUN, C-FOS and CAVIN1, were highly expressed in AS and regulated the osteogenesis of mesenchymal stem cells. A novel subcluster of early-stage neutrophils, CD99_G1, was elevated in AS. The proinflammatory characteristics of monocyte dendritic cell progenitor-recombinant adiponectin receptor 2 monocytic cells were explored. Interactions between Adipo-CAR cells, CD99_G1 neutrophils and other cell types were mapped by identifying ligand-receptor pairs, revealing the recruitment characteristics of CD99_G1 neutrophils by Adipo-CAR cells and the pathogenesis of osteogenesis induced in AS. CONCLUSIONS: Our results revealed the dynamics of cell subpopulations, gene expression and intercellular interactions during AS pathogenesis. These findings provide new insights into the cellular and molecular mechanisms of osteogenesis and will benefit the development of novel therapeutic strategies.

CX3CL1 promotes M1 macrophage polarization and osteoclast differentiation through NF-κB signaling pathway in ankylosing spondylitis in vitro.

BACKGROUND: Ankylosing spondylitis (AS) is an autoimmune disease with a genetic correlation and is characterized by inflammation in the axial skeleton and sacroiliac joints. Many AS patients also have inflammatory bowel diseases (IBD), but the underlying causes of intestinal inflammation and osteoporosis in AS are not well understood. CX3CL1, a protein involved in inflammation, has been found to be up-regulated in AS patients and AS-model mice. METHODS: The authors investigated the effects of CX3CL1 on AS by studying its impact on macrophage polarization, inflammation factors, and osteoclast differentiation. Furthermore, the effects of inhibiting the NF-κB pathway and blocking CX3CL1 were assessed using BAY-117082 and anti-CX3CL1 mAb, respectively. AS model mice were used to evaluate the effects of anti-CX3CL1 mAb on limb thickness, spine rupture, and intestinal tissue damage. RESULTS: The authors found that CX3CL1 increased the expression of M1-type macrophage markers and inflammation factors, and promoted osteoclast differentiation. This effect was mediated through the NF-κB signaling pathway. Inhibition of the NF-κB pathway prevented M1-type macrophage polarization, reduced inflammation levels, and inhibited osteoclast differentiation. Injection of anti-CX3CL1 mAb alleviated limb thickness, spine rupture, and intestinal tissue damage in AS model mice by inhibiting M1-type macrophage polarization and reducing intestinal tissue inflammation. CONCLUSIONS: The study demonstrated that up-regulated CX3CL1 promotes M1-type macrophage polarization and osteoclast differentiation through the NF-κB signaling pathway. Inhibition of this pathway and blocking CX3CL1 can alleviate inflammation and bone destruction in AS. These findings contribute to a better understanding of the pathogenesis of AS and provide a basis for clinical diagnosis and treatment.

PDGFRB as a potential therapeutic target of ankylosing spondylitis: validation following bioinformatics analysis.

Ankylosing spondylitis (AS) is a chronic, progressive, and inflammatory disease that mainly affects the central axis joint. Although this disease has already been well documented and studied, its pathogenesis is still not well understood. This study aimed to screen and identify key candidate genes involved in the progression of AS. For this purpose, expression profiles of GSE39340 and GSE41038 were downloaded from the Gene Expression Omnibus and displayed in the form of volcano plots and heatmaps. Differentially expressed genes (DEGs) were identified by the Limma package in R and functional enrichment analyses were performed. Moreover, STRING and Cytoscape were utilized to construct protein-protein interaction (PPI) networks and screen significant modules. Immunohistochemistry (IHC) in tissue chips of AS and normal human synovial tissues was performed to confirm the major proteins associated with its development. Western blotting (WB) and alizarin red staining were applied to validate the expression level of platelet-derived growth factor receptor beta (PDGFRB) and function during osteogenesis differentiation of fibroblasts in AS. A total of 256 DEGs were screened, including 191 up-regulated genes and 65 down-regulated genes. The enriched functions of these identified genes mainly included adherens junction, focal adhesion, and cell-substrate adherens junction. The pathways most highly associated with the progression of AS were TGF-ß signaling pathway, the Hippo signaling pathway, and the AGE-RAGE signaling pathway. In addition, IHC showed that mitogen-activated protein kinase 1 (MAPK1), C-X-C motif chemokine receptor 4 (CXCR4), and PDGFRB were highly expressed in AS. PDGFRB was found upregulated during osteogenesis of fibroblasts and stimulates osteogenesis in AS. These findings may improve our understanding of the molecular mechanisms controlling AS. Pharmacological targeting of PDGFRB may initiate a possible suppression of bone formation in AS.

NEDD4 Depletion Inhibits Hepatocellular Carcinoma Growth via Targeting PTEN.

BACKGROUND/AIMS: Neural precursor cell-expressed developmentally down-regulated gene 4 (NEDD4) plays an important role in tumor cell growth, yet its role in hepatocellular carcinoma (HCC) remains unclear. This study is to establish NEDD4 as a prognostic biomarker by which the survival of HCC patients can be predicted and to reveal the role of NEDD4 in hepatocellular carcinoma cell growth. METHODS: The expression of NEDD4 in 219 HCC specimens was assessed by immunohistochemistry. Postoperative overall survival and time to recurrence were evaluated by univariate and multivariate analyses. The roles of NEDD4 in hepatocellular carcinoma cell proliferation and invasion were determined. RESULTS: The patients with low NEDD4 expression tumors had an average cumulative survival of 64.9 ± 6.5 months during follow-up while the patients with high NEDD4 expression tumors had an average cumulative survival of 20.3 ± 15.8 months. NEDD4 silencing inhibited Huh7 cell proliferation and altered cell cytoskeletal assembly, and NEDD4 depletion furthermore seemed to suppress cell migration and invasion. A possible molecular mechanism for the observed effects might be that NEDD4 silence led to an increase in PTEN (phosphatase and tensin homologue) expression, which in turn resulted in the inactivation of STAT3, AKT, and ERK1/2. CONCLUSION: Our findings indicate that NEDD4 may participate in the HCC progression and may therefore be a potential target for HCC therapy.

CDKN1A regulation on chondrogenic differentiation of human chondrocytes in osteoarthritis through single-cell and bulk sequencing analysis.

Objective: Chondrocyte death is the hallmark of cartilage degeneration during osteoarthritis (OA). However, the specific pathogenesis of cell death in OA chondrocytes has not been elucidated. This study aims to validate the role of CDKN1A, a key programmed cell death (PCD)-related gene, in chondrogenic differentiation using a combination of single-cell and bulk sequencing approaches. Design: OA-related RNA-seq data (GSE114007, GSE55235, GSE152805) were downloaded from Gene Expression Omnibus database. PCD-related genes were obtained from GeneCards database. RNA-seq was performed to annotate the cell types in OA and control samples. Differentially expressed genes (DEGs) among those cell types (scRNA-DEGs) were screened. A nomogram of OA was constructed based on the featured genes, and potential drugs targeting the featured genes were predicted. The presence of key genes was confirmed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), Western blot (WB), and immunohistochemistry (IHC). Micromass culture and Alcian blue staining were used to determine the effect of CDKN1A on chondrogenesis. Results: Six cell types, namely HomC, HTC, RepC, preFC, FC, and RegC, were annotated in scRNA-seq data. Five featured genes (JUN, CDKN1A, HMGB2, DDIT3, and DDIT4) were screened by multiple biological information analysis methods. TAXOTERE had the highest ability to dock with DDIT3. Functional analysis indicated that CDKN1A was enriched in processes related to collagen catabolism and acts as a positive regulator of autophagy. Additionally, CDKN1A was found to be associated with several KEGG pathways, including those involved in acute myeloid leukemia and autoimmune thyroid disease. CDKN1A was confirmed down-regulated in the joint tissues of OA mouse model and OA model cell. Inhibiting the expression of CDKN1A can significantly suppress the differentiation of OA chondrocytes. Conclusion: Our findings highlight the critical role of CDKN1A in promoting cartilage formation in both in vivo and in vitro and suggest its potential as a therapeutic target for OA treatment.

Diagnostic value of anti-Kaiso autoantibody in axial spondyloarthritis.

Objective: Axial spondyloarthritis (axSpA) is a chronic rheumatic disease predominantly characterized by inflammation and progressive structural damage. Patients are often diagnosed very late, which delays the optimal treatment period. Early diagnosis of axSpA, especially non-radiographic axSpA (nr-axSpA), remains a major challenge. This study aimed to investigate the diagnostic value of anti-Kaiso autoantibodies in axSpA and their correlation with clinical disease indicators. Methods: Two pooled serum samples (seven patients with nr-axSpA and seven healthy controls) were profiled using HuProt arrays to investigate the diagnostic value of autoantibodies in nr-axSpA. Levels of anti-Kaiso autoantibodies in patients with axSpA and controls were determined using the Meso Scale Discovery assay system. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic performance of anti-Kaiso autoantibodies in axSpA. Pearson's correlation was used to assess the correlation between anti-Kaiso autoantibodies and clinical parameters. Results: Seven candidate autoantibodies were present in the serum of patients with nr-axSpA. The levels of anti-Kaiso autoantibodies were significantly higher in the nr-axSpA group than in the other groups. It can differentiate nr-axSpA from ankylosing spondylitis (AS), healthy controls, and rheumatoid arthritis. The level of early-stage AS among patients with nr-axSpA decreased when they progressed to the late stage. Of all patients with axSpA, serum anti-Kaiso autoantibody levels were positively correlated with the C-reactive protein level and the Bath Ankylosing Spondylitis Disease Activity Index score and negatively correlated with disease duration. Conclusion: Anti-Kaiso autoantibody may be a valuable diagnostic biomarker for early-stage AS in the nr-axSpA period and may be a potential therapeutic target.

EDIL3 knockdown inhibits retinal angiogenesis through the induction of cell cycle arrest in vitro.

Pathological retinal angiogenesis is one of the most common causes of blindness, with limited treatment options being currently available. Epidermal growth factor (EGF)­like repeat and discoidin I­like domain­containing protein 3 (EDIL3) has been reported to serve an important role in embryonic vasculogenesis and tumor angiogenesis; however, its implication in retinal angiogenesis has yet to be elucidated. The present study aimed to investigate the putative roles of EDIL3 in retinal endothelial cells. RNA interference was used to disrupt the expression of EDIL3 in human retinal endothelial cells (HRECs) in vitro, and the resulting effects were examined. Cell proliferation was assessed using cell counting kit­8 reagent, Cell migration was investigated using a transwell chamber and a tube formation assay was used to study angiogenic capability in vitro. Flow cytometry was used to detect the cell cycle distribution and western blotting was used to study protein expression. The present results demonstrated that silencing EDIL3 expression significantly impaired the proliferative, migratory and tube forming capabilities of HRECs. Furthermore, EDIL3 knockdown was revealed to induce cell cycle arrest at the G1 phase. Western blot analysis suggested that the possible mechanisms underlying the antiproliferative effects of EDIL3 silencing may involve the inhibition of EGF receptor-mediated pathways, and the suppression of cyclin D1 and cyclin E1 expression in HRECs. In conclusion, the findings of the present study suggested that EDIL3 may be implicated in retinal angiogenesis, and may have potential as a novel therapeutic target for the treatment of pathological angiogenesis.