关键词： Asporin   Endochondral growth   Growth plate   Mandibular condylar cartilage   TGF-β  

摘要： Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time. © 2017 The Authors

关键词： asporin gene   association study   Mexican Mestizo population   osteoarthritis of the knee  

摘要： BackgroundAsporin is a novel extracellular matrix protein (ECM) with an important role in the development of osteoarthritis (OA), because it has been reported that functional polymorphisms in the aspartic acid repeat (D) of the asporin gene (ASPN) are associated with susceptibility to OA. AimThis study was planned to investigate the association of the ASPN polymorphism with primary OA of the knee in a Mexican population, including several countryside regions. MethodsWe conducted a case-control study in which 93 cases with primary OA of the knee and 118 controls were included. Cases included patients > 40 years of age, with a body mass index (BMI) 27 and a radiologic score for OA of the knee of 2. Controls were subjects > 40 years of age with a radiologic score of < 2. The D repeat polymorphism was genotyped and logistic regression was developed to evaluate risk magnitude. ResultsThe D14 allele was more common in our cases and was associated with an increased risk for developing OA, while the frequencies of the remaining alleles did not exhibit differences. ConclusionOur data suggest that the D14 allele of the ASPN polymorphism could exert an influence on primary OA of the knee etiology in a Mexican Mestizo population.

关键词： Aspartic acid   Osteoarthritis   Knee   Gender   Ethnicity   Meta-analysis   Polymorphism  

摘要： Background: Knee osteoarthritis (KOA) is the most prevalent form of knee joint disease and characterized by the progressive degeneration of articular cartilage. Although pathology of KOA remains unknown, genetic factors are considered to be the major cause. Asporin is a group of biologically active components of extracellular matrix (ECM) in articular cartilage, and asporin gene (ASPN) D-repeat polymorphism was reported to be associated with KOA. Thus, our meta-analysis is aimed at investigation of the association between asporin D-repeat polymorphism and susceptibility of KOA. Methods: We gathered data from MEDLINE, Embase, OVID, and ScienceDirect to search relevant published epidemiological studies through April 2017. Compared with previous studies, our meta-analysis is the first study to investigate the association of ASPN D15, D16, and D17 alleles and KOA susceptibility by ethnic- and sex-stratified subgroup analysis. Results: We found no significant association between D15 allele and susceptibility to KOA (OR = 1.05, 95% CI 0.95-1.17) in overall population. The same results were observed in the analysis of D16 (OR = 1.01, 95% CI 0.80-1.28) and D17 alleles (OR = 1.28, 95% CI 0.91-1.80). The ethnic- and sex-subgroup analyses did not alter the ORs. However, significant association was detected in the sensitivity analysis of D17 in overall population (OR = 1.05, 95% CI 0.95-1.17) and Asian population (OR = 1.78, 95% CI 1.02-3.11, P < 0.05). Conclusion: Our results indicated that D-repeat polymorphism of ASPN may not play a major role in susceptibility of KOA in ethnic- and sex-specific analysis. Because of the limitations of the present meta-analysis, firm conclusions could not be drawn based on the current evidence, and further studies are required to detect genuine role of ASPN.

关键词： Asporin   Nlrp3 inflammasome   CAECs   ROS  

摘要： OBJECTIVE Recent studies have demonstrated that the Nlrp3 inflammasome serve as a central role in the pathogenesis of cardiovascular diseases and endothelial dysfunction occurs in association with several cardiovascular risk *** the demonstrated anti-inflammatory effects of aspirin,the present study was designed to test whether aspirin diminish NLRP3 inflammasome activation and prevent endothelium injury and associated coronary artery damage during *** Mouse carotid arterial endothelial cells(CAECs)were cultured and treated with 0.1-3 mmol·L^(-1) of aspirin in response to LPS(2μg·mL^(-1))*** 24 h,the Nlrp3 inflammasome complexes consist of varied proteins were analyzed by *** and T-AOC in the supernatant was detected by *** reactive oxygen species(ROS)generation for 24 h was observed by DCF *** mice were treated with aspirin(12.5 mg·kg^(-1) per day,62.5 mg·kg^(-1) per day,125 mg·kg^(-1) per day)and dexamethasone(0.0182 mg·kg^(-1)per day)for 7 *** level of IL^(-1)β,IL^(-1)8 protein was detected by *** Immunofluorescence results showed the colocalization of Nlrp3 with ASC or caspase 1decrease in a concentration-dependent ***,the expression of Nlrp3 and caspase 1protein was decreased with the concentration of aspirin,but no changes the expression of ASC ***3 protein levels in CAECs were 0.33-0.8-fold and cle-caspase 1 protein levels in CAECs were 0.48-1-fold compared to those in LPS stimulation when treated with 0.1-3 mmol·L^(-1) aspirin for 24 h(P<0.01).Aspirin significantly antagonized the effect of LPS on NO(1.22-1.91-fold that of LPS stimulation,P<0.01)and T-AOC expression(1.02-1.90-fold that of LPS stimulation,P<0.01).As the different concentration of aspirin treated,the generation of ROS was 0.51-1.10-fold that of LPS stimulation(P<0.01).In vivo data shown the level of IL^(-1)β,IL^(-1)8 protein from serum are in concordance with the level of Nlrp3 inflammasome activation.

关键词： Collagens   Mouse models   Extracellular matrix proteins   Disaccharides   Extracellular matrix   Genetically modified animals   Dermis   Proteoglycans  

摘要： The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs' effects on collagen and extracellular matrix in vivo have been elucidated;one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn(-/-) mouse model and the investigation of the Aspn(-/-) skin phenotype. Functionally, Aspn(-/-) mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn(-/-) mice (not statistically significant). Several matrix genes were upregulated, including collagens (Col1a1, Col1a2, Col3a1), matrix metalloproteinases (Mmp2, Mmp3) and lysyl oxidases (Lox, Loxl2), while lysyl hydroxylase (Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn(-/-) skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn(-/-) skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties.

关键词： Asporin   Pancreatic cancer   Invasion   Migration   EMT  

摘要： Pancreatic cancer is histopathologically characterized by excessive desmoplasia induced by pancreatic stellate cells (PSCs). Asporin, an extracellular matrix (ECM) protein, is highly expressed in cancer associated fibroblasts (CAFs). Asporin expression in PSCs and its roles in PSC-pancreatic cancer cell (PCC) interaction remain unclear. The present study firstly showed that Asporin is highly expressed in activated PSCs and is involved in PSC-mediated invasion and migration of PCCs. Exogenous Asporin interacted with the transmembrane receptor CD44 on PCCs to activate NF-kappa B/p65 and promoted the epithelial mesenchymal transition (EMT) in PCCs. Furthermore, AKT and ERK pathways participated in Asporin/CD44-induced NF-kappa B/p65 activation in pancreatic cancer. Asporin had similar effects on PCCs via an autocrine mechanism. Consistent with our in vitro experiments, we showed that Asporin in peritumoral stroma of pancreatic cancer tissues was associated with poor clinical outcome. In conclusion, this is the first study to show that Asporin promotes EMT, invasion, and migration of PCCs by activating CD44-AIT/ERK-NF-kappa B pathway in paracrine and autocrine manners. Moreover, our results indicate that Asporin may be a prognostic marker and suggest that targeting the tumor microenvironment represents a promising therapeutic strategy in pancreatic cancer. (C) 2017 Elsevier B.V. All rights reserved.

关键词： 椎间盘   转化生长因子β1   细胞外基质   信号传导  

摘要： 目的探讨asporin在椎间盘基质合成和分泌中的分子调控机理，明确其在椎间盘退行性病变中的作用。方法取因严重椎间盘突出症（有典型临床症状、体征及Pfirrmann’s Ⅲ级以上）入院行椎间融合术患者的椎间盘组织标本共8例，其中男6例，女2例，年龄11-28岁，平均（20．25±3．37）岁。利用酶消化法提取椎间盘组织中的原代细胞，在藻酸盐珠中悬浮培养2周后播种于培养皿中。细胞予以不同浓度TGF-β1刺激6、12、18和24h，提取细胞RNA后行实时荧光定量PCR，检测asporin的mRNA表达；细胞经siRNA沉默内源性asporin表达后，以TGF—β1刺激细胞24h，提取细胞RNA行实时荧光定量PCR，检测asporin、Ⅱ型胶原蛋白和蛋白聚糖的mRNA表达；细胞经特异性p38抑制剂或ERK抑制剂处理12h后，以TGF-β1刺激细胞24h，提取细胞蛋白质后行蛋白印迹实验，检测asporin蛋白水平。结果在原代椎间盘细胞实验中，TGF-β1诱导下asporin表达水平显著增高，TGF—β1呈现剂量与时间正依赖关系，刺激24h后不同浓度（5，10，15ng／m1）之间有显著差异，分别为2．754±0．24、3．651±0．319和4．583±0．38（F=24．782，P=0．001）。经敲除内源性asporin表达后，TGF-β1诱导合成的基质蛋白，即蛋白聚糖（t=7．387，P=0.002）和Ⅱ型胶原蛋白（t=4．443，P=0．011）显著增加。利用特异性p38抑制剂阻断p38磷酸化后，TGF-β1对asporin的诱导作用明显地被抑制。结论在椎间盘细胞中asporin与TGF-β1呈现功能反馈环：TGF—β1上调asporin表达，而asporin通过负反馈抑制TGF-β1信号传导通路，进而抑制TGF-β介导的细胞外基质的合成；在椎间盘细胞中TGF-β1通过p38途径上调asporin表达。

摘要： Disc degeneration (DD) is a multifaceted chronic process that alters the structure and function of intervertebral discs. The pathophysiology of degeneration is not completely understood, but the consensus is that changes in genes encoding extracellular matrix (ECM) proteins in the disc are the leading factors contributing to DD. Asporin is an ECM protein that has been shown to be increased in degenerated intervertebral discs, but little is known about how asporin is regulated during DD. In exploring the intricate mechanism, we confirmed that asporin was abundantly increased in patients' degenerated nucleus pulposus. Consistently, the increased asporin expression with degeneration was also proved by rabbit intervertebral disc degeneration (IDD) model. Mechanistically, IL-1 beta upregulated asporin expression by activating the p65 pathway in human nucleus pulposus cells. Furthermore, p65 mediated asporin expression by binding to -41/-31 bp on asporin promoter. Functionally, asporin was the intermediator of IL-1 beta-inhibited aggrecan and collagen. expression and played a negative role in TGF-beta- induced aggrecan and collagen. formation in human nucleus pulposus cells. Therefore, identifying asporin as a negative regulator of aggrecan and collagen. and elucidating its induction mechanisms in human nucleus pulposus cells provides new insight for asporin induction during IDD.

关键词： prostate cancer   tumour stroma   stromal markers   asporin  

摘要： Background: Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes. Methods: We have used Kaplan-Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells. Results: We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan-Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p(53) inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines. Conclusions: Our results suggest