ADAMTS2 promotes radial migration by activating TGF-ß signaling in the developing neocortex.

The mammalian neocortex is formed by sequential radial migration of newborn excitatory neurons. Migrating neurons undergo a multipolar-to-bipolar transition at the subplate (SP) layer, where extracellular matrix (ECM) components are abundantly expressed. Here, we investigate the role of the ECM at the SP layer. We show that TGF-ß signaling-related ECM proteins, and their downstream effector, p-smad2/3, are selectively expressed in the SP layer. We also find that migrating neurons express a disintegrin and metalloproteinase with thrombospondin motif 2 (ADAMTS2), an ECM metalloproteinase, just below the SP layer. Knockdown and knockout of Adamts2 suppresses the multipolar-to-bipolar transition of migrating neurons and disturbs radial migration. Time-lapse luminescence imaging of TGF-ß signaling indicates that ADAMTS2 activates this signaling pathway in migrating neurons during the multipolar-to-bipolar transition at the SP layer. Overexpression of TGF-ß2 in migrating neurons partially rescues migration defects in ADAMTS2 knockout mice. Our data suggest that ADAMTS2 secreted by the migrating multipolar neurons activates TGF-ß signaling by ECM remodeling of the SP layer, which might drive the multipolar to bipolar transition.

Hypoxic regulation of ADAMTS-2 and -3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells.

ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.

Identification of TGF-ß-related genes in cardiac hypertrophy and heart failure based on single cell RNA sequencing.

BACKGROUND: Heart failure (HF) remains a huge medical burden worldwide. Pathological cardiac hypertrophy is one of the most significant phenotypes of HF. Several studies have reported that the TGF-ß pathway plays a double-sided role in HF. Therefore, TGF-ß-related genes (TRGs) may be potential therapeutic targets for cardiac hypertrophy and HF. However, the roles of TRGs in HF at the single-cell level remain unclear. METHOD: In this study, to analyze the expression pattern of TRGs during the progress of cardiac hypertrophy and HF, we used three public single-cell RNA sequencing datasets for HF (GSE161470, GSE145154, and GSE161153), one HF transcriptome data (GSE57338), and one hypertrophic cardiomyopathy transcriptome data (GSE141910). Weighted gene co-expression network analysis (WGCNA), functional enrichment analysis and machine learning algorithms were used to filter hub genes. Transverse aortic constriction mice model, CCK-8, wound healing assay, quantitative real-time PCR and western blotting were used to validate bioinformatics results. RESULTS: We observed that cardiac fibroblasts (CFs) and endothelial cells showed high TGF-ß activity during the progress of HF. Three modules (royalblue, brown4, and darkturquoize) were identified to be significantly associated with TRGs in HF. Six hub genes (TANC2, ADAMTS2, DYNLL1, MRC2, EGR1, and OTUD1) showed anomaly trend in cardiac hypertrophy. We further validated the regulation of the TGF-ß-MYC-ADAMTS2 axis on CFs activation in vitro. CONCLUSIONS: This study identified six hub genes (TANC2, ADAMTS2, DYNLL1, MRC2, EGR1, and OTUD1) by integrating scRNA and transcriptome data. These six hub genes might be therapeutic targets for cardiac hypertrophy and HF.

Effect of pegbelfermin on NASH and fibrosis-related biomarkers and correlation with histological response in the FALCON 1 trial.

Background & Aims: FALCON 1 was a phase IIb study of pegbelfermin in patients with non-alcoholic steatohepatitis (NASH) and stage 3 fibrosis. This FALCON 1 post hoc analysis aimed to further assess the effect of pegbelfermin on NASH-related biomarkers, correlations between histological assessments and non-invasive biomarkers, and concordance between the week 24 histologically assessed primary endpoint response and biomarkers. Methods: Blood-based composite fibrosis scores, blood-based biomarkers, and imaging biomarkers were evaluated for patients with available data from FALCON 1 at baseline through week 24. SomaSignal tests assessed protein signatures of NASH steatosis, inflammation, ballooning, and fibrosis in blood. Linear mixed-effect models were fit for each biomarker. Correlations and concordance were assessed between blood-based biomarkers, imaging, and histological metrics. Results: At week 24, pegbelfermin significantly improved blood-based composite fibrosis scores (ELF, FIB-4, APRI), fibrogenesis biomarkers (PRO-C3 and PC3X), adiponectin, CK-18, hepatic fat fraction measured by MRI-proton density fat fraction, and all four SomaSignal NASH component tests. Correlation analyses between histological and non-invasive measures identified four main categories: steatosis/metabolism, tissue injury, fibrosis, and biopsy-based metrics. Concordant and discordant effects of pegbelfermin on the primary endpoint vs. biomarker responses were observed; the most clear and concordant effects were on measures of liver steatosis and metabolism. A significant association between hepatic fat measured histologically and by imaging was observed in pegbelfermin arms. Conclusions: Pegbelfermin improved NASH-related biomarkers most consistently through improvement of liver steatosis, though biomarkers of tissue injury/inflammation and fibrosis were also improved. Concordance analysis shows that non-invasive assessments of NASH support and exceed the improvements detected by liver biopsy, suggesting that greater consideration should be given to the totality of available data when evaluating the efficacy of NASH therapeutics. Clinical trial number: Post hoc analysis of NCT03486899. Impact and implications: FALCON 1 was a study of pegbelfermin vs. placebo in patients with non-alcoholic steatohepatitis (NASH) without cirrhosis; in this study, patients who responded to pegbelfermin treatment were identified through examination of liver fibrosis in tissue samples collected through biopsy. In the current analysis, non-invasive blood- and imaging-based measures of fibrosis, liver fat, and liver injury were used to determine pegbelfermin treatment response to see how they compared with the biopsy-based results. We found that many of the non-invasive tests, particularly those that measured liver fat, identified patients who responded to pegbelfermin treatment, consistent with the liver biopsy findings. These results suggest that there may be additional value in using data from non-invasive tests, along with liver biopsy, to evaluate how well patients with NASH respond to treatment.

Overexpression of ADAMTS-2 in tumor cells and stroma is predictive of poor clinical prognosis in gastric cancer.

ADAMTS-2 is a member of the ADAMTS family and is a procollagen N-proteinase. The objective of our research is to explore the prognostic significance of ADAMTS-2 in gastric carcinoma. A total of 655 samples with full clinicopathological data were investigated in this study. Tissue microarray immunohistochemistry analysis was used to analyze the relationship between clinicopathological characteristics and ADAMTS-2 expression. Oncomine and Kaplan-Meier plotters were performed for the relationship analysis between prognosis and ADAMTS-2 expression in patients with gastric cancer. Compared with that of normal tissues, the ADAMTS-2 protein expression was remarkably higher in gastric cancer cells and fibroblast cells. The results of univariate analysis indicated that the expression of ADAMTS-2 in tumor cells and fibroblast cells, Laurén classification, TNM grade, and carcinoembryonic antigen level in gastric cancer were all correlated with overall survival. The results of multivariate analysis indicated that the high expression of ADAMTS-2 in gastric cancer cells and fibroblast cells both were independent prognostic factors. Therefore, ADAMTS-2 may be a potential biomarker for assessing the prognosis of gastric carcinoma.

The role of ADAMTS-2, collagen type-1, TIMP-3 and papilin levels of uterosacral and cardinal ligaments in the etiopathogenesis of pelvic organ prolapse among women without stress urinary incontinence.

OBJECTIVE(S): To investigate the potential role of 'a disintegrin-like and metalloproteinase with thrombospondin type motifs-2 (ADAMTS-2), collagen type-1, tissue inhibitor of metalloproteinases-3 (TIMP-3) and papilin' levels in the uterosacral ligament (USL) and cardinal ligament (CL) of the uterus on the etiopathogenesis of pelvic organ prolapse (POP) among postmenopausal women without stress urinary incontinence (SUI). STUDY DESIGN: A total of 45 postmenopausal women, 22 diagnosed as POP stage III-IV and 23 age- and body mass index (BMI)-matched controls referred for hysterectomy due to POP or benign gynecological disease, respectively, were recruited prospectively for our study. The biopsies of the USL and CL were obtained during hysterectomy. ADAMTS-2, collagen type-1, TIMP-3 and papilin levels were determined by enzyme-linked immunosorbent assay (ELISA) method after tissue homogenization. We excluded patients who smoked or presented with SUI. RESULTS: There were no differences in terms of demographic features including age, BMI, obesity, duration of menopause, gravidity, parity, delivery modes and family history for POP between the POP and non-POP groups. Significant differences in the levels of ADAMTS-2, collagen type-1, TIMP-3 and papilin of USL were noted among the groups. Females with POP had lower levels of ADAMTS-2, collagen type-1, TIMP-3 and papilin in the USL compared to non-POP females. All investigated markers in the CL were also decreased in the POP group, but this relationship was not statistically significant. When age, duration of menopause, gravidity, parity and obesity were taken as covariates, only the USL papilin levels were negatively predictive for the development of POP. CONCLUSION(S): ADAMTS-2, collagen type-1, TIMP-3 and papilin levels of the USL play essential roles in the etiopathogenesis of POP among postmenopausal women without SUI. Moreover, significantly decreased USL papilin levels in females with POP suggest the importance of the USL and the impact of papilin on the development of POP.

Critical Role of ADAMTS2 (A Disintegrin and Metalloproteinase With Thrombospondin Motifs 2) in Cardiac Hypertrophy Induced by Pressure Overload.

ADAMTS2 (A Disintegrin and Metalloproteinase With Thrombospondin Motifs 2) is recognized as a metalloproteinase that promotes the cleavage of amino propeptides of types I, II, III, and V procollagens. However, the role of ADAMTS2 in the heart has not yet been defined. Herein, we observed the upregulated expression of ADAMTS2 in failing human hearts and hypertrophic murine hearts. Mice lacking ADAMTS2 display exacerbated cardiac hypertrophy on pressure overload-induced hypertrophic response, whereas mice with cardiac-specific overexpression of ADAMTS2 display alleviation of this detrimental phenotype. Consistent with these results, in vitro loss or gain of function experiments in neonatal rat cardiomyocytes confirmed that ADAMTS2 negatively regulates cardiomyocyte hypertrophy in response to Ang II. Mechanistically, blockage of the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)-dependent signaling pathway with specific inhibitors both in vivo and in vitro could rescue the aggravated hypertrophic response to the loss of ADAMTS2. Collectively, we propose that ADAMTS2 regulates the hypertrophic response through inhibiting the activation of the PI3K/AKT-dependent signaling pathway. Because ADAMTS2 is an extracellular protein, it could be effectively manipulated using pharmacological means to modulate cardiac hypertrophy.

Induction of human ADAMTS-2 gene expression by IL-1α is mediated by a multiple crosstalk of MEK/JNK and PI3K pathways in osteoblast like cells.

Up-regulation of ADAMTS genes with proinflammatory cytokines is important for some pathological conditions such as osteoarthritis (OA) that is a disease based on ECM degradation in cartilage. IL-1α is a proinflammatory cytokine and important both to normal and pathophysiologic conditions in cartilage and bone. Effects of some proinflammatory cytokines such as TNF-α and IL-1ß on the some members of ADAMTS family have been investigated in some chondrocyte tissues or cell lines. However the effect of the IL-1α on the expression of ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cell lines, remains unclear. Therefore, the aim of this study is to investigate the effect of IL-1α on ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cells, Saos-2 and MG-63. The present study, for the first time, demonstrated that IL-1α increases ADAMTS-2 and ADAMTS-3 gene expressions in both Saos-2 and MG-63 cells. Having correlation to mRNA induction, the upregulation of ADAMTS-2,-3 protein levels by IL-1α stimulation is also observed. The inhibition studies showed that this upregulation occurred at the level of transcription, and there was no effect of IL-1α on ADAMTS-2 mRNA half-life in Saos-2 cells. Transactivation potential of IL-1α on ADAMTS-2 promoter was investigated by transient transfection assay. Specifically, IL-1α strongly increased -658/+112 and -530/+112 ADAMTS-2 promoter constructs. Further, we analyzed signaling pathways involved in ADAMTS-2 induction. Pathway inhibition studies revealed that this upregulation depends on the activation of MEK, JNK and PI3K pathways. These findings suggested that IL-1α is a strong positive regulator of ADAMTS-2 and ADAMTS-3 expression. These findings would provide novel insight into the pathophysiology of OA.

Effect of insulin on the mRNA expression of procollagen N-proteinases in chondrosarcoma OUMS-27 cells.

Chondrosarcoma is one of the most common bone tumors, and at present, there is no non-invasive treatment option for this cancer. The chondrosarcoma OUMS-27 cell line produces proteoglycan and type II, IX, and XI collagens, which constitutes cartilage tissue. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases are a group of secreted proteases, which include the procollagen N-proteinases ADAMTS-2, -3 and -14. These procollagen N-proteinases perform a role in the processing of procollagens to collagen and the maturation of type I collagen. The present study aimed to improve the understanding of the causes of metastasis, local invasion and resistance to chemo- and radiotherapy in chondrosarcoma, as well as the effect of insulin on cancer cells. The present study was designed to reveal the effects of insulin on procollagen N-proteinases in chondrosarcoma OUMS-27 cells. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) alone or in DMEM containing 10 µg/ml insulin. The medium was changed every other day for 11 days. The cells were harvested on days 1, 3, 7 and 11, and total RNA isolation was performed immediately following harvesting. The expression levels of ADAMTS2, ADAMTS3 and ADAMTS14 mRNA were estimated by reverse transcription-quantitative polymerase chain reaction using appropriate primers. ADAMTS2 mRNA expression was found to be decreased on day 7 (P=0.028) and increased at day 11 compared with the control group (P=0.016). The increase in mRNA concentration at day 11 was significantly different compared to the concentrations on days 3 (P=0.047) and 7 (P=0.008). The expression of ADAMTS3 mRNA decreased immediately subsequent to insulin induction on day 1 compared with the control group (P=0.008). The most evident decrease in mRNA concentration was seen at day 7 subsequent to insulin induction (P=0.008). The present results demonstrated that ADAMTS2 and ADAMTS3 may perform a role in the invasion and metastasis of tumors, and may also possess proteolytic activity that results in the breakdown of the extracellular matrix (ECM). Insulin itself can modulate the biosynthesis of ECM macromolecules that are altered in diabetes through various pathways.

Gene expression profiling of craniofacial fibrous dysplasia reveals ADAMTS2 overexpression as a potential marker.

Fibrous dysplasia (FD) as an abnormal bone growth is one of the common fibro-osseous leasions (FOL) in oral and maxillofacial region, however, its etiology still remains unclear. Here, we performed gene expression profiling of FD using microarray analysis to explore the key molecule events in FD development, and develop potential diagnostic markers or therapeutic targets for FD. We found that 1,881 genes exhibited differential expression with more than two-fold changes in FD compared to normal bone tissues, including 1,200 upregulated genes and 681 downregulated genes. Pathway analysis indicated that obviously activated pathways are Ribosome and ECM-receptor interaction pathways; downregulated pathways are "Hepatitis C" and "cancer" signaling pathways. We further validated the expression of ADAMTS2, one of most differentiated expressed genes, by Immunohistochemistry (IHC) in 40 of FD cases. Results showed that ADAMTS2 was significantly overexpressed in FD tissues, but rarely expressed in normal bone tissues, suggesting that ADAMTS2 could be a potential biomarker for FD. Thus, this study uncovered differentially expressed candidate genes in FD, which provides pilot data for understanding FD pathogenesis, and developing novel biomarkers for diagnosis and targeting of FD.

Sexual differences of imprinted genes' expression levels.

In mammals, genomic imprinting has evolved as a dosage-controlling mechanism for a subset of genes that play critical roles in their unusual reproduction scheme involving viviparity and placentation. As such, many imprinted genes are highly expressed in sex-specific reproductive organs. In the current study, we sought to test whether imprinted genes are differentially expressed between the two sexes. According to the results, the expression levels of the following genes differ between the two sexes of mice: Peg3, Zim1, Igf2, H19 and Zac1. The expression levels of these imprinted genes are usually greater in males than in females. This bias is most obvious in the developing brains of 14.5-dpc embryos, but also detected in the brains of postnatal-stage mice. However, this sexual bias is not obvious in 10.5-dpc embryos, a developmental stage before the sexual differentiation. Thus, the sexual bias observed in the imprinted genes is most likely attributable by gonadal hormones rather than by sex chromosome complement. Overall, the results indicate that several imprinted genes are sexually different in terms of their expression levels, and further suggest that the transcriptional regulation of these imprinted genes may be influenced by unknown mechanisms associated with sexual differentiation.

Ehlers-Danlos Syndrome Type VIIC: A Mexican Case Report.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective tissue disorders whose primary clinical features include soft and extensible skin, articular hypermobility and tissue fragility. EDS type VIIC or 'human dermatosparaxis' is an autosomal recessive disease characterized by severe skin fragility and sagging redundant skin (major criteria) with a soft, doughy texture, easy bruising, premature rupture of fetal membranes and large hernias (minor criteria). Dermatosparaxis (meaning 'tearing of skin'), which has been described in several non-human species, is a disorder of the connective tissue resulting from a deficiency of the enzyme that cleaves the registration peptide off the N-terminal end of collagen after it has been secreted from fibroblasts. We describe a Mexican case from consanguineous parents with all the phenotypical characteristics previously described, plus skeletal abnormalities.