Circ_0104652 Promotes the Proliferation and Migration of ox-LDL-Stimulated Vascular Smooth Muscle Cells via Stabilizing ADAMTS7 and HMGB1.

BACKGROUND: Atherosclerosis (AS) stands as the primary contributor to cardiovascular disease, a pervasive global health concern. Extensive research has underscored the pivotal role of circular RNAs (circRNAs) in cardiovascular disease development. However, the specific functions of numerous circRNAs in AS remain poorly understood. METHODS: Quantitative real-time PCR analysis revealed a significant upregulation of circ_0104652 in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs). Loss-of-function experiments were subsequently employed to assess the impact of circ_0104652 on ox-LDL-induced VSMCs. RESULTS: Silencing circ_0104652 was found to impede the proliferation and migration while promoting the apoptosis of ox-LDL-stimulated VSMCs. Mechanistic assays unveiled that circ_0104652 stabilized ADAM metallopeptidase with thrombospondin type 1 motif 7 (ADAMTS7) and high mobility group box 1 (HMGB1) by recruiting eukaryotic translation initiation factor 4A3 (EIF4A3) protein. Rescue assays further confirmed that circ_0104652 exerted its influence on ox-LDL-induced VSMC proliferation through modulation of ADAMTS7 and HMGB1. CONCLUSIONS: This study elucidates the role of the circ_0104652/EIF4A3/ADAMTS7/HMGB1 axis in ox-LDL-stimulated VSMCs, providing valuable insights into the intricate mechanisms involved.

BAY-9835: Discovery of the First Orally Bioavailable ADAMTS7 Inhibitor.

The matrix metalloprotease ADAMTS7 has been identified by multiple genome-wide association studies as being involved in the development of coronary artery disease. Subsequent research revealed the proteolytic function of the enzyme to be relevant for atherogenesis and restenosis after vessel injury. Based on a publicly known dual ADAMTS4/ADAMTS5 inhibitor, we have in silico designed an ADAMTS7 inhibitor of the catalytic domain, which served as a starting point for an optimization campaign. Initially our inhibitors suffered from low selectivity vs MMP12. An X-ray cocrystal structure inspired us to exploit amino acid differences in the binding site of MMP12 and ADAMTS7 to improve selectivity. Further optimization composed of employing 5-membered heteroaromatic groups as hydantoin substituents to become more potent on ADAMTS7. Finally, fine-tuning of DMPK properties yielded BAY-9835, the first orally bioavailable ADAMTS7 inhibitor. Further optimization to improve selectivity vs ADAMTS12 seems possible, and a respective starting point could be identified.

ADAMTS7 Attenuates House Dust Mite-Induced Airway Inflammation and Th2 Immune Responses.

PURPOSE: ADAMTS7 is a secreted metalloproteinase enzyme and proteoglycan associated with the early progression of coronary artery disease. However, there is limited information regarding the role of ADAMTS7 in lung adaptive immunity and inflammation. Thus, we sought to assess whether ADAMTS7 expression in the lung modulates house dust mite (HDM)-induced airway inflammation and Th2 immune response. METHODS: The role of ADAMTS7 in HDM-induced airway disease was assessed in ADAMTS7-deficient (ADAMTS7-/-) mice and compared with the wild-type control mice by flow cytometry, ELISA, and histopathology. Furthermore, the antigen priming capability of dendritic cells (DC) was determined ex vivo by employing coculture with CD4+ OT-II cells. RESULTS: ADAMTS7-/- mice develop an augmented eosinophilic airway inflammation, mucous cell metaplasia, and increased Th2 immune response to inhaled HDM. In addition, allergen uptake by lung DC and migration to draining mediastinal lymph node were significantly increased in ADAMTS7-/- mice, which shows an enhanced capacity to mount allergen-specific T-cell proliferation and effector Th2 cytokine productions. We propose that the mechanism by which ADAMTS7 negatively regulates DC function involves attenuated antigen uptake and presentation capabilities, which reduces allergic sensitization and Th2 immune responses in the lung. CONCLUSION: In aggregate, we provide compelling evidence that ADAMTS7 plays a pivotal role in allergic airway disease and Th2 immunity and would be an attractive target for asthma.

Development of a fluorogenic ADAMTS-7 substrate.

The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC50 values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-ß-binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.

Coronary Disease Association With ADAMTS7 Is Due to Protease Activity.

[Figure: see text].

Age-associated changes in the transcriptomes of non-cultured adipose-derived stem cells from young and old mice assessed via single-cell transcriptome analysis.

Adipose-derived stem cells (ASCs) exhibit self-renewal and pluripotency. The differentiation potency of ASCs has been reported to deteriorate with aging; however, relevant studies used ASCs that were isolated and subcultured several times. It is still unclear whether subcultured ASCs accurately reflect the in vivo state. To address this question, we used freshly isolated stromal vascular fractions (SVFs) and performed comprehensive single-cell transcriptome analysis. In this study, we identified three cell populations as putative ASC candidates in SVFs and three novel ASC-related genes: Adamts7, Snai2, and Tgfbr1, that are reported to be negative regulators of cell differentiation. Moreover, we identified age-associated high gene expression levels of Adamts7, Egfr, and Igfbp4 in the earliest differentiation stage of ASCs. These results suggest that aging may make it impossible to maintain the stringency of the regulation of the expression of some genes related to ASC differentiation.

ADAMTS7 degrades Comp to fuel BMP2-dependent osteogenic differentiation and ameliorate oncogenic potential in osteosarcomas.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, with a high metastatic potential. Despite dramatic changes in OS treatments over the past decades, their efficiency still remains limited, with severe complications and adverse side effects. Key mechanisms underlining tumorigenesis, metastasis and chemotherapy resistance are currently lacking, in turn hindering any progress with respect to developing effective and safe therapeutic strategies against OS. Recently, ADAMTS7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was shown to be involved in osteogenic differentiation-related pathological processes. ADAMTS7 promotes vascular calcification via disturbing the balance between osteogenic bone morphogenetic protein (BMP)2 (regulating osteogenic differentiation and bone formation during development) and its natural inhibitor cartilage oligomeric matrix protein (Comp). Hence, in the present study, we aimed to investigate the role of ADAMTS7 in the pathological process of OS. We first revealed that ADAMTS7 was decreased in OS tissues. Lower expression of ADAMTS7 was correlated with poor histological differentiation and an advanced clinical stage of OS. Through loss- and gain-function analysis, we further revealed that ADAMTS7 attenuated cell proliferation, migration and invasion, at the same time as promoting the expression of osteogenic differentiation markers in two OS cell lines: MG63 and SAOS2. Moreover, Comp was responsible for the effects of ADAMTS7 on OS pathogenesis by reinforcing cell osteogenic differentiation mediated by BMP2 in vitro. In conclusion, ADAMTS7-mediated degradation of Comp may provide a potential therapeutic target for the treatment of OS.

Hypomethylation of DNA promoter upregulates ADAMTS7 and contributes to HTR-8/SVneo and JEG-3 cells abnormalities in pre-eclampsia.

INTRODUCTION: Accumulating evidences have suggested a crucial role of epigenetics in the initiation and progression of pre-eclampsia (PE). Here, we studied the expression of the metalloproteinase ADAMTS7 and the methylation level of its promoter in PE placentas and investigated ADAMTS7 role in the pathogenesis of PE. METHODS: We first explored ADAMTS7 expression in PE and normal placentas by reverse transcription quantitative PCR (RT-qPCR), western blot, and immunohistochemistry. Methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) were performed to evaluate the methylation status of ADAMTS7 promoter. Treatment with 5'-Aza was used to induce demethylation and thereby to explore the direct relationship between promoter methylation and ADAMTS7 expression. CCK8 assay, colony formation assay, and trans-well assay were conducted to assess the viability, migration, and invasion of HTR-8/SVneo and JEG-3 cells. RESULTS: Our results showed that ADAMTS7 expression was upregulated in PE placentas. Methylation analysis revealed a hypomethylated status of ADAMTS7 promoter regions in PE placenta tissues. Besides, demethylation induced by 5'-Aza directly restored ADAMTS7 expression in trophoblast cells. Finally, overexpression of ADAMTS7 inhibited viability, migration, and invasion of HTR-8/SVneo and JEG-3 cells, while silence of ADAMTS7 by RNA interference reciprocally facilitated cell viability, migration and invasion in vitro. DISCUSSION: Upregulation of ADAMTS7 by promoter hypomethylation in placenta might contribute to the etiology of PE via suppressing cell functions of trophoblasts.

ADATMS-7 regulates the focal adhesion kinase signaling and promotes invasiveness of trophoblasts in early pregnancy.

INTRODUCTION: ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be associated with cell migration and invasion. However, its function on trophoblasts remains unknown. In this study, we are aimed to investigate the role of ADAMTS-7 on trophoblasts in human first trimester gestation. METHODS: The expression of ADAMTS-7 in trophoblasts and HTR8/SVneo cells is examined by immunohistochemistry and quantitative real-time PCR. BrdU incorporation and Annexin V/PI staining are utilized to measure the effect of ADAMTS-7 on the proliferation and apoptosis of HTR8/SVneo cells, respectively. In addition, we detect the role of ADAMTS-7 on the invasion ability of HTR8/SVneo cells using matrigel invasion assays. The activation of focal adhesion kinase (FAK) and integrinß1 induced by ADAMTS-7 were determined by Western blot. RESULTS: ADAMTS-7 and its substrate cartilage oligomeric matrix protein (COMP) were expressed in both primary human trophoblasts and human trophoblast cell lines. TGF-ß1 induced a continuous and significant decrease of ADAMTS-7. Inversely, IL-1ß up-regulated the ADAMTS-7 level in a dosage dependent manner. In addition, knockdown of ADAMTS-7 inhibited the growth and invasion of HTR8/SVneo cells. To the contrary, ADAMTS-7 overexpression promoted the growth and invasion of HTR8/SVneo cells. ADAMTS-7 knockdown led to a decreased level of FAK Tyr-397 phosphorylation. DISCUSSION: Our results suggest that ADAMTS-7 may regulate trophoblasts invasion through focal adhesion kinase (FAK) signaling.

ADAMTS7: Recombinant Protein Expression and Purification.

ADAMTS7 is a secreted protease that is predominantly expressed in tissues of the cardiovascular system and tendon. Although recent evidence suggests that it may play a role in the etiology of coronary artery disease, its physiological function and substrates are unknown. The enzyme undergoes extensive posttranslational modifications, including chondroitin sulfate attachment, N and O-linked glycosylation, and a two-step activation process. For the benefit of scientists who study the function of ADAMTS7 and its role in disease, this chapter provides an introduction to the chemical and functional properties of the various ADAMTS7 domains, as well as a protocol for the recombinant expression and purification of ADAMTS7.

Genetic variants of ADAMTS7 confer risk for ischaemic stroke in the Chinese population.

Large-scale genome-wide association analyses show an association between ADAMTS7 variations and coronary risk. However, the link between ADAMTS7 variability and ischaemic stroke (IS) has yet to be determined. This study evaluated ADAMTS7 variants with respect to the risk of IS. Genetic association analyses were performed in two independent case-control cohorts with 1279 patients with IS and 1268 age-matched healthy controls. Four variant genotypes of the ADAMTS7 gene were identified using the Multiplex SNaPshot assay. The rs3825807, rs11634042, and rs7173743 variants of ADAMTS7 were related to lower IS risk in both initial and replication cohort. The G-T-T-C and G-T-C-C haplotypes are significantly less prevalent in the IS group than in the control group. Further stratification according to IS subtypes indicated that carriers with the variant alleles of the rs3825807, rs11634042 and rs7173743 variants of ADAMTS7conferred a lower risk of developing large-artery atherosclerosis stroke subtype. Also, the mutated rs3825807 G allele, as well as the mutated rs11634042 T allele of ADAMTS7, are linked to a significant reduction of ADAMTS7 in patients with IS. Our findings confirm the role of ADAMTS7 in the pathophysiology of IS, with potentially significant implications for the prevention, treatment, and development of novel therapies for IS.

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast-derived ADAMTS-7 and -12.

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS-7 and -12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK-Runx2 axis and Wnt/ß-catenin signalling in ADAMTS-12 and ADAMTS-7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL-1ß or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS-12 expression in OA-SF, also reducing Fn-fs-induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS-7 and COMP degradation in OA-SF as well. In addition, Wnt7B expression was induced by IL-1ß and by itself, also increasing ADAMTS-7. Our results could contribute to the development of disease-modifying OA drugs targeting ADAMTS-7 and -12 for the prevention of extracellular matrix components degradation like COMP.

Upregulation of ADAMTS­7 and downregulation of COMP are associated with spontaneous abortion.

A disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS­7) has been revealed to serve an important role in inflammation­associated diseases. However, the role of ADAMTS­7 in spontaneous abortion (SA) remains unclear. In the present study, human and mouse decidual tissues were used to detect the expression of ADAMTS­7 and cartilage oligomeric matrix protein (COMP) in mice with lipopolysaccharide (LPS)­induced abortion (10 mice/group), and in SA humans and the corresponding control group (21 participants in the SA group and 15 participants in the control group). The results revealed that ADAMTS­7 expression was upregulated and that COMP expression was downregulated in the mouse decidual tissue of the LPS­induced abortion group, when compared with that of the normal control group. The results were further confirmed by western blot analysis and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis, which revealed increased ADAMTS­7 and decreased COMP expression at the protein and mRNA levels in mice treated with LPS. Additionally, the expression of ADAMTS­7 was negatively correlated with the expression of COMP in mice, with a correlation coefficient of ­0.936 (P<0.001). In addition, the expression of ADAMTS­7 and COMP exhibited was similar in the decidual tissue of SA patients when compared with the levels observed in the tissues of the normal control participants, as demonstrated by increased ADAMTS­7 expression and decreased COMP expression. Western blotting and RT­qPCR analysis revealed that ADAMTS­7 was increased and COMP was decreased in the decidual tissue of SA subjects. The correlation analysis of ADAMTS­7 and COMP in human decidual tissue also revealed a similar result, with a correlation coefficient of ­0.836 (P<0.001). The results of the present study demonstrated that ADAMTS­7 was upregulated and COMP was downregulated in the decidual tissues of humans and mice with SA, and a negative correlation was identified between the expression levels of ADAMTS­7 and COMP, thereby providing novel evidence for a better understanding of the pathogenesis of SA, which may lead to improvements in the clinical pregnancy outcomes of these individuals.

The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate, tendon-specific protective mechanism against heterotopic ossification.

Heterotopic ossification (HO) is a significant clinical problem with incompletely resolved mechanisms. Here, the secreted metalloproteinases ADAMTS7 and ADAMTS12 are shown to comprise a unique proteoglycan class that protects against a tendency toward HO in mouse hindlimb tendons, menisci, and ligaments. Adamts7 and Adamts12 mRNAs were sparsely expressed in murine forelimbs but strongly coexpressed in hindlimb tendons, skeletal muscle, ligaments, and meniscal fibrocartilage. Adamts7-/- Adamts12-/- mice, but not corresponding single-gene mutants, which demonstrated compensatory upregulation of the intact homolog mRNA, developed progressive HO in these tissues after 4 months of age. Adamts7-/- Adamts12-/- tendons had abnormal collagen fibrils, accompanied by reduced levels of the small leucine-rich proteoglycans (SLRPs) biglycan, fibromodulin, and decorin, which regulate collagen fibrillogenesis. Bgn-/0 Fmod-/- mice are known to have a strikingly similar hindlimb HO to that of Adamts7-/- Adamts12-/- mice, implicating fibromodulin and biglycan reduction as a likely mechanism underlying HO in Adamts7-/- Adamts12-/- mice. Interestingly, degenerated human biceps tendons had reduced ADAMTS7 mRNA compared with healthy biceps tendons, which expressed both ADAMTS7 and ADAMTS12. These results suggest that ADAMTS7 and ADAMTS12 drive an innate pathway protective against hindlimb HO in mice and may be essential for human tendon health.

Expression of ADAMTS-7 in myocardial dystrophy associated with white muscle disease in lambs.

The aim of the present study was to investigate the role of ADAMTS-7 gene in the pathogenesis of myocardial dystrophy associated with white muscle disease (WMD) in lambs. A total of 217 cardiac tissue samples from lambs with WMD were used in the study. Histopathological sections of the samples were stained with hematoxylin-eosin (HE) and examined using Western-blot, real-time PCR (RT-PCR) and immunohistochemistry for ADAMTS-7 gene expression, and the findings were statistically evaluated. Histopathological examinations revealed fibrosis associated with hyalinization, necrosis and granular calcifications in cardiomyocytes. Western blot and RT-PCR showed a statistically significant upregulation of ADAMTS-7 (p<0.05) (p<0.05). Immunohistochemical analyses showed that immunopositive cell numbers significantly high for ADAMTS-7 (p<0.05). The study has revealed that ADAMTS-7 gene is significantly expressed in myocardial dystrophy associated with WMD in addition to its role in the pathogenesis of this disease.

Genetic variants rs1994016 and rs3825807 in ADAMTS7 affect its mRNA expression in atherosclerotic occlusive peripheral arterial disease.

AIM: Peripheral artery disease (PAD) is a vascular disease affecting peripheral circulation. Recently, genome-wide association studies revealed a relationship between single nucleotide polymorphisms (SNPs) in ADAMTS7 (a disintegrin and metalloprotease with thrombospondin motif 7) and atherosclerosis. In this study, we aimed to determine ADAMTS7 expression in peripheral blood mononuclear cells (PBMCs) and the frequency of ADAMTS7 rs1994016 and rs3825807 polymorphisms in a sample of Turkish patients with PAD, and to evaluate the association of matrix metalloproteinase (MMP) levels with PAD development. METHODS: In this case-control study, ADAMTS7mRNA and protein expression was determined using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, respectively, and rs1994016 and rs3825807 variants in ADAMTS7 were determined by real-time PCR in 115 PAD patients and 116 healthy controls. Plasma levels of nine MMPs were determined using a multiplex immunoassay system. RESULTS: ADAMTS7mRNA levels were significantly higher in PAD patients than in controls (t=-2.75, P=.007). There was no significant difference in the frequencies of rs1994016 and rs3825807 between PAD patients and controls (P>.05). In PAD patients, ADAMTS7mRNA levels were significantly increased for the CC genotype of rs1994016 (t=-2.31, P=.026) and TT genotype of rs3825807 (t=-2.23, P=.032). Furthermore, plasma levels of MMP-1, MMP-3, MMP-7, MMP-10, MMP-12, and MMP-13 were significantly higher in PAD patients than in controls (P<.05). CONCLUSION: This is the first report of the relationship between PAD and ADAMTS7 expression and the effects of the rs1994016 and rs3825807 variants on PAD development. ADAMTS7 may be associated with PAD development.

Upregulation of ADAMTS­7 and downregulation of COMP are associated with aortic aneurysm.

Aortic aneurysm (AA) remains a fatal condition with high rates of morbidity and mortality, and the associated underlying mechanism influencing its pathology remains to be elucidated. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­7 has previously been demonstrated to be involved in the pathogenesis of vascular atherosclerosis via degradation of cartilage oligomeric matrix protein (COMP). The ADAMTS­7/COMP pathway may therefore act as a potential therapeutic target for vascular disorders. To the best of the author's knowledge, the present study aimed to investigate for the first time, the expression of ADAMTS­7 and COMP in human AA. Human aortic aneurysm samples were collected from patients with AA (n=24), and ascending aorta control samples were harvested from dilated cardiomyopathy patients who underwent heart transplantation (n=18). Expression levels of ADAMTS­7 and matrix metalloproteinase­9 were significantly increased in the AA group, as detected by immunohistochemistry (P<0.05). The COMP protein level was markedly decreased in the AA group when compared with the control group, as demonstrated via immunohistochemistry and western blot analysis (P<0.05). The findings suggest that upregulation of ADAMTS­7 and downregulation of COMP are associated with induction of human AA. ADAMTS­7/COMP pathway may provide therefore act as a potential therapeutic target in human AA for efficient, optimal treatment interventions in the future.

ADAMTS-7 is associated with a high-risk plaque phenotype in human atherosclerosis.

Several large-scale genome-wide association studies have identified single-nucleotide polymorphisms in the genomic region of A Disintegrin And Metalloproteinase with ThromboSpondin type 1 repeats (ADAMTS)-7 and associations to coronary artery disease. Experimental studies have provided evidence for a functional role of ADAMTS-7 in both injury-induced vascular neointima formation and development of atherosclerotic lesions. However, whether ADAMTS-7 is associated with a specific plaque phenotype in humans has not been investigated. Carotid plaques (n = 206) from patients with and without cerebrovascular symptoms were analyzed for expression of ADAMTS-7 by immunohistochemistry and correlated to components associated with plaque vulnerability. Plaques from symptomatic patients showed increased levels of ADAMTS-7 compared with lesions from asymptomatic patients. High levels of ADAMTS-7 correlated with high levels of CD68-staining and lipid content, but with low smooth muscle cell and collagen content, which together are characteristics of a vulnerable plaque phenotype. ADAMTS-7 levels above median were associated with increased risk for postoperative cardiovascular events. Our data show that ADAMTS-7 is associated with a vulnerable plaque phenotype in human carotid lesions. These data support previous observations of a potential proatherogenic role of ADAMTS-7.