Diagnostic value of serum COMP and ADAMTS7 for intervertebral disc degeneration.

OBJECTIVE: Intervertebral disc degeneration (IVDD) is a major cause of morbidity and disability. Our study aimed to investigate the potential of cartilage oligomeric matrix protein (COMP) and ADAMTS7 (A disintegrin and metalloproteinases with thrombospondin motifs 7) as biomarkers for IVDD together with their functional relationship. METHODS: IVD tissues and peripheral blood samples were collected from IVDD rabbit models over 1-4 weeks. Tissues and blood samples were also collected from clinical patients those were stratified into four equal groups according to Pfirrmann IVDD grading (I-V) with baseline data collected for each participant. COMP and ADAMTS7 expression were analyzed and biomarker characteristics were assessed using linear regression and receiver operating curve (ROC) analyses. RESULTS: COMP and ADAMTS7 expression increased in tissues and serum during IVDD progression. Serum COMP (sCOMP) and serum ADAMTS7 (sADAMTS7) levels increased in a time-dependent manner following IVD damage in the rabbit model while significant positive correlations were detected between sCOMP and sADAMTS7 and Pfirrmann grade in human subjects. ROC analysis showed that combining sCOMP and sADAMTS7 assay results produced an improved diagnostic measure for IVDD compared to individual sCOMP or sADAMTS7 tests. In vitro assays conducted on human cell isolates revealed that COMP prevented extracellular matrix degradation and antagonized ADAMTS7 expression although this protective role was uncoupled under microenvironmental conditions mimicking IVDD. CONCLUSIONS: Increases in circulating COMP and ADAMTS7 correlate with IVDD progression and may play regulatory roles. Assays for sCOMP and/or sADAMTS7 levels can discriminate between healthy subjects and IVDD patients, warranting further clinical assessment.

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.

The Association of ADAMTS7 Gene Polymorphisms with the Risk of Coronary Artery Disease Occurrence and Cardiovascular Survival in the Polish Population: A Case-Control and a Prospective Cohort Study.

The aim of this study was to investigate whether the polymorphisms of the ADAMTS7 gene affect the risk of occurrence and mortality due to CAD. The study group included 231 patients diagnosed with CAD and 240 control blood donors. The genotyping of specified polymorphisms, i.e., rs1994016, rs3825807, and rs7173743, was performed using the TaqMan-PCR. We found that the C allele carriers of the rs1994016 and A allele carriers of the rs3825807 polymorphisms increased the risk of CAD, respectively: OR = 1.72, p = 0.036; OR = 1.64, p = 0.04. Moreover, we studied the biological interactions of specified variants, i.e., rs3825807, rs1994016, and rs7173743, and previously approved risk factors of CAD. We demonstrated here that selected polymorphisms of ADAMTS7 increased the risk of CAD altogether with abnormalities of total cholesterol and LDL concentrations in serum. Although survival analyses did not reveal statistical significance, we observed a trend for the AA genotype of the rs3825807 ADAMTS7, which may predispose to death due to CAD in a 5-year follow-up. In conclusion, the ADAMTS7 polymorphisms investigated in this study may increase the risk of occurrence and/or death due to CAD in the Polish population.

ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.

BACKGROUND: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis. METHODS: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. RESULTS: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site. CONCLUSIONS: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.

ADAMTS7: a Novel Therapeutic Target in Atherosclerosis.

PURPOSE OF REVIEW: Genome-wide association studies have repeatedly linked the metalloproteinase ADAMTS7 to coronary artery disease. Here we aim to highlight recent findings surrounding the human genetics of ADAMTS7, novel mouse models that investigate ADAMTS7 function, and potential substrates of ADAMTS7 cleavage. RECENT FINDINGS: Recent genome-wide association studies in coronary artery disease have replicated the GWAS signal for ADAMTS7 and shown that the signal holds true even across different ethnic groups. However, the direction of effect in humans remains unclear. A recent novel mouse model revealed that the proatherogenicity of ADAMTS7 is derived from its catalytic functions, while at the translational level, vaccinating mice against ADAMTS7 reduced atherosclerosis. Finally, in vitro proteomics approaches have identified extracellular matrix proteins as candidate substrates that may be causal for the proatherogenicity of ADAMTS7. ADAMTS7 represents an enticing target for therapeutic intervention. The recent studies highlighted here have replicated prior findings, confirming the genetic link between ADAMTS7 and atherosclerosis, while providing further evidence in mice that ADAMTS7 is a targetable proatherogenic enzyme.

The rs3825807 Polymorphism of ADAMTS7 as a Potential Genetic Marker for Myocardial Infarction in Slovenian Subjects with Type 2 Diabetes Mellitus.

BACKGROUND: A disintegrin and metalloprotease with thrombospondin motif 7 (ADAMTS-7) was reported to play a role in the migration of vascular smooth muscle cells and neointimal formation. The object of the study was to investigate the association between the rs3825807 polymorphism of ADAMTS7 and myocardial infarction among patients with type 2 diabetes mellitus in a Slovenian cohort. METHODS: 1590 Slovenian patients with type 2 diabetes mellitus were enrolled in this retrospective cross-sectional case-control study. In total, 463 had a history of recent myocardial infarction, and 1127 of the subjects in the control group had no clinical signs of coronary artery disease. Genetic analysis of an rs3825807 polymorphism of ADAMTS7 was performed with logistic regression. RESULTS: Patients with the AA genotype had a higher prevalence of myocardial infarction than those in the control group in recessive [odds ratio (OR) 1.647; confidence interval (CI) 1.120-2.407; p = 0.011] and co-dominant (OR 2.153; CI 1.215-3.968; p = 0.011) genetic models. CONCLUSION: We found a statistically significant association between rs3825807 and myocardial infarction in a cohort of Slovenian patients with type 2 diabetes mellitus. We report that the AA genotype might be a genetic risk factor for myocardial infarction.

ADAMTS7-Mediated Complement Factor H Degradation Potentiates Complement Activation to Contributing to Renal Injuries.

BACKGROUND: The dysfunction of complement factor H (CFH), the main soluble complement negative regulator, potentiates various complement-induced renal injuries. However, insights into the underlying mechanism of CFH dysfunction remain limited. In this study, we investigated whether extracellular protease-mediated degradation accounts for CFH dysfunction in complement-mediated renal injuries. METHODS: An unbiased interactome of lupus mice kidneys identified CFH-binding protease. In vitro cleavage assay clarified CFH degradation. Pristane-induced SLE or renal ischemia-reperfusion (I/R) injury models were used in wild-type and ADAMTS7-/- mice. RESULTS: We identified the metalloprotease ADAMTS7 as a CFH-binding protein in lupus kidneys. Moreover, the upregulation of ADAMTS7 correlated with CFH reduction in both lupus mice and patients. Mechanistically, ADAMTS7 is directly bound to CFH complement control protein (CCP) 1-4 domain and degraded CCP 1-7 domain through multiple cleavages. In mice with lupus nephritis or renal I/R injury, ADAMTS7 deficiency alleviated complement activation and related renal pathologies, but without affecting complement-mediated bactericidal activity. Adeno-associated virus-mediated CFH silencing compromised these protective effects of ADAMTS7 knockout against complement-mediated renal injuries in vivo. CONCLUSION: ADAMTS7-mediated CFH degradation potentiates complement activation and related renal injuries. ADAMTS7 would be a promising anticomplement therapeutic target that does not increase bacterial infection risk.

Genome-wide meta-analysis identifies new candidate genes for sickle cell disease nephropathy.

Sickle cell disease nephropathy (SCDN), a common SCD complication, is strongly associated with mortality. Polygenic risk scores calculated from recent transethnic meta-analyses of urinary albumin-to-creatinine ratio and estimated glomerular filtration rate (eGFR) trended toward association with proteinuria and eGFR in SCD but the model fit was poor (R2 < 0.01), suggesting that there are likely unique genetic risk factors for SCDN. Therefore, we performed genome-wide association studies (GWAS) for 2 critical manifestations of SCDN, proteinuria and decreased eGFR, in 2 well-characterized adult SCD cohorts, representing, to the best of our knowledge, the largest SCDN sample to date. Meta-analysis identified 6 genome-wide significant associations (false discovery rate, q ≤ 0.05): 3 for proteinuria (CRYL1, VWF, and ADAMTS7) and 3 for eGFR (LRP1B, linc02288, and FPGT-TNNI3K/TNNI3K). These associations are independent of APOL1 risk and represent novel SCDN loci, many with evidence for regulatory function. Moreover, GWAS SNPs in CRYL1, VWF, ADAMTS7, and linc02288 are associated with gene expression in kidney and pathways important to both renal function and SCD biology, supporting the hypothesis that SCDN pathophysiology is distinct from other forms of kidney disease. Together, these findings provide new targets for functional follow-up that could be tested prospectively and potentially used to identify patients with SCD who are at risk, before onset of kidney dysfunction.

Peptide Vaccine Against ADAMTS-7 Ameliorates Atherosclerosis and Postinjury Neointima Hyperplasia.

BACKGROUND: The metalloprotease ADAMTS-7 (a disintegrin and metalloproteinase with thrombospondin type 1 motif 7) is a novel locus associated with human coronary atherosclerosis. ADAMTS-7 deletion protects against atherosclerosis and vascular restenosis in rodents. METHODS: We designed 3 potential vaccines consisting of distinct B cell epitopic peptides derived from ADAMTS-7 and conjugated with the carrier protein KLH (keyhole limpet hemocyanin) as well as aluminum hydroxide as an adjuvant. Arterial ligation or wire injury was used to induce neointima in mice, whereas ApoE-/- and LDLR-/- (LDLR [low-density lipoprotein receptor]) mice fed a high-fat diet were applied to assess atherosclerosis. In addition, coronary stent implantation was performed on vaccine-immunized Bama miniature pigs, followed by optical coherence tomography to evaluate coronary intimal hyperplasia. RESULTS: A vaccine, ATS7vac, was screened out from 3 candidates to effectively inhibit intimal thickening in murine carotid artery ligation models after vaccination. As well, immunization with ATS7vac alleviated neointima formation in murine wire injury models and mitigated atherosclerotic lesions in both hyperlipidemic ApoE-/- and LDLR-/- mice without lowering lipid levels. Preclinically, ATS7vac markedly impeded intimal hyperplasia in swine stented coronary arteries, but without significant immune-related organ injuries. Mechanistically, ATS7vac vaccination produced specific antibodies against ADAMTS-7, which markedly repressed ADAMTS-7-mediated COMP (cartilage oligomeric matrix protein) and TSP-1 (thrombospondin-1) degradation and subsequently inhibited vascular smooth muscle cell migration but promoted re-endothelialization. CONCLUSIONS: ATS7vac is a novel atherosclerosis vaccine that also alleviates in-stent restenosis. The application of ATS7vac would be a complementary therapeutic avenue to the current lipid-lowering strategy for atherosclerotic disease.

Long non­coding RNA LINC01960­201 hinders decidualization of endometrial stromal cell in endometriosis: Relevance to endometrial receptivity.

Emerging data have indicated that long non­coding RNAs (lncRNA) are associated with the pathogenesis of endometriosis. However, few are associated with endometriosis­associated infertility. In addition, to the best of our knowledge, the role of lncRNAs in decidual formation during the window of implantation with endometriosis has not been reported to date. Based on our previous results, the aim of the present study was to explore the role of lncRNA long intergenic non­protein coding RNA (LINC)01960­201 in in vitro decidualization of endometrial stromal cells in endometriosis during the window of implantation, as well as to explore the biological function of LINC01960­201, and the regulation of a disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS7), hsa­microRNA (miR)­760 and hsa­miR­608 in the decidualization of endometrial stromal cells with endometriosis. Using miRanda, PITA and RNAhybrid, the present study predicted which miRs share the common target gene ADAMTS7 with LINC01960­201 and the existence of regulatory targets. Dual luciferase vectors were constructed to extract the plasmids and measure the relative fluorescence values in order to estimate target regulatory association between LINC01960­201, ADAMTS7 and miRs. Mid­secretory endometrial tissues were collected from women with endometriosis­associated infertility. From these tissues, endometrial stromal cells were extracted and cultured as primary cultures. Medroxyprogesterone acetate (MPA) and 8­Bromoadenosine 3',5'­cyclic monophosphate (8­Br­cAMP) were added to induce in vitro decidualization, and to knockdown LINC01960­201 and transfect a hsa­miR­608 mimic at the same time. Reverse transcription­quantitative PCR and western blotting were conducted to compare the difference in gene expression between the experimental and negative control groups. No regulatory sites between LINC01960­201 and hsa­miR­608 were identified; however, potential regulatory sites were detected between hsa­miR­608 and the 3'­untranslated region (UTR) of ADAMTS7, whereas neither the 3'­UTR of LINC01960­201 or the 3'­UTR of ADAMTS7 had any regulatory targets with hsa­miR­760. During the process of decidualization of endometrial stromal cells by in vitro induction, the expression of hsa­miR­608 in the knockdown group was significantly higher compared with that of the negative control group after LINC01960­201­knockdown, and the expression of ADAMTS7 in the transfection group was significantly lower compared with that of the negative control group after hsa­miR­608 mimic transfection. In conclusion, it was hypothesized that LINC01960­201 played a notable regulatory role in the decidualization of endometrial stromal cells in women with endometriosis during the window of implantation, and its abnormal expression may lead to the decline of endometrial receptivity and recurrent abortions.

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.

Genome-wide pleiotropy analysis of coronary artery disease and pneumonia identifies shared immune pathways.

Coronary artery disease (CAD) remains the leading cause of death despite scientific advances. Elucidating shared CAD/pneumonia pathways may reveal novel insights regarding CAD pathways. We performed genome-wide pleiotropy analyses of CAD and pneumonia, examined the causal effects of the expression of genes near independently replicated SNPs and interacting genes with CAD and pneumonia, and tested interactions between disruptive coding mutations of each pleiotropic gene and smoking status on CAD and pneumonia risks. Identified pleiotropic SNPs were annotated to ADAMTS7 and IL6R. Increased ADAMTS7 expression across tissues consistently showed decreased risk for CAD and increased risk for pneumonia; increased IL6R expression showed increased risk for CAD and decreased risk for pneumonia. We similarly observed opposing CAD/pneumonia effects for NLRP3. Reduced ADAMTS7 expression conferred a reduced CAD risk without increased pneumonia risk only among never-smokers. Genetic immune-inflammatory axes of CAD linked to respiratory infections implicate ADAMTS7 and IL6R, and related genes.

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease.

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

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.

Investigating ADAMTS7 and ADAMTS12 levels in prediabetic and Type 2 diabetic patients.

Background: This study aims to investigate the role of ADAMTS7 and ADAMTS12 on atherosclerosis and inflammation in prediabetic and diabetic patients. Patients & methods: Serum ADAMTS7 and ADAMTS12 levels were compared with the atherosclerotic and inflammatory markers in diabetic (n = 65, female 30.9%, mean age = 53 years), prediabetic (n = 55, female 36.6%, mean age = 49 years) and control groups (n = 55, females 32.5%, mean age = 49 years). Serum ADAMTS levels were determined by a human enzyme-liked immunoassay. Results: In terms of ADAMTS7, there was no significant difference between diabetic, prediabetic and control groups (50.93, 44.34, 59.07, respectively; p > 0.05). ADAMTS12 is lower in diabetics (p < 0.05), whereas it is similar in prediabetics and controls (14.53, 20.76, 25.05, respectively; p > 0.05). ADAMTS7 and ADAMTS12 levels did not differ in diabetic nephropathy, retinopathy and neuropathy (p > 0.05). Conclusion: While ADAMTS12 was significantly lower in diabetics and prediabetics, ADAMTS7 and ADAMTS12 were not related to diabetic complications (nephropathy, retinopathy and neuropathy).

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.