Coronary Disease Association With ADAMTS7 Is Due to Protease Activity

Abstract

Rationale: Despite contemporary therapy, coronary artery disease (CAD) remains a leading cause of mortality. Genetic variants at ADAMTS7 have been associated with CAD and the loss of ADAMTS7 is protective for atherosclerosis. ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) is a secreted metalloproteinase and complex proteoglycan, yet the mechanism linking ADAMTS7 to CAD risk remains unresolved. Objective: To investigate the role of ADAMTS7 catalytic function in vascular smooth muscle cellular migration and during atherosclerosis. Methods and Results: We established a new purification strategy for full-length mouse ADAMTS7 and demonstrated the loss of activity in the catalytic mutant form of ADAMTS7. To test if the enzymatic activity of ADAMTS7 mediates atherosclerosis, we generated a catalytically inactive mutant mouse allele and compared it to the Adamts7 knockout. Using two models of atherosclerosis, we found that reducing either ADAMTS7 dosage or catalytic function decreased the burden of atherosclerosis. We demonstrate impaired vascular smooth muscle migration in both Adamts7 catalytic mutant and null cells using a lateral migration wound healing assay. Expression of the wild-type allele rescued the migration phenotype in Adamts7 null cells while expression of the catalytic mutant protein did not. We then characterized a human ADAMTS7 coding variant rs3825807 (Ser214Pro) associated with reduced CAD risk. This variant had a hypomorphic effect on ADAMTS7 secretion and migration of vascular smooth muscle cells (VSMC), findings consistent with our mouse studies. Conclusions: We demonstrated that loss of ADAMTS7 catalytic function protects against atherosclerosis via phenotype switch of VSMCs and that the atherosclerosis protective effects could be mediated by a loss-of-function coding variant associated with CAD risk. In aggregate, we provide compelling evidence that dosage of ADAMTS7 and catalytic function are responsible for the atherosclerotic phenotype, suggesting that the catalytic domain would be an attractive therapeutic target for CAD.

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

“…1 B and supplemental Table S5 ). It is notable that the predominant TAILS-identified ADAMTS7 autocleavage site is identical to the mucin domain autocleavage product we previously reported ( 28 ) and serves to validate our approach for identifying ADAMTS7-dependent cleavage sites.

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“…We obtained commercially purified full-length epitope tagged HA-EFEMP1 and combined it with our purified full-length mouse ADAMTS7 S3A WT or ADAMTS7 S3A EQ for 4 h at 37 °C. The engineered ADAMTS7 S3A constructs alter the CS-GAG attachment consensus from “SGSGS” to “AGAGA” to prevent GAG-modification and allow for full-length ADAMTS7 purification as previously described ( 28 ). Mobility of EFEMP1 was detected by Western blot using samples in reducing or nonreducing conditions.…”

“…ADAMTS7 Expression, Cell Culture and Preparation of the Secreted Proteins -Fulllength mouse Adamts7 1-1857 (Uniprot Q68SA9, wild type (WT) or E373Q (EQ) catalytic mutant) with a carboxyl terminal 3xFLAG tag was used to generate custom adenoviruses as previously described (28). Adeno-CMV-Luciferase (Luc) from Vector Biolabs was used as a negative control.…”

“…To validate EFEMP1 cleavage in a binary assay, purified recombinant human HA-tagged EFEMP1/Fibulin-3 (R&D, 8416-FB) provided in PBS was dialyzed into TBS pH 8.0 to prevent precipitation with the CaCl 2 in the assay buffer. Purified mouse Adamts7 WT S3A 3xFlag contained a 250 kDa full-length form and 150 kDa truncated Flag tagged form enriched in later size-exclusion chromatography fractions as previously described ( 28 ). For the mouse ADAMTS7 S3A constructs, the conserved Ser991 CS-GAG attachment site and adjacent Ser989 and Ser993 from the SGSGS motif were mutated to alanine (S989A/S991A/S993A or AGAGA) to facilitate protein purification.…”

“…1). Control groups contained either the expression of a non-specific luciferase or an inactive EQ catalytic mutant protease with a glutamate to glutamine substitution in the HExxH metalloproteinase active site (28). We used three different experimental designs to perform TAILS discovery experiments in human coronary artery smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) (supplemental Fig.…”