Coronary Disease Association With ADAMTS7 Is Due to Protease Activity

Abstract

[Figure: see text].

Keywords: atherosclerosis; coronary artery disease; phenotype; protease; wound healing.

Figure 1:. Full-length mouse ADAMTS7 purification and in vitro TSP1 cleavage.

A, ADAMTS7 protein domains and locations of the glutamate to glutamine (EQ) catalytic mutant and serine to alanine (S3A) substitutions to prevent proteoglycan (PG) attachment. Abbreviated ADAMTS7 domains: signal peptide (SP), disintegrin (Dis), thrombospondin repeats (T), cysteine-rich (Cys-rich), protease and lacunin (PL). B, expression of full-length (FL) mouse and human ADAMTS7 3xFlag proteins in the whole cell lysate and secreted in the media under reducing conditions, detected by western blot using the M2-HRP antibody. High molecular weight proteoglycan (PG) species are marked in orange. A longer exposure of the conditioned media was required to visualize the secreted human ADAMTS7 proteins. C, full-length ADAMTS7 two step purification workflow used to purify WT S3A active enzyme and EQ S3A negative control protein. D, Thrombospondin1 in vitro cleavage by purified full-length mouse ADAMTS7 WT protein (* indicates co-purified auto-cleavage band). Western blots under reducing conditions to resolve proteolytic TSP1 bands generated by active ADAMTS7 WT purified enzyme. Presence of the E373Q catalytic mutation in the purified full-length mouse ADAMTS7 EQ protein ablated the catalytic activity.

Figure 2:. Generation and characterization of the mouse Adamts7 E373Q catalytic mutant allele.

A, Structural context of the ADAMTS7 HExxH to HQxxH catalytic mutation. Crystal structures from ADAMTS4 WT (PDB:4WKI) and ADAMTS4 EQ (PDB: 2RJP) were aligned and annotated in PyMOL to highlight the catalytic residues and visualize the Zinc metal in the active site. The E to Q substitution preserves the tertiary structure of ADAMTS4 and the residues “VAHELGH” in the active site are conserved in ADAMTS7. B, Schematic of the Adamts7 E373Q catalytic mutant allele within exon 7. To generate the mutant allele, c. 1117G->C (p. E373Q) mutation at the Adamts7 catalytic domain and c. 1113C->T (p. A371A) to disrupt the PAM site were induced by CRISPR Homology-Directed Repair (HDR). C, Sanger sequencing of genomic tail DNA PCR and heart mRNA RT-PCR from WT and heterozygous +/E373Q mice. Representative forward reads show no evidence of allelic expression imbalance at the two nucleotide substitutions. D, Adamts7 mRNA expression level in the heart from WT, Adamts7 −/− (KO) and homozygous E373Q/E373Q (EQ/EQ) mice were measured by real time quantitative polymerase chain reaction (qPCR) using 2 TaqMan probe sets (exon 4–5 boundary and exon 23–24 boundary). n=4 per group. E, Adamts12 mRNA expression levels were analyzed by real time qPCR in the heart and aorta harvested from WT, KO and EQ/EQ mice. n=4 per group. A Kruskal-Wallis test with Dunn’s multiple comparison test was applied to D and E.

Figure 3.. Transient expression of ADAMTS7 during atherogenesis.

ADAMTS7 expression reported by the LacZ gene trap tm1b allele in heterozygotes during atherogenesis at 6, 8, 12 and 16 weeks post infection with AAV8-PCSK9. A, Stages examined in the atherogenic model post infection with AAV8-PCSK9. B, Quantitative analysis of β-gal positive cells in the plaque (n=3 to 5 per group, data points represent individual animals). A Kruskal-Wallis test with Dunn’s multiple comparison test was applied, *P<0.05. C-F, Representative photomicrographs of β-gal staining in the aortic sinus at the indicated weeks after AAV8-PCSK9 injection.

Figure 4.. Decreased plaque formation in Adamts7 knockouts and in homozygous catalytic mutants in the AAV-PCSK9 atherogenic mouse model.

At the age of 10 weeks, mice were injected with rAAV8-D377Y-mPCSK9 and challenged a western diet for an additional 16 weeks to stimulate atherosclerosis. The mice were euthanized at 26 weeks of age for evaluation of atherosclerosis in the aortic arch and aortic root. A, B, D and E, Representative photomicrographs of Oil-Red O staining and quantitative analysis of atherosclerotic lesion area in the aortas from littermate controls and Adamts7 KO (D-E, males, n=13 to 15 per group; females, n=17 to 23 per group). G, H, J and K, From a separate +/EQ x +/EQ cross, littermate controls and EQ/EQ homozygotes were evaluated for atherosclerosis (J-K, males, n=10 to 14 per group; females, n=11 to 14 per group). C,F,I and L, Representative sections and quantitative analyses of α-SMA positive smooth muscle cells in the aortic sinus (n=10 to 12 per group). Data points represent individual animals, error bars indicate means ±SEM. Two-way ANOVA with Sidak’s multiple comparison test was applied to D, E, J and K. A two-tailed Student’s test was applied to F, which showed normality and Mann-Whitney test was applied to L, which did not pass a normality test. Normality was tested by Kolmogorov-Smirnov test. *P<0.05.

Figure 5:. Adamts7 dosage and catalytic dependent effects on the ApoE KO atherogenic background.

Mice from heterozygous intercrosses for the tm1b loss of function allele or the E373Q catalytic mutant allele, were challenged with 10 weeks of high fat diet to stimulate atherosclerosis on the ApoE KO background. The mice were euthanized at 20 weeks of age for evaluation of atherosclerosis. A and D, Representative photomicrographs of Oil-Red O staining and quantitative analysis of atherosclerotic lesion area in the aortic arch. B, male WT vs Adamts7 +/− (HET) vs Adamts7 −/− (KO), n=14 to 18 per group; C, female WT vs HET vs KO, n=10 to 19 per group; E, male WT vs +/EQ vs EQ/EQ, n=9 to 14 per group; F, female WT vs +/EQ vs EQ/EQ, n=11 to 17 per each group. Data points represent individual animals, error bars indicate means ±SEM, and two-way ANOVA with Tukey’s test was applied. Normality was tested by Kolmogorov-Smirnov test. *P<0.05, **P<0.01.

Figure 6.. ADAMTS7 catalytic function is responsible for ADAMTS7-mediated VSMC migration.

A-D, Migration of primary VSMCs from Adamts7 −/− (KO) and homozygous EQ/EQ catalytic mutant mice was assessed by wound healing assay. A and C, Representative photomicrographs of the wound at 0 and 12 hours in indicated genotypes of VSMCs are shown. Solid line indicates wound edge. Dotted line indicates migration edge. B and D, The distance of migration is quantified at 4, 8 and 12 hours. n=8 to 9 per group. E, Representative images of western blot in conditioned media and cell lysates from adenovirus-infected primary Adamts7 KO VSMCs. Cell lysates and conditioned media collected from Ad-Luciferase-, Ad-mAdamts7 (mAts7)-WT- and Ad-mAts7-E373Q-infected primary Adamts7 KO VSMCs were subjected to western blot analysis with anti-Flag antibody. F, Migration of primary Adamts7 KO VSMCs infected with Ad-Luciferase, Ad-mAdamts7 WT and Ad-E373Q was assessed by wound healing assay. The mean distance of migration is quantified at 4, 8 and 12 hours. n= 8 to 9 per group. Error bars indicate ±SEM. Two-way RM ANOVA with Sidak’s multiple comparisons test was applied to B, D. *P<0.05. Two-way RM ANOVA with Tukey’s test was applied to F. Normality was tested by Kolmogorov-Smirnov test. **P<0.01, (Ad-Luciferase vs Ad-mAts7 WT), ^#P<0.05 (Ad-mAts7-WT vs Ad-mAts7–373Q).

Figure 7.. ADAMTS7 Ser214 CAD risk variant rs3825807 displays increased secretion and function compared to the Pro214 protective variant or catalytic mutant.

A-C, Representative images and quantifications of western blots from HEK 293 cells transfected with empty vector control, full-length ADAMTS7 risk variant Ser214 or Pro214. A, Full-length ADAMTS7 3xFlag protein was detected by anti-Flag antibodies from conditioned media and cell lysates. B-C, Relative band intensities were quantified from n=4 per group. D-F, Representative images and quantifications of western blot in conditioned media and cell lysates from adenovirus-infected HCA-SMCs infected with Ad-Luciferase (Luc), Ad-ADAMTS7-Ser214 or Ad-ADAMTS7-Pro214. D, Full-length ADAMTS7 3xFlag protein was detected by western blot in conditioned media and cell lysates. NS (non-specific) band migrating faster than ADAMTS7-Flag is present in Luc control. E-F, Relative band intensities were quantified (n=5 per group). Error bars indicate means ±SEM, Mann-Whitney test was applied to B, C, E and F, *P<0.05, **P<0.01. G, Migration of primary Adamts7 KO VSMCs infected with Ad-Luciferase, Ad-ADAMTS7-Ser214 (WT), Ad-ADAMTS7-Pro214 or the Ad-ADAMTS7 catalytic mutant E389Q was assessed by wound healing assay. Representative photomicrographs of the wound at 0 and 12 hours in VSMCs infected with indicated adenovirus are shown. Solid line indicates wound edge. Dotted line indicates migration edge. H, Migration was quantified at 4, 8 and 12 hours (n= 8 to 9 per group, two-way RM ANOVA with Tukey’s test. Normality was tested by Kolmogorov-Smirnov test. *P<0.05, **P<0.01 for LUC vs Ser214).