ADAMTS7, a target in atherosclerosis, cooperates with its homolog ADAMTS12 to protect against myxomatous valve degeneration

Figures

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  Fig. 1. Adamts7 and Adamts12 are coordinately expressed in aortic and mitral valves and show compensatory upregulation in gene knockouts. A. Adamts7 and Adamts12 expression is revealed in embryonic and 3-week-old mouse hearts by LacZ (blue) and RNA in situ hybridization (red), respectively. Both genes are expressed in aortic and mitral valves during development and maturation. n=4 biological replicates and two technical replicates for each time point and stain. B. qRT-PCR analysis of Adamts7 and Adamts12 mRNAs in wild type, Adamts7−/− and Adamts12−/− mice shows compensatory upregulation of each in the absence of the other. N = 3 biological replicates and three technical replicates. One way ANOVA was used for statistical analysis. Error bars represent SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. E, embryonic day; PND, postnatal day. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Adamts7−/−;Adamts12−/− aortic valves are enlarged and disorganized postnatally. A–B. Hematoxylin and eosin stained two-month (2 M) and four-month-old (4 M) aortic valves from Adamts7−/−;Adamts12−/− aortic valves (A) have increased thickness and area with reduced cell number, as compared to control (B). Note Adamts7−/− aortic valves have increased area and decreased cell density as compared to control (B). n = 11 wild type, 7 Adamts7−/−, 7 Adamts12−/−, 7 Adamts7−/−;Adamts12−/−. Duplicate analysis was performed. Error bars represent SEM. One-way ANOVA followed by Tukey test was used for statistical analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. C. Synchrotron imaging reveals thickened aortic valve leaflets as compared to control. The images are color coded to illustrate the relative valve thickness. Coronary arteries are shown in red at the top of the aortic root. D. Four-month-old histological sections of Adamts7−/−;Adamts12−/− aortic valves show disorganized collagen distribution (yellow staining, left panels; Picrosirius red stain (red), right panels) and proteoglycan staining (Movat Pentachrome stain (blue, left panels); alcian blue staining (blue), right panels) and loss of…

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  Fig. 3. Assessment of systolic flow through pulse wave-Doppler analysis. A. Representative transaortic valve Doppler images (top panels) and color M-mode ultrasound (middle panels). Note regurgitant (red colored) flow in Adamts7−/−;Adamts12−/− mice as compared to control (middle panels). Representative two-dimensional M-mode ultrasound images of control and Adamts7−/−;Adamts12−/− aortic valves (bottom panels) illustrating thicker Adamts7−/−;Adamts12−/− valves (white arrows). B. Aortic peak velocity and pressure gradient (top panels) are increased in Adamts7−/−;Adamts12−/− mice. Assessment of cardiac systolic function in the left ventricle of wild type and Adamts7−/−;Adamts12−/− mice shows reduced percent ejection fraction and percent fractional shortening in Adamts7−/−;Adamts12−/− mice (bottom panels) as compared to control. n = 10 wild type, 9 Adamts7−/−;Adamts12−/−. Each measurement was performed in duplicate. Error bars represent SEM. An unpaired Student t-test was used for statistical analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

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  Fig. 4. Enlargement of Adamts7−/−;Adamts12−/− aortic valves is initiated during development. A. E18.5 Adamts7−/−;Adamts12−/− aortic valves are enlarged, as assessed by hematoxylin and eosin-stained sections, have disorganized collagen (picrosirius red), increased proteoglycans (alcian blue and Movat pentachrome). The dashed line demarcates collagen. Arrowheads show the elastic fibers in wild type valves and lack of elastin in Adamts7−/−;Adamts12−/− aortic valves. The analysis used n=4 of each genotype and two technical replicates for each stain. B. Adamts7−/−;Adamts12−/− aortic valves have increased thickness and length and reduced cell number, as compared to control. n = 7 wild type, 10 Adamts7−/−;Adamts12−/−. Each stain was done on two or more sections from each heart. Error bars represent SEM. An unpaired Student t-test was used for statistical analysis. *p ≤ 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 5. Adamts7−/−;Adamts12−/− aortic valves have increased proteoglycan and glycosaminoglycan staining, with reduced versican proteolysis. Newborn and four-month-old histological sections of Adamts7−/−;Adamts12−/− aortic valves show increased versican staining and reduced staining intensity of cleaved versican (DPEAAE) as compared to littermate controls. HABP and CLP accumulated in Adamts7−/−;Adamts12−/− aortic valves with ectopic COMP in newborn and adult and ectopic aggrecan in adult aortic valves. n=4 mice of each genotype, with two technical replicates for each genotype and time point. Error bars represent SEM. A paired Student t-test was used for statistical analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

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  Fig. 6. Identification of ADAMTS7 and ADAMTS12 substrates. A. Conditioned medium collected from Adamts7−/−;Adamts12−/− mouse embryonic fibroblasts transfected with ADAMTS7, ADAMTS12 or empty vector and TAILS workflow showing sample generation and subsequent handling. B. Cleavage sites of periostin by ADAMTS7 and ADAMTS12. C. Annotated MS2 spectrum examples of ADAMTS7 and ADAMTS12-generated dimethyl peptides, showing b-(N-terminus preserved) and y-type (C-terminus preserved) ions generated by amide bond cleavage during collisional-induced dissociation.

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  Fig. 7. Verified targets of ADAMTS7 and ADAMTS12. A. Increased FBN1, FN1, and periostin staining in 4-month-old Adamts7−/−;Adamts12−/− aortic valves. B. pSMAD2 staining, indicative of aberrant TGFβ staining, is increased in newborn and adult Adamts7−/−;Adamts12−/− aortic valve leaflets. n=4 mice of each genotype, with two technical replicates for each genotype and time point. Error bars represent SEM. A paired Student t-test was used for statistical analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.