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

Abstract

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.

Keywords: aorta; atherosclerosis; disintegrins; mice; solubility.

Graphical Abstract

Figure 1. Proteome profiling of atherosclerotic aortas in Apoe-/- (n=3) and Apoe-/-Adamts7-/- (n=4) mice.

A. Volcano plot displaying proteins that were more abundant in Apoe-/-Adamts7- /- mice (right) as compared to Apoe-/- mice (left). Red and blue dots represent proteins which were significantly different between the genotypes after correction for multiple testing. B. The known ADAMTS-7 targets Comp and thrombospondin 1 (Thbs1) were detected at numerically lower levels in aortic tissue of Apoe-/- as compared to Apoe-/-Adamts7-/- mice. C. As putative novel targets of ADAMTS-7, we observed higher levels for the endogenous tissue inhibitors of MMPs (Timp) Timp-2 and, in particular, Timp-1 (Timp2 was detected in only one of three aortas of Apoe-/- mice). Unpaired t-tests with permutation-based FDR correction for multiple hypothesis testing.

Figure 2. Binding of TIMP-1 to ADAMTS-7.

A. ADAMTS-7 constructs. We cloned full-length ADAMTS-7 containing a C-terminal V5-tag (ADAMTS7-V5) and two constructs lacking the C-terminal disintegrin domain and thrombospondin repeats (_ΔTSPrADAMTS7-V5) or the N-terminal catalytic domain (_ΔcatADAMTS7-V5), respectively. The constructs were ectopically expressed in HEK293 cells. B. Co-IP of ADAMTS-7 and TIMP-1-HA. After precipitation of TIMP-1-HA, ADAMTS7-V5 was detectable. C, D. Binding of TIMP-1-HA to different parts of ADAMTS-7. After precipitation of TIMP-1-HA, _ΔTSPrADAMTS7-V5 was detectable (C) while in contrast _ΔcatADAMTS7-V5 was not detectable (D) indicating binding of TIMP-1 to the N-terminal part containing the catalytic domain.

Figure 3. Degradation of TIMP-1 in the presence of ADAMTS-7.

A, B. In vitro degradation assay. TIMP-1-HA was either overexpressed with full-length ADAMTS7-V5 (TS7-V5), the C-terminal part lacking the N-terminal catalytic domain (_ΔcatTS7-V5) or an empty vector (mock). ADAMTS-7 constructs and TIMP-1 were detected by immunoblotting in cell lysates (CL) and the supernatant (SN). Vinculin served as housekeeping control. After co-expression with fulll-length ADAMTS7-V5, less TIMP-1-HA was detectable compared to mock or _ΔcatTS7-V5 co-expression. Four independent experiments. C, D. Fluorescence intensity of TIMP-1-GFP after co-expression with an empty vector (mock), TS7-V5 or _ΔcatTS7-V5 indicates reduced TIMP-1 levels in the presence of full-length ADAMTS-7. Five independent experiments. Scale bar: 100 μm. E, F. GFP-positive cells after overexpression of TIMP-1-GFP with either empty vector (mock), TS7-V5 or _ΔcatTS7-V5. Presence of full-length ADAMTS-7 leads to reduced TIMP-1-GFP fluorescence. Six independent experiments. One-way ANOVA with Sidak’s multiple comparisons test. Data are mean and s.e.m.

Figure 4. Influence of ADAMTS-7 on inhibition of MMP-9 by TIMP-1.

A. Endogenous MMP-2-/MMP-9-activity of hCASMC in the presence (TS7-V5) or absence (mock transfection) of full-length ADAMTS-7. Presence of full-length ADAMTS-7 reduced the inhibitory potential of TIMP-1. Nine (TS7-V5) and ten (mock) independent experiments. Student’s t-test. B-D. Gel zymography with endogenous (pro-) MMP-2 and MMP-9 in the presence of either mock or TS7-V5 (B). Quantification of MMP-9 and MMP-2 bands reveals higher MMP-9 (C) and MMP-2 activity (D) in the presence of full-length ADAMTS-7, respectively. Eight independent experiments. Mann-Whitney (C) and Student’s t-test (D). E. Reduced inhibition of recombinant MMP-9 (rMMP-9) by TIMP-1 in the presence (TS7-V5) as compared to the absence (mock) of full-length ADAMTS-7. Five independent experiments. Student’s t-test. F, G. Secondary to precipitating FLAG-tagged MMP-9 (MMP-9-FLAG) more TIMP-1-HA could be recovered in the presence of full-length ADAMTS-7 (TS7-V5) as compared to _ΔcatTS7-V5. Representative Western Blot. Three independent experiments. Student’s t-test. H, I. Picosirius red staining of collagen fibers in aortic root sections of Apoe-/- (n=14) and Apoe-/- Adamts7-/- mice (n=9) which were fed a Western diet. Mice lacking Adamts-7 displayed increased collagen content as compared to Apoe-/- mice. Student’s t-test. Scale bar, 500 μM. Data are mean and s.e.m.

Figure 5. ADAMTS7 expression in human atherosclerotic carotid plaques.

ADAMTS7 mRNA was detected at higher levels in caps of unstable (n=10) as compared to caps of stable plaques (n=10). Mann-Whitney test.

Figure 6. ADAMTS-7-TIMP-1 screening assay.

A. Left: After binding of ADAMTS-7 and TIMP-1 and excitation of cryptate linked to ADAMTS-7, FRET takes place between cryptate and d2. The ratio between emission of d2 and cryptate is used as the readout for interaction between ADAMTS-7 and TIMP-1. Right: 665/620 nm ratios for ADAMTS-7 and TIMP-1 and the negative controls, i.e., TIMP-1 or ADAMTS-7 alone. Four (TS7-FLAG-Crypt.+mock) and six (TS7-FLAG-Crypt.+TIMP-1-HA-d2, mock+TIMP1-HA-d2) independent experiments. One-way ANOVA with Sidak’s multiple comparisons test. B. Competition assay. Left: In the presence of untagged TIMP-1, emission of cryptate at 620 nm wavelength increases and FRET-induced emission of d2 at 665 nm wavelength decreases. Right: 665/620 nm ratios for ADAMTS-7 and TIMP-1 in the presence (400 or 800 ng) or absence (0 ng) of untagged TIMP-1. Seven independent experiments. One-way ANOVA with post-test for linear trend.