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bioRxiv2023

The novel coronary artery disease risk factor ADAMTS-7 modulates atherosclerotic plaque formation by degradation of TIMP-1

BackgroundThe ADAMTS7 locus was genome-wide significantly associated with coronary artery disease (CAD). Lack of the extracellular matrix (ECM) protease ADAMTS-7 was shown to reduce atherosclerotic plaque formation. ObjectiveTo identify molecular mechanisms and downstream targets of ADAMTS-7 mediating risk of atherosclerosis. MethodsTargets 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, co-immunoprecipitation, and Forster resonance energy transfer (FRET)-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. ResultsIn 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 tissue inhibitor of metalloproteases 1 (Timp-1). In co-immunoprecipitation 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 matrix metalloprotease 9 (MMP-9) As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after 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. In order to facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim to decrease TIMP-1 degradation, we designed a FRET-based assay targeting the ADAMTS-7 catalytic site. ConclusionADAMTS-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 genome-wide significantly associated with CAD. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for reduction of atherosclerosis-related events.

molecular biology↗

bioRxiv2021

MIF-2/D-DT is an atypical atherogenic chemokine that promotes advanced atherosclerosis and hepatic lipogenesis

Atherosclerosis is the underlying cause of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. It is a lipid-triggered chronic inflammatory condition of the arterial vascular wall that is driven by various inflammatory pathways including atherogenic cytokines and chemokines. D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2 (MIF-2), belongs to the MIF protein family, which is best known for its pathogenic role in a variety of inflammatory and immune conditions including CVDs. While MIF is well known as a promoter of atherogenic processes, MIF-2 has not been studied in atherosclerosis. Here, we investigated atherosclerosis in hyperlipidemic Mif-2-/-Apoe-/- mice and studied the role of MIF-2 in various atherogenic assays in vitro. We found that global Mif-2 deficiency as well as its pharmacological blockade by 4-CPPC protected against atherosclerotic lesion formation and vascular inflammation in models of early and advanced atherogenesis. On cellular level, MIF-2 promoted monocyte migration in 2D and 3D and monocyte arrest on aortic endothelial monolayers, promoted B-cell chemotaxis in vitro and B-cell homing in vivo, and increased macrophage foam cell formation. Dose curves and direct comparison in a 3D migration set-up suggest that MIF-2 may be a more potent chemokine than MIF for monocytes and B cells. We identify CXCR4 as a novel receptor for MIF-2. The evidence relies on a CXCR4 inhibitor, CXCR4 internalization experiments, MIF-2/CXCR4 binding studies by yeast-CXCR4 transformants, and fluorescence spectroscopic titrations with a soluble CXCR4 surrogate. Of note, Mif-2-/-Apoe-/- mice exhibited decreased plasma cholesterol and triglyceride levels, lower body weights, smaller livers, and profoundly reduced hepatic lipid accumulation compared to Apoe-/- mice. Mechanistic experiments in Huh-7 hepatocytes suggest that MIF-2 regulates the expression and activation of sterol-regulatory element binding protein-1 and -2 (SREBP-1, SREBP-2) to induce lipogenic downstream genes such as FASN and LDLR, while it attenuated the activation of the SREBP inhibiting AMPK pathway. Studies using receptor Inhibitors showed that SREBP activation and hepatic lipoprotein uptake by MIF-2 is mediated by both CXCR4 and CD74. Lastly and in line with a combined role of MIF-2 in vascular inflammation and hepatic lipid accumulation, MIF-2 was found to be profoundly upregulated in unstable human carotid plaques, underscoring a critical role for MIF-2 in advanced stages of atherosclerosis. Together, these data identify MIF-2 as a novel atherogenic chemokine and CXCR4 ligand that not only promotes lesion formation and vascular inflammation but also strongly affects hepatic lipogenesis in an SREBP-mediated manner, possibly linking atherosclerosis and hepatic steatosis.

immunology↗