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

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.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation.

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr-/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr-/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.

cGMP Signaling in Cardiovascular Diseases: Linking Genotype and Phenotype.

Cyclic guanosine 3',5'-monophosphate (cGMP) is the key second messenger molecule in nitric oxide signaling. Its rapid generation and fate, but also its role in mediating acute cellular functions has been extensively studied. In the past years, genetic studies suggested an important role for cGMP in affecting the risk of chronic cardiovascular diseases, for example, coronary artery disease and myocardial infarction. Here, we review the role of cGMP in atherosclerosis and other cardiovascular diseases and discuss recent genetic findings and identified mechanisms. Finally, we highlight open questions and promising research topics.

Functional Characterization of the GUCY1A3 Coronary Artery Disease Risk Locus.

BACKGROUND: A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the α1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. METHODS: Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses. RESULTS: Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3 expression. Ex vivo-studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxide donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta. CONCLUSIONS: Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.

Genetics links between transforming growth factor ß pathway and coronary disease.

Recent genome-wide association studies broadened our view on the complex genetic roots of coronary artery disease (CAD). The subsequent challenge is now to elucidate the mechanisms linking genetic variants with disease risk. Here, we focus on the transforming growth factor beta (TGF-ß) pathway and review genetic as well as comprehensive annotation and systems biology analyses to provide further links between TGF-ß, the SMAD family and CAD risk. Indeed, it has long been shown that the TGF-ß signaling pathway plays a role in the pathophysiology of endothelial and vascular smooth muscle cells. Nevertheless, the precise mechanisms leading to CAD are still vague, due to the vast number of mediated effects and multi-functional and context-dependent biology of the TGF-ß signaling. While previous reviews addressed these issues, less attention was paid to the genetic underpinnings in the signaling process, which are of parallel importance and essence regarding the transduction and functioning of the whole pathway. Here, we provide an overview of how the so far identified genetic risk loci are involved in the regulation of the canonical TGF-ß - SMAD signaling pathway, which might give further mechanistic insights into CAD risk.

Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the ß1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the ß1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants.

Role of sGC-dependent NO signalling and myocardial infarction risk.

The NO/cGMP pathway plays an important role in many physiological functions and pathophysiological conditions. In the last few years, several genetic and functional studies pointed to an underestimated role of this pathway in the development of atherosclerosis. Indeed, several genetic variants of key enzymes modulating the generation of NO and cGMP have been strongly associated with coronary artery disease and myocardial infarction risk. In this review, we aim to place the genomic findings on components of the NO/cGMP pathway, namely endothelial nitric oxide synthase, soluble guanylyl cyclase and phosphodiesterase 5A, in context of preventive and therapeutic strategies for treating atherosclerosis and its sequelae.

Molecular variants of soluble guanylyl cyclase affecting cardiovascular risk.

Soluble guanylyl cyclase (sGC) is the physiological receptor for nitric oxide (NO) and NO-releasing drugs, and is a key enzyme in several cardiovascular signaling pathways. Its activation induces the synthesis of the second messenger cGMP. cGMP regulates the activity of various downstream proteins, including cGMP-dependent protein kinase G, cGMP-dependent phosphodiesterases and cyclic nucleotide gated ion channels leading to vascular relaxation, inhibition of platelet aggregation, and modified neurotransmission. Diminished sGC function contributes to a number of disorders, including cardiovascular diseases. Knowledge of its regulation is a prerequisite for understanding the pathophysiology of deficient sGC signaling. In this review we consolidate the available information on sGC signaling, including the molecular biology and genetics of sGC transcription, translation and function, including the effect of rare variants, and present possible new targets for the development of personalized medicine in vascular diseases.