Robert C. Bauer

Genetic Basis of Atherosclerosis: Insights From Mice and Humans

Atherosclerosis is a complex and heritable disease involving multiple cell types and the interactions of many different molecular pathways. The genetic and molecular mechanisms of atherosclerosis have, in part, been elucidated by mouse models; at least 100 different genes have been shown to influence atherosclerosis in mice. Importantly, unbiased genome-wide association studies have recently identified a number of novel loci robustly associated with atherosclerotic coronary artery disease. Here, we review the genetic data elucidated from mouse models of atherosclerosis, as well as significant associations for human coronary artery disease. Furthermore, we discuss in greater detail some of these novel human coronary artery disease loci. The combination of mouse and human genetics has the potential to identify and validate novel genes that influence atherosclerosis, some of which may be candidates for new therapeutic approaches.

Genetic Ablation of Adamts13 Gene Dramatically Accelerates the Formation of Early Atherosclerosis in a Murine Model

Objective—ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves von Willebrand factor, thereby modulating thrombosis and inflammation. Low plasma ADAMTS13 activity is associated with cardiovascular events, including myocardial and cerebral infarction. Here, we investigated the role of ADAMTS13 in the development of early atherosclerosis in a murine model. Methods and Results—Apolipoprotein E–null (ApoE−/−) and Adamts13-null (Adamts13−/−) ApoE−/− mice were fed with a high-fat Western diet for 12 weeks. Atherosclerotic lesions in the aorta and aortic roots were quantified after staining. Leukocyte rolling and adhesion onto cremaster venules after oxidative injury were determined by intravital microscopy. Although plasma cholesterol levels were largely similar in both groups, the extent of atherosclerotic lesions in the aorta en face and in the aortic roots in the Adamts13−/−ApoE−/− mice increased ≈5.5-fold (P=0.0017) and ≈6.1-fold (P=0.0037), respectively. In addition, the ratio of plasma high- to low-molecular-weight von Willebrand factor multimers increased ≈3-fold. The leukocyte rolling velocities were significantly reduced (P<0.001), with an increased number of leukocyte rolling (P=0.0026) and macrophage infiltration into the atherosclerotic lesions in the Adamts13−/−ApoE−/− mice. Conclusion—Our results suggest that ADAMTS13 plays a critical role in modulating the development of early atherosclerosis, likely through the proteolytic cleavage of ultra-large von Willebrand factor multimers, thereby inhibiting platelet deposition and inflammation.

Knockout of Adamts7, a Novel Coronary Artery Disease Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Functional validation of new pathways in lipoprotein metabolism identified by human genetics.

Purpose of review Recent genome-wide association studies (GWAS) have identified approximately 100 genomic loci that are associated with plasma lipid traits, two-thirds of which had never been previously associated with lipoprotein metabolism. Identification of the causal genes and variants, functional validation of these genes and biological pathways, and elucidation of molecular mechanisms is required and poses a daunting task. Recent findings Human genetics have been used to recently ‘validate’ genes, such as LIPG, SCARB1 and ANGPTL3, which were previously implicated in lipoprotein metabolism through classical wet bench approaches. Additionally, many novel genes have been identified as associated with plasma lipid traits by GWAS, though only relatively few have been functionally validated through targeted sequencing and genetic manipulation in cells and animals. These types of studies have defined new roles in lipid metabolism for the novel lipid genes SORT1 and TRIB1. These examples demonstrate the ways in which human genetics can validate candidate genes, as well as provide a novel discovery that requires functional validation at the bench, and point towards a more complete understanding of the molecular physiology of lipoprotein metabolism. Summary This review summarizes recent developments in the use of human genetics to validate candidate genes in lipoprotein metabolism as well as in the functional validation of novel GWAS loci associated with plasma lipid traits.

Tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα

Variants near the gene TRIB1 are significantly associated with several plasma lipid traits, circulating liver enzymes, and the development of coronary artery disease in humans; however, it is not clear how its protein product tribbles-1 regulates lipid metabolism. Here, we evaluated mice harboring a liver-specific deletion of Trib1 (Trib1_LSKO) to elucidate the role of tribbles-1 in mammalian hepatic lipid metabolism. These mice exhibited increased hepatic triglyceride (TG) content, lipogenic gene transcription, and de novo lipogenesis. Microarray analysis revealed altered transcription of genes that are downstream of the transcription factor C/EBPα, and Trib1_LSKO mice had increased hepatic C/EBPα protein. Hepatic overexpression of C/EBPα in WT mice phenocopied Trib1_LSKO livers, and hepatic knockout of Cebpa in Trib1_LSKO mice revealed that C/EBPα is required for the increased lipogenesis. Using ChIP-Seq, we found that Trib1_LSKO mice had increased DNA-bound C/EBPα near lipogenic genes and the Trib1 gene, which itself was transcriptionally upregulated by C/EBPα overexpression. Together, our results reveal that tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα, which in turn transcriptionally upregulates Trib1. These data suggest an important role for C/EBPα in mediating the lipogenic effects of hepatic Trib1 deletion and provide insight into the association between TRIB1 and plasma lipids, and liver traits in humans.

From Loci to Biology: Functional Genomics of Genome-Wide Association for Coronary Disease

Genome-wide association studies have provided a rich collection of ≈ 58 coronary artery disease (CAD) loci that suggest the existence of previously unsuspected new biology relevant to atherosclerosis. However, these studies only identify genomic loci associated with CAD, and many questions remain even after a genomic locus is definitively implicated, including the nature of the causal variant(s) and the causal gene(s), as well as the directionality of effect. There are several tools that can be used for investigation of the functional genomics of these loci, and progress has been made on a limited number of novel CAD loci. New biology regarding atherosclerosis and CAD will be learned through the functional genomics of these loci, and the hope is that at least some of these new pathways relevant to CAD pathogenesis will yield new therapeutic targets for the prevention and treatment of CAD.

Therapeutic Targets of Triglyceride Metabolism as Informed by Human Genetics

Human genetics has contributed to the development of multiple drugs to treat hyperlipidemia and coronary artery disease (CAD), most recently including antibodies targeting PCSK9 to reduce LDL cholesterol. Despite these successes, a large burden of CAD remains. Genetic and epidemiological studies have suggested that circulating triglyceride (TG)-rich lipoproteins (TRLs) are a causal risk factor for CAD, presenting an opportunity for novel therapeutic strategies. We discuss recent unbiased human genetics testing, including genome-wide association studies (GWAS) and whole-genome or -exome sequencing, that have identified the lipoprotein lipase (LPL) and hepatic lipogenesis pathways as important mechanisms in the regulation of circulating TRLs. Further strengthening the causal relationship between TRLs and CAD, findings such as these may provide novel targets for much-needed potential therapeutic interventions.

Knockout of Adamts7, A Novel CAD Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Tribbles-1: a novel regulator of hepatic lipid metabolism in humans.

The protein tribbles-1, encoded by the gene TRIB1, is increasingly recognized as a major regulator of multiple cellular and physiological processes in humans. Recent human genetic studies, as well as molecular biological approaches, have implicated this intriguing protein in the aetiology of multiple human diseases, including myeloid leukaemia, Crohns disease, non-alcoholic fatty liver disease (NAFLD), dyslipidaemia and coronary artery disease (CAD). Genome-wide association studies (GWAS) have repeatedly identified variants at the genomic TRIB1 locus as being significantly associated with multiple plasma lipid traits and cardiovascular disease (CVD) in humans. The involvement of TRIB1 in hepatic lipid metabolism has been validated through viral-mediated hepatic overexpression of the gene in mice; increasing levels of TRIB1 decreased plasma lipids in a dose-dependent manner. Additional studies have implicated TRIB1 in the regulation of hepatic lipogenesis and NAFLD. The exact mechanisms of TRIB1 regulation of both plasma lipids and hepatic lipogenesis remain undetermined, although multiple signalling pathways and transcription factors have been implicated in tribbles-1 function. Recent reports have been aimed at developing TRIB1-based lipid therapeutics. In summary, tribbles-1 is an important modulator of human energy metabolism and metabolic syndromes and worthy of future studies aimed at investigating its potential as a therapeutic target.

Genetic manipulation of the ApoF/Stat2 locus supports an important role for type I interferon signaling in atherosclerosis

Apolipoprotein F (ApoF) is a sialoglycoprotein that is a component of the HDL and LDL fractions of human serum. We sought to test the hypothesis that ApoF plays an important role in atherosclerosis in mice by modulating lipoprotein function. Atherosclerosis was assessed in male low density lipoprotein receptor knockout (Ldlr KO) and ApoF/Ldlr double knockout (DKO) mice fed a Western diet for 16 weeks. ApoF/Ldlr DKO mice showed a 39% reduction in lesional area by en face analysis of aortas (p < 0.05), despite no significant differences in plasma lipid parameters. ApoF KO mice had reduced expression of Interferon alpha (IFNα) responsive genes in liver and spleen, as well as impaired macrophage activation. Interferon alpha induced gene 27 like 2a (Ifi27l2a), Oligoadenylate synthetases 2 and 3 (Oas2 and Oas3) were significantly reduced in the ApoF KO mice relative to wild type controls. These effects were attributable to hypomorphic expression of Stat2 in the ApoF KO mice, a critical gene in the Type I IFN pathway that is situated just 425 base pairs downstream of ApoF. These studies implicate STAT2 as a potentially important player in atherosclerosis, and support the growing evidence that the Type I IFN pathway may contribute to this complex disease.