Juyong Brian Kim

Effect of 9p21.3 Coronary Artery Disease Locus Neighboring Genes on Atherosclerosis in Mice

Background— The human 9p21.3 chromosome locus has been shown to be an independent risk factor for atherosclerosis in multiple large-scale genome-wide association studies, but the underlying mechanism remains unknown. We set out to investigate the potential role of the 9p21.3 locus neighboring genes, including Mtap, the 2 isoforms of Cdkn2a, p16Ink4a and p19Arf, and Cdkn2b, in atherosclerosis using knockout mice models. Methods and Results— Gene-targeted mice for neighboring genes, including Mtap, Cdkn2a, p19Arf, and Cdkn2b, were each bred to mice carrying the human APO*E3 Leiden transgene that sensitizes the mice for atherosclerotic lesions through elevated plasma cholesterol. We found that the mice heterozygous for Mtap developed larger lesions compared with wild-type mice (49623±21650 versus 18899±9604 &mgr;m2 per section [mean±SD]; P=0.01), with morphology similar to that of wild-type mice. The Mtap heterozygous mice demonstrated changes in metabolic and methylation profiles and CD4+ cell counts. The Cdkn2a knockout mice had smaller lesions compared with wild-type and heterozygous mice, and there were no significant differences in lesion size in p19Arf and Cdkn2b mutants compared with wild type. We observed extensive, tissue-specific compensatory regulation of the Cdkn2a and Cdkn2b genes among the various knockout mice, making the effects on atherosclerosis difficult to interpret. Conclusions— Mtap plays a protective role against atherosclerosis, whereas Cdkn2a appears to be modestly proatherogenic. However, no relation was found between the 9p21 genotype and the transcription of 9p21 neighboring genes in primary human aortic vascular cells in vitro. There is extensive compensatory regulation in the highly conserved 9p21 orthologous region in mice.The 9p21.3 region of the genome has been identified as the locus with strongest association to coronary artery disease (CAD) and myocardial infarction (MI) in multiple independent large scale genome-wide association studies (GWAS).1-3 The locus is within a 58kb region that is devoid of protein coding genes, suggestive of a regulatory function (Figure 1). Interestingly, the neighboring genes in the region include well-known tumor suppressor genes, including CDKN2A and CDKN2B.4-6 The CDKN2A locus encodes a cyclin-dependent protein kinase (CDK) inhibitory protein (CKI), p16INK4A, and a p53-regulatory protein, p19ARF. The CDKN2B gene encodes another CKI, p15INK4B. Another gene in the region is methylthioadenosine phosphorylase (MTAP), which encodes a ubiquitously expressed metabolic enzyme S-methyl-5′-thioadenosine phosphorylase7 that processes the polyamine biosynthesis byproduct in the methionine salvage pathway. Loss or inactivation of MTAP has frequently been observed in a number of different human tumors, and it has been shown to have a tumor suppressive role in a mice model.8 Figure 1 The landscape of the 9p21.3 region Multiple studies demonstrated a potential role for cell cycle regulatory mechanisms in atherosclerosis progression. Previously, the master tumor suppressor gene p53 has been implicated in the development of atherosclerosis in apolipoprotein E (ApoE)-null mice9, 10, affecting both cell proliferation and apoptosis within the atheroma. Another tumor suppressor gene, p21Waf1, was also shown to increase the atheroma size in ApoE-null mice11, whereas the tumor suppressor p27Kip1 was shown to protect against atherosclerosis.12 Correlations of the 9p21 locus SNP genotype to differential expression of the neighboring genes have been observed in several studies with inconsistent findings.13-15 A knockout (KO) mouse model involving the entire region orthologous to the 9p21.3 CAD locus showed significant decreases in the expressed levels of Cdkn2a and Cdkn2b, and increased proliferation of primary smooth muscle cells (SMC) and mouse embryonic fibroblasts (MEF), although an effect on atherosclerosis in vivo was not demonstrated.16 Mice deficient in the p19Arf gene were found to have increased atherosclerotic lesions in an ApoE null background with significant attenuation of apoptosis in lesions as well as in cultured primary macrophages and vascular smooth muscle cells.17 However, to date no observation regarding atherosclerotic phenotype has been made involving the other neighboring genes. We set out to survey the 9p21.3 orthologous region using knockout mice models to systematically address the role of the neighboring protein-coding genes in atherosclerosis. We chose the APOE*3 Leiden sensitizing model because it is dominant, simplifying the construction of the models, and also because it exhibits relatively modest elevations of cholesterol, more realistically modeling the human disease than other widely used models.

Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

Coronary artery disease (CAD) is the leading cause of mortality and morbidity, driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies have identified >150 loci associated with CAD and myocardial infarction susceptibility in humans. A majority of these variants reside in non-coding regions and are co-inherited with hundreds of candidate regulatory variants, presenting a challenge to elucidate their functions. Herein, we use integrative genomic, epigenomic and transcriptomic profiling of perturbed human coronary artery smooth muscle cells and tissues to begin to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps, we prioritize 64 candidate variants and perform allele-specific binding and expression analyses at seven top candidate loci: 9p21.3, SMAD3, PDGFD, IL6R, BMP1, CCDC97/TGFB1 and LMOD1. We validate our findings in expression quantitative trait loci cohorts, which together reveal new links between CAD associations and regulatory function in the appropriate disease context.

Paraoxonase-2 Modulates Stress Response of Endothelial Cells to Oxidized Phospholipids and a Bacterial Quorum–Sensing Molecule

Objective—Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and these bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAECs) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression. Methods and Results—Using expression profiling and network modeling, we identified the unfolded protein response (UPR), cell cycle genes, and the mitogen-activated protein kinase signaling pathway to be heavily involved in the HAEC response to 3OC12-HSL. The network also showed striking similarities to a network created based on HAEC response to Ox-PAPC, a major component of minimally modified low-density lipoprotein. HAECs in which PON2 was silenced by small interfering RNA showed increased proinflammatory response and UPR when treated with 3OC12-HSL or Ox-PAPC. Conclusion—3OC12-HSL and Ox-PAPC influence similar inflammatory and UPR pathways. Quorum sensing molecules, such as 3OC12-HSL, contribute to the proatherogenic effects of chronic infection. The antiatherogenic effects of PON2 include destruction of quorum sensing molecules.

Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci

To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

Genetics and Genomics of Coronary Artery Disease.

Coronary artery disease (or coronary heart disease), is the leading cause of mortality in many of the developing as well as the developed countries of the world. Cholesterol-enriched plaques in the heart’s blood vessels combined with inflammation lead to the lesion expansion, narrowing of blood vessels, reduced blood flow, and may subsequently cause lesion rupture and a heart attack. Even though several environmental risk factors have been established, such as high LDL-cholesterol, diabetes, and high blood pressure, the underlying genetic composition may substantially modify the disease risk; hence, genome composition and gene-environment interactions may be critical for disease progression. Ongoing scientific efforts have seen substantial advancements related to the fields of genetics and genomics, with the major breakthroughs yet to come. As genomics is the most rapidly advancing field in the life sciences, it is important to present a comprehensive overview of current efforts. Here, we present a summary of various genetic and genomics assays and approaches applied to coronary artery disease research.

TCF21 and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells

Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor TCF21 as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of TCF21 expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of AHR, its heterodimerization partner ARNT, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including IL1A, MMP1, and CYP1A1. TCF21 was shown to bind in AHR, ARNT and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD.

Anti-Inflammatory Strategies for Plaque Stabilization after Acute Coronary Syndromes

Despite dramatic advances in standard of care, the risk of recurrent myocardial infarction early after an acute coronary syndrome (ACS) remains high. This period of elevated risk after a cardiovascular event is associated with an acute inflammatory response. While post-ACS inflammation correlates with the risk for recurrent events and is likely to play a causal role in this period, the precise pathophysiologic mechanisms have been unclear. Recent studies have proposed that the cardiac event itself activates the sympathetic nervous system to directly mobilize hematopoietic stem cells to differentiate into inflammatory monocytes, acutely infiltrate plaque, and lead to recurrent plaque rupture. Here, we summarize the existing and emerging evidence implicating post-ACS activation of systemic inflammation in the progression of atherosclerosis, and identify possible targets for therapeutic intervention. We highlight experimental therapies and ongoing clinical studies that will validate these targets.

Dyslipidemia and cardiovascular diseases.

Introduction Despite the progress made in the management of hypercholesterolemia with statins, events are reduced by only 30% [1 ]. Protecting the other 70% requires, additionally, interventions including adjunctive use of anti-inflammatory or immunologic treatments. The results of the statin trials seriously underestimate the ultimate potential of cholesterol-lowering therapy. Experts suggest continuing and more extensive use of lipid-improving regimens and intervention at an earlier stage [1 ].

GDF-15 (Growth Differentiation Factor 15) Is Associated With Lack of Ventricular Recovery and Mortality After Transcatheter Aortic Valve Replacement

Background— Recent data suggest that circulating biomarkers may predict outcome in patients undergoing transcatheter aortic valve replacement (TAVR). We examined the association between inflammatory, myocardial, and renal biomarkers and their role in ventricular recovery and outcome after TAVR. Methods and Results— A total of 112 subjects undergoing TAVR were included in the prospective registry. Plasma levels of B-type natriuretic peptide, hs-TnI (high-sensitivity troponin I), CRP (C-reactive protein), GDF-15 (growth differentiation factor 15), GAL-3 (galectin-3), and Cys-C (cystatin-C) were assessed before TAVR and in 100 sex-matched healthy controls. Among echocardiographic parameters, we measured global longitudinal strain, indexed left ventricular mass, and indexed left atrial volume. The TAVR group included 59% male, with an average age of 84 years, and 1-year mortality of 18%. Among biomarkers, we found GDF-15 and CRP to be strongly associated with all-cause mortality (P<0.001). Inclusion of GDF-15 and CRP to the Society of Thoracic Surgeons score significantly improved C index (0.65–0.79; P<0.05) and provided a category-free net reclassification improvement of 106% at 2 years (P=0.01). Among survivors, functional recovery in global longitudinal strain (>15% improvement) and indexed left ventricular mass (>20% decrease) at 1 year occurred in 48% and 22%, respectively. On multivariate logistic regression, lower baseline GDF-15 was associated with improved global longitudinal strain at 1 year (hazard ratio=0.29; P<0.001). Furthermore, improvement in global longitudinal strain at 1 month correlated with lower overall mortality (hazard ratio=0.45; P=0.03). Conclusions— Elevated GDF-15 correlates with lack of reverse remodeling and increased mortality after TAVR and improves risk prediction of mortality when added to the Society of Thoracic Surgeons score.

Coronary artery disease associated transcription factor TCF21 regulates smooth muscle precursor cells that contribute to the fibrous cap

TCF21 is a basic helix–loop–helix transcription factor that has recently been implicated as contributing to susceptibility to coronary heart disease based on genome wide association studies. In order to identify transcriptionally regulated target genes in a major disease relevant cell type, we performed siRNA knockdown of TCF21 in in vitro cultured human coronary artery smooth muscle cells and compared the transcriptome of siTCF21 versus siCONTROL treated cells. The raw (FASTQ) as well as processed (BED) data from 3 technical replicates per treatment has been deposited with Gene Expression Omnibus (GSE44461).