RATIONALE:Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes.OBJECTIVE:To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs.METHODS AND RESULTS:We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans.CONCLUSIONS:Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Overnutrition increases the proliferation of hematopoietic stem and multipotent progenitor cells (HSPCs) and overproduction of monocyte, resulting in the development of low-grade inflammatory and metabolic phenotypes. One molecule that regulates hematopoietic stem cell proliferation is heparin-binding EGF-like growth factor (HB-EGF). Cellular and oxidative stresses sensitively upregulate the HB-EGF gene expression in the cells. Hypothesis: In this study, we tested the hypothesis that HB-EGF is a crucial regulator for the production of myeloids during metabolic stress associated with overnutrition. Methods and Results: The administration of the HB-EGF antisense oligonucleotide (ASO), which is adequately distributed in bone marrow, in the LDLR KO mice under the Western diet (21% fat and 0.2% cholesterol) downregulated the number of leucocytes and myeloids (neutrophils and monocytes) in peripheral bloodstream. Flow cytometry analysis result indicated that the antisense also downregulated the number of myeloid progenitors (MPCs, CMPs, and GMPs) in the bone marrow and spleen tissues. The HB-EGF knockdown in a mouse system using the loxP-Cre approach also effectively induced downregulation of quantity of myeloid progenitors in the bone marrow and spleen tissues. The HB-EGF antisense effectively reduced the CD68 macrophage content in the liver in the LDLR KO mice under the Western diet. The HB-EGF antisense protected against the development of metabolic disease phenotypes (hyperlipidemia, atherosclerosis, and insulin resistance) in the mice. Conclusion: The results from this study consistently suggested that HB-EGF is a critical regulator for the myeloid production during the metabolic stress environment associated with overnutrition. The study also shows the applicability of the targeting of HB-EGF to prevent or reverse metabolic diseases associated with low-grade inflammation.

BACKGROUND AND AIMS:Heparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model.METHODS AND RESULTS:The male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances.CONCLUSION:The HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

Objective The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Approach and results Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. Conclusion This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

Objective: Elevation of apoB-containing lipoproteins is a well-established risk factor for the development of atherosclerosis. Previous reports showed that expression of heparin-binding EGF-like growth factor (HBEGF), a ligand of epidermal growth factor receptor (EGFR), is associated with atherosclerosis development. In this study, we examined in vivo effects of HBEGF targeting on hyperlipidemia-induced atherosclerosis by suppressing HBEGF expression using antisense oligonucleotide (ASO). Methods and Results: Female and male LDLR deficient mice were fed a high fat diet (HFD; 21% fat, 0.2% cholesterol) throughout the study. After 8 weeks of HFD feeding, mice were injected intraperitoneally with either control or HBEGF ASOs weekly for 12 weeks. At termination, we measured circulating lipid concentrations and atherosclerotic lesion size in the aorta. Compared to control ASO group, HBEGF ASO group had a significant reduction of circulating total cholesterol, triglyceride, and apoB-containing lipoprotein concentrations but no change of high-density lipoprotein (HDL) concentration. Importantly, HBEGF ASO injection significantly suppressed atherosclerosis in the aortic arch, thoracic, and abdominal aorta. HBEGF ASO suppressed sterol synthetic gene expression in the liver but elevated lipid contents in the liver. HBEGF gene silencing in a liver cell system induced downregulation of sterol regulatory element binding protein (SREBP) target genes including LDLR and Insig1. Conclusion: Targeting HBEGF using ASOs is an efficient approach to suppress dyslipidemia and hyperlipidemia-induced atherosclerosis. The differential gene expression analysis suggests that HBEGF ASO administration suppresses SREBP-regulated gene expression in the liver leading to downregulation of circulating cholesterol and TG concentrations.

Objective: Elevation of ApoB-containing lipoproteins in circulation a well-established risk factor for the development of cardiovascular diseases. HB-EGF, which is an endogenous ligand of EGFR and ...

Although much progress has been made in identifying the mechanisms that trigger endothelial activation and inflammatory cell recruitment during atherosclerosis, less is known about the intrinsic pathways that counteract these events. Here we identified NOTCH1 as an antagonist of endothelial cell (EC) activation. NOTCH1 was constitutively expressed by adult arterial endothelium, but levels were significantly reduced by high-fat diet. Furthermore, treatment of human aortic ECs (HAECs) with inflammatory lipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [Ox-PAPC]) and proinflammatory cytokines (TNF and IL1β) decreased Notch1 expression and signaling in vitro through a mechanism that requires STAT3 activation. Reduction of NOTCH1 in HAECs by siRNA, in the absence of inflammatory lipids or cytokines, increased inflammatory molecules and binding of monocytes. Conversely, some of the effects mediated by Ox-PAPC were reversed by increased NOTCH1 signaling, suggesting a link between lipid-mediated inflammation and Notch1. Interestingly, reduction of NOTCH1 by Ox-PAPC in HAECs was associated with a genetic variant previously correlated to high-density lipoprotein in a human genome-wide association study. Finally, endothelial Notch1 heterozygous mice showed higher diet-induced atherosclerosis. Based on these findings, we propose that reduction of endothelial NOTCH1 is a predisposing factor in the onset of vascular inflammation and initiation of atherosclerosis.

Objective: Heparin-binding EGF-like growth factor (HB-EGF) is a receptor ligand that regulates inflammatory gene expression in the human aortic endothelial cells. In this study, we examined the effects of targeting HB-EGF using an antisense oligonucleotide (ASO) in the development of atherosclerosis. Methods and Results: Control and HB-EGF ASOs were injected intraperitoneally (i.p.; weekly; Dose: 50 mg/kg/wk) for 12 weeks into LDLR deficient mice fed a fat-enriched diet (21% fat; 0.2% cholesterol). Compared with the control ASO injected group (N=13), the HB-EGF ASO injected group (N=15) had efficiently reduced atherosclerosis in the arch of aorta (p<0.0001). The HB-EGF ASO also significantly reduced plasma cholesterol concentration (p = 0.0397). We observed that the VLDL was consistently reduced in all HB-EGF ASO injected mice tested. In liver tissue, the HB-EGF ASO induced significant downregulation of gene expressions of the sterol synthetic pathway as determined by a microarray and DAVID functional analysis (adjusted p = 5.5 x 10-9)). The HB-EGF ASO also induced lipid droplet accumulation in liver tissues as determined by histological analysis with liver tissue sections. Conclusions: This study indicates that HB-EGF is a key mediator for atherosclerosis and the targeting of HB-EGF is an efficient approach to inhibit lesion formation in the aorta. Downregulation of the sterol synthesis pathway and reduction of VLDL particles in the blood may contribute to the protective effects of HB-EGF ASO.

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.

Atherosclerosis is the main underlying cause of major cardiovascular diseases such as stroke and heart attack. Oxidized phospholipids such as oxidized 1-palmitoyl-2-arachidonoyl-sn-Glycero-3-phosphorylcholine (OxPAPC) accumulate in lesions of and promote atherosclerosis. OxPAPC activates endothelial cells, a critical early event of atherogenesis. Epoxyisoprostane E2 (EI) is an oxidized fatty acid contained at the sn-2 position of 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine (PEIPC), the most active component of OxPAPC in regulating inflammation. OxPAPC and its components including PEIPC activate endothelial cells to express an array of genes in different categories including oxidative stress response genes such as tumor suppressor gene OKL38 and Heme oxygenase-1 (HO-1). EI can be released by lipase from PEIPC. In this study, we examined the ability of EI to stimulate oxidative stress response in endothelial cells. EI released from OxPAPC and synthetic EI stimulated the expression of oxidative stress response gene OKL38 and antioxidant gene HO-1. Treatment of endothelial cells with EI increased the production of superoxide. NADPH oxidase inhibitor Apocynin and superoxide scavenger N-acetyl-cysteine (NAC) significantly attenuated EI-stimulated expression of OKL38 and HO-1. We further demonstrated that EI activated oxidative stress-sensitive transcription factor Nrf2. Silencing of Nrf2 with siRNA significantly reduced EI stimulated expression of OKL38 and HO-1. Thus, we demonstrated that EI induced oxidative stress in endothelial cells leading to increased expression of oxidative stress response gene OKL38 and HO-1 via Nrf2 signaling pathway relevant to atherosclerosis.

Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phospholcholine (OxPAPC) and its component phospholipids accumulate in atherosclerotic lesions and regulate the expression of >1,000 genes, many proatherogenic, in human aortic endothelial cells (HAECs). In contrast, there is evidence in the literature that HDL protects the vasculature from inflammatory insult. We have previously shown that in HAECs, HDL attenuates the expression of several proatherogenic genes regulated by OxPAPC and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. We now demonstrate that HDL reverses >50% of the OxPAPC transcriptional response. Genes reversed by HDL are enriched for inflammatory and vascular development pathways, while genes not affected by HDL are enriched for oxidative stress response pathways. The protective effect of HDL is partially mimicked by cholesterol repletion and treatment with apoA1 but does not require signaling through scavenger receptor class B type I. Furthermore, our data demonstrate that HDL protection requires direct interaction with OxPAPC. HDL-associated platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes short-chain bioactive phospholipids in OxPAPC; however, inhibiting PAF-AH activity does not prevent HDL protection. Our results are consistent with HDL sequestering specific bioactive lipids in OxPAPC, thereby preventing their regulation of select target genes. Overall, this work implicates HDL as a major regulator of OxPAPC action in endothelial cells via multiple mechanisms.

Maintenance of endothelial homeostasis is critical in the prevention of vascular disorders including atherosclerosis. While we have a good understanding of the mechanisms that promote endothelial activation, less understood are the mechanisms that counterbalance this state. We found that inducible deletion of Notch1 in the endothelium of adult mice led to the accumulation of CD45+ cells in the subendothelial space of large arteries. NOTCH1 is constitutively expressed in adult arteries of mouse and human in vivo but it is excluded from veins. Together these observations led us to speculate that NOTCH1 might actively suppress the acquisition of a pro-inflammatory state in adult endothelium of large arteries. In fact, knockdown of NOTCH1 in human aortic endothelial cells (HAECs) in vitro triggered the expression of inflammatory cytokines in the absence of any other stimuli. Furthermore exposure of HAECs to oxidized phospholipids (OxPAPC) resulted in a rapid reduction of the expression levels of NOTCH1 and several NOTCH1-target genes. Using gene expression microarrays, we found that approximately 25% of the genes differentially expressed after exposure to OxPAPC (by 20%) were similarly regulated (ie either proportionally down or upregulated) when NOTCH1 was knockdown in HAECs, suggesting that these two events might be linked. Among these genes, CXCL1, a molecule previously shown to facilitate monocyte recruitment and binding to the endothelium, was increased in both conditions. Upregulation of CXCL1 upon exposure to OxPAPC was also consistently observed in a cohort of cells isolated from 147 individual donors and suppression of NOTCH1 also led to increase of the chemokine at the mRNA and protein levels. These data indicated that constant NOTCH1 expression was required for a homeostatic anti-inflammatory phenotype in the endothelium. We conclude that NOTCH1 may provide inherent protection to the endothelium during the initial stages of atherosclerosis by repressing the recruitment of inflammatory cells. Thus, decline of endogenous endothelial NOTCH1 levels is likely to constitute a predisposing factor for atherosclerosis.

Recent genome-wide association studies (GWAS) have identified 35 loci that significantly associate with coronary artery disease (CAD) susceptibility. The majority of the genes represented in these loci have not previously been studied in the context of atherosclerosis. To characterize the roles of these candidate genes in the vessel wall, we determined their expression levels in endothelial, smooth muscle, and macrophage cells isolated from healthy, prelesioned, and lesioned mouse aortas. We also performed expression quantitative locus (eQTL) mapping of these genes in human endothelial cells under control and proatherogenic conditions. Of the 57 genes studied, 31 were differentially expressed in one or more cell types in disease state in mice, and the expression levels of 8 were significantly associated with the CAD SNPs in human cells, 7 of which were also differentially expressed in mice. By integrating human and mouse results, we predict that PPAP2B, GALNT4, MAPKAPK5, TCTN1, SRR, SNF8, and ICAM1 play a causal role in the susceptibility to atherosclerosis through a role in the vasculature. Additionally, we highlight the genetic complexity of a subset of CAD loci through the differential expression of multiple candidate genes per locus and the involvement of genes that lie outside linkage disequilibrium blocks.

Objective— Endothelial cells are central to the initiation of atherosclerosis, yet there has been limited success in studying their gene expression in the mouse aorta. To address this, we developed a method for determining the global transcriptional changes that occur in the mouse endothelium in response to atherogenic conditions and applied it to investigate inflammatory stimuli. Approach and Results— We characterized a method for the isolation of endothelial cell RNA with high purity directly from mouse aortas and adapted this method to allow for the treatment of aortas ex vivo before RNA collection. Expression array analysis was performed on endothelial cell RNA isolated from control and hyperlipidemic prelesion mouse aortas, and 797 differentially expressed genes were identified. We also examined the effect of additional atherogenic conditions on endothelial gene expression, including ex vivo treatment with inflammatory stimuli, acute hyperlipidemia, and age. Of the 14 most highly differentially expressed genes in endothelium from prelesion aortas, 8 were also perturbed significantly by ≥1 atherogenic conditions: 2610019E17Rik, Abca1, H2-Ab1, H2-D1, Pf4, Ppbp, Pvrl2 , and Tnnt2 . Conclusions— We demonstrated that RNA can be isolated from mouse aortic endothelial cells after in vivo and ex vivo treatments of the murine vessel wall. We applied these methods to identify a group of genes, many of which have not been described previously as having a direct role in atherosclerosis, that were highly regulated by atherogenic stimuli and may play a role in early atherogenesis.

The goal of these studies was to determine the effect of 5,6-epoxyisoprostane, EI, on human aortic endothelial cells (HAEC). EI can form as a phospholipase product of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine, PEIPC, a proinflammatory molecule that accumulates in sites of inflammation where phospholipases are also increased. To determine the effect of EI on HAEC, we synthesized several stereoisomers of EI using a convergent approach from the individual optically pure building blocks, the epoxyaldehydes 5 and 6 and the bromoenones 14 and 16. The desired stereoisomer of EI can be prepared from these materials in only six operations, and thus, large amounts of the product can be obtained. The trans/trans isomers had the most potent activity, suggesting specificity in the interaction of EI with the cell surface. EI has potent anti-inflammatory effects in HAEC. EI strongly inhibits the production of MCP-1, a major monocyte chemotactic factor, and either decreases or minimally increases the levels of 10 proinflammatory molecules increased by PEIPC. EI also strongly down-regulates the inflammatory effects of IL-1β, a major inflammatory cytokine. Thus EI, a hydrolytic product of PEIPC, has potent anti-inflammatory function.