RNA interference-based therapies for the control of atherosclerosis risk factors.

PURPOSE OF REVIEW: Atherosclerosis, characterized by lipid accumulation and chronic inflammation in the arterial wall, is the leading causes of death worldwide. The purpose of this article is to review the status of RNA interference (RNAi) based therapies in clinical trials for the treatment and prevention of atherosclerosis risk factors. RECENT FINDINGS: There is a growing interest on using RNAi technology for the control of atherosclerosis risk factors. Current clinical trials utilizing RNAi for atherosclerosis are targeting lipid metabolism regulating genes including proprotein convertase subtilisin/kexin 9, apolipoprotein C-III, lipoprotein (a) and angiopoietin-like protein 3. Currently, three RNAi-based drugs have been approved by U.S. Food and Drug Administration, but there are several therapies in clinical trials at the moment, and potentially entering the market in near future. In addition, recent preclinical studies on regulating vascular inflammation have shown promising results. SUMMARY: In recent years, RNAi based technologies and therapies have been intensively developed for the treatment of atherosclerosis risk factors, such as hyperlipidemia and vascular inflammation. Multiple potential therapeutic targets have emerged, and many of the reported clinical trials have already been successful in plasma lipid lowering. The scope of RNAi therapies is well recognized and recent approvals are encouraging for the treatment of cardiovascular and metabolic disorders.

Deletion of Lymphangiogenic and Angiogenic Growth Factor VEGF-D Leads to Severe Hyperlipidemia and Delayed Clearance of Chylomicron Remnants.

Objective- Dyslipidemia is one of the key factors behind coronary heart disease. Blood and lymphatic vessels play pivotal roles in both lipoprotein metabolism and development of atherosclerotic plaques. Recent studies have linked members of VEGF (vascular endothelial growth factor) family to lipid metabolism, but the function of VEGF-D has remained unexplored. Here, we investigated how the deletion of VEGF-D affects lipid and lipoprotein metabolism in atherogenic LDLR-/- ApoB100/100 mice. Approach and Results- Deletion of VEGF-D (VEGF-D-/-LDLR-/-ApoB100/100) led to markedly elevated plasma cholesterol and triglyceride levels without an increase in atherogenesis. Size distribution and hepatic lipid uptake studies confirmed a delayed clearance of large chylomicron remnant particles that cannot easily penetrate through the vascular endothelium. Mechanistically, the inhibition of VEGF-D signaling significantly decreased the hepatic expression of SDC1 (syndecan 1), which is one of the main receptors for chylomicron remnant uptake when LDLR is absent. Immunohistochemical staining confirmed reduced expression of SDC1 in the sinusoidal surface of hepatocytes in VEGF-D deficient mice. Furthermore, hepatic RNA-sequencing revealed that VEGF-D is also an important regulator of genes related to lipid metabolism and inflammation. The lack of VEGF-D signaling via VEGFR3 (VEGF receptor 3) led to lowered expression of genes regulating triglyceride and cholesterol production, as well as downregulation of peroxisomal ß-oxidation pathway. Conclusions- These results demonstrate that VEGF-D, a powerful lymphangiogenic and angiogenic growth factor, is also a major regulator of chylomicron metabolism in mice.

The follow-up of progressive hypertrophic cardiomyopathy using magnetic resonance rotating frame relaxation times.

Magnetic resonance rotating frame relaxation times are an alternative non-contrast agent choice for the diagnosis of chronic myocardial infarct. Fibrosis typically occurs in progressive hypertrophic cardiomyopathy. Fibrosis has been imaged in myocardial infarcted tissue using rotating frame relaxation times, which provides the possibility to follow up progressive cardiomyopathy without contrast agents. Mild and severe left ventricular hypertrophy were induced in mice by transverse aortic constriction, and the longitudinal rotating frame relaxation times (T1ρ ) and relaxation along the fictitious field (TRAFF2 , TRAFF3 ) were measured at 5, 10, 24, 62 and 89 days after transverse aortic constriction in vivo. Myocardial fibrosis was verified using Masson's trichrome staining. Increases in the relative relaxation time differences of T1ρ , together with TRAFF2 and TRAFF3 , between fibrotic and remote tissues over time were observed. Furthermore, TRAFF2 and TRAFF3 showed higher relaxation times overall in fibrotic tissue than T1ρ . Relaxation time differences were highly correlated with an excess of histologically verified fibrosis. We found that TRAFF2 and TRAFF3 are more sensitive than T1ρ to hypertrophic cardiomyopathy-related tissue changes and can serve as non-invasive diagnostic magnetic resonance imaging markers to follow up the mouse model of progressive hypertrophic cardiomyopathy.

Shame in social interaction: Descriptions of experiences of shame by participants with high or low levels of narcissistic traits.

In this study, we investigate how personal experiences about shameful events are described in face-to-face social interaction, and how these stories differ between participants who have either high or low levels of narcissistic personality traits. The dataset consists of 22 dyadic conversations where the participants describe events where they felt ashamed of themselves. We found the narratives to vary in terms of five dimensions. With narcissistic individuals, the default narrative tended to exhibit a cluster of characteristics that gather at one end of these dimensions: (1) weak expressions of shame; (2) located in the story-world; (3) low level of reflexivity as well as; (4) responsibility of the described event; and (5) a general level of description. We discuss the findings in relation to sociological and psychological theories of shame and suggest that individuals with narcissistic personality traits are more inclined to use suppressive conversational practices in their treatment of shame, thus providing a "window" to these interactional practices.

Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice.

The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic ß-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.

Lymphatic insufficiency leads to distinct myocardial infarct content assessed by magnetic resonance TRAFFn, T1ρ and T2 relaxation times.

The role of cardiac lymphatics in the pathogenesis of myocardial infarction (MI) is unclear. Lymphatic system regulates cardiac physiological processes such as edema and tissue fluid balance, which affect MI pathogenesis. Recently, MI and fibrosis have been assessed using endogenous contrast in magnetic resonance imaging (MRI) based on the relaxation along a fictitious field with rank n (RAFFn). We extended the RAFFn applications to evaluate the effects of lymphatic insufficiency on MI with comparison to longitudinal rotating frame (T1ρ) and T2 relaxation times. MI was induced in transgenic (TG) mice expressing soluble decoy VEGF receptor 3 that reduces lymphatic vessel formation and their wild-type (WT) control littermates for comparison. The RAFFn relaxation times with rank 2 (TRAFF2), and rank 4 (TRAFF4), T1ρ and T2 were acquired at time points 0, 3, 7, 21 and 42 days after the MI at 9.4 T. Infarct sizes were determined based on TRAFF2, TRAFF4, T1ρ and T2 relaxation time maps. The area of differences (AOD) was calculated based on the MI areas determined on T2 and TRAFF2, TRAFF4 or T1ρ relaxation time maps. Hematoxylin-eosin and Sirius red stained histology sections were prepared to confirm MI locations and sizes. MI was detected as increased TRAFF2, TRAFF4, T1ρ and T2 relaxation times. Infarct sizes were similar on all relaxation time maps during the experimental period. Significantly larger AOD values were found together with increased AOD values in the TG group compared to the WT group. Histology confirmed these findings. The lymphatic deficiency was found to increase cardiac edema in MI. The combination of TRAFF2 (or TRAFF4) and T2 characterizes MI and edema in the myocardium in both lymphatic insufficiency and normal mice without any contrast agents.

Deletion of the murine ortholog of human 9p21.3 locus promotes atherosclerosis by increasing macrophage proinflammatory activity.

Background: Several genome-wide association studies have reported a risk locus for coronary artery disease (CAD) in the 9p21. 3 chromosomal region. This region encodes a lncRNA in the INK4 locus (ANRIL) and its genetic variance has a strong association with CAD, but its mechanisms in atherogenesis remain unclear. Objectives: This study aimed to investigate the role of the murine ortholog of human 9p21.3 locus in atherogenesis in hypercholesterolemic mice. Methods: Murine 9p21.3 ortholog knockout mice (Chr4Δ70kb/Δ70kb ) were crossbred with Ldlr -/- ApoB 100/100 mice, and atherosclerotic plaque size and morphology were analyzed on a standard or a high-fat diet (HFD). The hematopoietic cell-specific effect of Chr4Δ70kb/Δ70kb on atherosclerotic plaque development was studied via bone marrow (BM) transplantation, where Chr4Δ70kb/Δ70kb or wild-type BM was transplanted into Ldlr -/- ApoB 100/100 mice. The role of Chr4Δ70kb/Δ70kb in macrophage M1/M2 polarization was studied. In addition, single-cell sequencing data from human and mouse atheroma were analyzed to show the expression profiles of ANRIL and its murine equivalent, Ak148321, in the plaques. Results: Both systemic and hematopoietic Chr4Δ70kb/Δ70kb increased atherosclerosis in Ldlr -/- ApoB 100/100 mice after 12 weeks of HFD. The systemic Chr4Δ70kb/Δ70kb also elevated the number of circulating leukocytes. Chr4Δ70kb/Δ70kb BMDMs showed enhanced M1 polarization in vitro. Single-cell sequencing data from human and mouse atheroma revealed that ANRIL and Ak148321 were mainly expressed in the immune cells. Conclusion: These data demonstrate that both systemic and BM-specific deletion of the murine 9p21.3 risk locus ortholog promotes atherosclerosis and regulates macrophage pro-inflammatory activity, suggesting the inflammation-driven mechanisms of the risk locus on atherogenesis.

SUR1-E1506K mutation impairs glucose tolerance and promotes vulnerable atherosclerotic plaque phenotype in hypercholesterolemic mice.

BACKGROUND AND AIMS: Diabetes is a major risk factor of atherosclerosis and its complications. The loss-of-function mutation E1506K in the sulfonylurea receptor 1 (SUR1-E1506K) induces hyperinsulinemia in infancy, leading to impaired glucose tolerance and increased risk of type 2 diabetes. In this study, we investigate the effect of SUR1-E1506K mutation on atherogenesis in hypercholesterolemic LDLR-/- mice. METHODS: SUR1-E1506K mutated mice were cross-bred with LDLR-/- mice (SUR1Δ/LDLR-/-), 6 months old mice were fed a western-diet (WD) for 6 months to induce advanced atherosclerotic plaques. At the age of 12 months, atherosclerosis and plaque morphology were analyzed and mRNA gene expression were measured from aortic sections and macrophages. Glucose metabolism was characterized before and after WD. Results were compared to age-matched LDLR-/- mice. RESULTS: Advanced atherosclerotic plaques did not differ in size between the two strains. However, in SUR1Δ/LDLR-/- mice, plaque necrotic area was increased and smooth muscle cell number was reduced, resulting in higher plaque vulnerability index in SUR1Δ/LDLR-/- mice compared to LDLR-/- mice. SUR1Δ/LDLR-/- mice exhibited impaired glucose tolerance and elevated fasting glucose after WD. The positive staining area of IL-1ß and NLRP3 inflammasome were increased in aortic sections in SUR1Δ/LDLR-/- mice compared to LDLR-/- mice, and IL-18 plasma level was elevated in SUR1Δ/LDLR-/- mice. Finally, the mRNA expression of IL-1ß and IL-18 were increased in SUR1Δ/LDLR-/- bone marrow derived macrophages in comparison to LDLR-/- macrophages in response to LPS. CONCLUSIONS: SUR1-E1506K mutation impairs glucose tolerance and increases arterial inflammation, which promotes a vulnerable atherosclerotic plaque phenotype in LDLR-/- mice.