Binod Aryal

Therapeutic Silencing of MicroRNA-33 Inhibits the Progression of Atherosclerosis in Ldlr−/− Mice—Brief Report

Objective—To study the efficacy of anti–miRNA-33 therapy on the progression of atherosclerosis. Approach and Results—Ldlr−/− mice were injected subcutaneously with PBS, control, or anti–miR-33 oligonucleotides weekly and fed a Western diet for 12 weeks. At the end of treatment, the expression of miR-33 target genes was increased in the liver and aorta, demonstrating effective inhibition of miR-33 function. Interestingly, plasma high-density lipoprotein (HDL)-cholesterol was significantly increased in anti–miR-33-treated mice but only when they were fed a chow diet. However, HDL isolated from anti–miR-33-treated mice showed an increase cholesterol efflux capacity compared with HDL isolated from nontargeting oligonucleotide-treated mice. Analysis of atherosclerosis revealed a significant reduction of plaque size and macrophage content in mice receiving anti–miR-33. In contrast, no differences in collagen content and necrotic areas were observed among the 3 groups. Conclusions—Long-term anti–miR-33 therapy significantly reduces the progression of atherosclerosis and improves HDL functionality. The antiatherogenic effect is independent of plasma HDL-cholesterol levels.

Noncoding RNAs and Atherosclerosis

Noncoding RNAs (ncRNAs) represent a class of RNA molecules that typically do not code for proteins. Emerging data suggest that ncRNAs play an important role in several physiological and pathological conditions such as cancer and cardiovascular diseases, including atherosclerosis. The best-characterized ncRNAs are the microRNAs which are small, approximately 22-nucleotide sequences of RNA that regulate gene expression at the posttranscriptional level through transcript degradation or translational repression. MicroRNAs control several aspects of atherosclerosis, including endothelial cell, vascular smooth cell, and macrophage functions as well as lipoprotein metabolism. Apart from microRNAs, recently ncRNAs, especially long ncRNAs, have emerged as important potential regulators of the progression of atherosclerosis. However, the molecular mechanism of their regulation and function as well as the significance of other ncRNAs such as small nucleolar RNAs during atherogenesis is largely unknown. In this review, we summarize the recent findings in the field, highlighting the importance of ncRNAs in atherosclerosis and discuss their potential use as therapeutic targets in cardiovascular diseases.

Micrornas and lipid metabolism

Purpose of review Work over the past decade has identified the important role of microRNAs (miRNAS) in regulating lipoprotein metabolism and associated disorders including metabolic syndrome, obesity, and atherosclerosis. This review summarizes the most recent findings in the field, highlighting the contribution of miRNAs in controlling LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) metabolism. Recent findings A number of miRNAs have emerged as important regulators of lipid metabolism, including miR-122 and miR-33. Work over the past 2 years has identified additional functions of miR-33 including the regulation of macrophage activation and mitochondrial metabolism. Moreover, it has recently been shown that miR-33 regulates vascular homeostasis and cardiac adaptation in response to pressure overload. In addition to miR-33 and miR-122, recent GWAS have identified single-nucleotide polymorphisms in the proximity of miRNA genes associated with abnormal levels of circulating lipids in humans. Several of these miRNAs, such as miR-148a and miR-128-1, target important proteins that regulate cellular cholesterol metabolism, including the LDL receptor (LDLR) and the ATP-binding cassette A1 (ABCA1). Summary MicroRNAs have emerged as critical regulators of cholesterol metabolism and promising therapeutic targets for treating cardiometabolic disorders including atherosclerosis. Here, we discuss the recent findings in the field, highlighting the novel mechanisms by which miR-33 controls lipid metabolism and atherogenesis, and the identification of novel miRNAs that regulate LDL metabolism. Finally, we summarize the recent findings that identified miR-33 as an important noncoding RNA that controls cardiovascular homeostasis independent of its role in regulating lipid metabolism.

Chronic miR‐29 antagonism promotes favorable plaque remodeling in atherosclerotic mice

Abnormal remodeling of atherosclerotic plaques can lead to rupture, acute myocardial infarction, and death. Enhancement of plaque extracellular matrix (ECM) may improve plaque morphology and stabilize lesions. Here, we demonstrate that chronic administration of LNA‐miR‐29 into an atherosclerotic mouse model improves indices of plaque morphology. This occurs due to upregulation of miR‐29 target genes of the ECM (col1A and col3A) resulting in reduced lesion size, enhanced fibrous cap thickness, and reduced necrotic zones. Sustained LNA‐miR‐29 treatment did not affect circulating lipids, blood chemistry, or ECM of solid organs including liver, lung, kidney, spleen, or heart. Collectively, these data support the idea that antagonizing miR‐29 may promote beneficial plaque remodeling as an independent approach to stabilize vulnerable atherosclerotic lesions.

Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis

Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide‐dependent serine‐threonine protein kinase, is highly express in insulin‐responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ‐line Akt2‐deficient mice develop similar atherosclerotic plaques as wild‐type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2‐/‐ mice to Ldlr‐/‐ mice results in marked reduction of the progression of atherosclerosis compared with Ldlr‐/‐ mice transplanted with wild‐type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein‐1 and macrophage colony‐stimulating factor). Moreover, Akt2‐/‐ macrophages accumulate less cholesterol and have an alternative activated or M2‐type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.—Rodlan, N., Chamorro‐Jorganes, A., Araldi, E., Wanschel, A. C., Aryal, B., Aranda, J. F., Goedeke, L., Salerno, A. G., Ramírez, C. M., Sessa, W. C., Suárez, Y., Fernández‐Hernando, C. Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis. FASEB J. 29, 597‐610 (2015). www.fasebj.org

ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and its absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation.

Therapeutic Silencing of MicroRNA-33 Inhibits the Progression of Atherosclerosis in Ldlr −/− Mice

Objective—To study the efficacy of anti–miRNA-33 therapy on the progression of atherosclerosis. Approach and Results—Ldlr−/− mice were injected subcutaneously with PBS, control, or anti–miR-33 oligonucleotides weekly and fed a Western diet for 12 weeks. At the end of treatment, the expression of miR-33 target genes was increased in the liver and aorta, demonstrating effective inhibition of miR-33 function. Interestingly, plasma high-density lipoprotein (HDL)-cholesterol was significantly increased in anti–miR-33-treated mice but only when they were fed a chow diet. However, HDL isolated from anti–miR-33-treated mice showed an increase cholesterol efflux capacity compared with HDL isolated from nontargeting oligonucleotide-treated mice. Analysis of atherosclerosis revealed a significant reduction of plaque size and macrophage content in mice receiving anti–miR-33. In contrast, no differences in collagen content and necrotic areas were observed among the 3 groups. Conclusions—Long-term anti–miR-33 therapy significantly reduces the progression of atherosclerosis and improves HDL functionality. The antiatherogenic effect is independent of plasma HDL-cholesterol levels.

Posttranscriptional regulation of lipid metabolism by non-coding RNAs and RNA binding proteins

Alterations in lipoprotein metabolism enhance the risk of cardiometabolic disorders including type-2 diabetes and atherosclerosis, the leading cause of death in Western societies. While the transcriptional regulation of lipid metabolism has been well characterized, recent studies have uncovered the importance of microRNAs (miRNAs), long-non-coding RNAs (lncRNAs) and RNA binding proteins (RBP) in regulating the expression of lipid-related genes at the posttranscriptional level. Work from several groups has identified a number of miRNAs, including miR-33, miR-122 and miR-148a, that play a prominent role in controlling cholesterol homeostasis and lipoprotein metabolism. Importantly, dysregulation of miRNA expression has been associated with dyslipidemia, suggesting that manipulating the expression of these miRNAs could be a useful therapeutic approach to ameliorate cardiovascular disease (CVD). The role of lncRNAs in regulating lipid metabolism has recently emerged and several groups have demonstrated their regulation of lipoprotein metabolism. However, given the high abundance of lncRNAs and the poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in controlling lipoprotein metabolism. In this review article, we summarize recent findings in the field and highlight the specific contribution of lncRNAs and RBPs in regulating lipid metabolism.

Non-coding RNA regulation of endothelial and macrophage functions during atherosclerosis

The endothelial lining can be viewed as the first line of defense against risk factors of vascular disease. Endothelial dysfunction is regarded as an initial event for atherogenesis since defects in vascular integrity and homeostasis are responsible for lipid infiltration and recruitment of monocytes into the vessel wall. Monocytes-turned-macrophages, which possess astounding inflammatory plasticity, perpetuate chronic inflammation and growth of atherosclerotic plaques and, are therefore central for the pathogenesis of atherosclerosis. Because endothelial cells and macrophages are key players during atherogenesis, it is crucial to understand the regulation of their functions in order to develop strategies to intervene disease progression. Interestingly, non-coding RNAs (ncRNAs), broad class of RNA molecules that do not code for proteins, are capable of reprogramming multiple cell functions and, thus, can be used as target agents. MicroRNAs are small ncRNAs whose roles in the regulation of vascular functions and development of atherosclerosis through post-transcriptional manipulation of gene expression have been widely explored. Recently, other ncRNAs including long noncoding RNAs (lncRNAs) have also emerged as potential regulators of these functions. However, given their poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in vascular biology. This review aims to provide a comprehensive perspective of ncRNA, mostly focusing in lncRNAs, mechanism of action and relevance in regulating lipid metabolism-independent endothelial and macrophages functions in the pathogenesis of atherosclerosis.

Absence of ANGPTL4 in adipose tissue improves glucose tolerance and attenuates atherogenesis

Alterations in ectopic lipid deposition and circulating lipids are major risk factors for developing cardiometabolic diseases. Angiopoietin-like protein 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL), controls fatty acid (FA) uptake in adipose and oxidative tissues and regulates circulating triacylglycerol-rich (TAG-rich) lipoproteins. Unfortunately, global depletion of ANGPTL4 results in severe metabolic abnormalities, inflammation, and fibrosis when mice are fed a high-fat diet (HFD), limiting our understanding of the contribution of ANGPTL4 in metabolic disorders. Here, we demonstrate that genetic ablation of ANGPTL4 in adipose tissue (AT) results in enhanced LPL activity, rapid clearance of circulating TAGs, increased AT lipolysis and FA oxidation, and decreased FA synthesis in AT. Most importantly, we found that absence of ANGPTL4 in AT prevents excessive ectopic lipid deposition in the liver and muscle, reducing novel PKC (nPKC) membrane translocation and enhancing insulin signaling. As a result, we observed a remarkable improvement in glucose tolerance in short-term HFD-fed AT-specific Angptl4-KO mice. Finally, lack of ANGPTL4 in AT enhances the clearance of proatherogenic lipoproteins, attenuates inflammation, and reduces atherosclerosis. Together, these findings uncovered an essential role of AT ANGPTL4 in regulating peripheral lipid deposition, influencing whole-body lipid and glucose metabolism and the progression of atherosclerosis.