Amarylis Wanschel

Mir-33 regulates cell proliferation and cell cycle progression

Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. microRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression, acting predominantly at posttranscriptional level. Recent work from our group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the Srebp genes, regulate cholesterol and fatty acid metabolism in concert with their host genes. Here, we show that hsa-miR-33 family members modulate the expression of genes involved in cell cycle regulation and cell proliferation. MiR-33 inhibits the expression of the cyclin-dependent kinase 6 (CDK6) and cyclin D1 (CCND1), thereby reducing cell proliferation and cell cycle progression. Overexpression of miR-33 induces a significant G1 cell cycle arrest in Huh7 and A549 cell lines. Most importantly, inhibition of miR-33 expression using 2’fluoro/methoxyethyl-modified (2’F/MOE-modified) phosphorothioate backbone antisense oligonucleotides improves liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these results suggest that Srebp/miR-33 locus may cooperate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

Netrin-1 promotes adipose tissue macrophage retention and insulin resistance in obesity

During obesity, macrophage accumulation in adipose tissue propagates the chronic inflammation and insulin resistance associated with type 2 diabetes. The factors, however, that regulate the accrual of macrophages in adipose tissue are not well understood. Here we show that the neuroimmune guidance cue netrin-1 is highly expressed in obese but not lean adipose tissue of humans and mice, where it directs the retention of macrophages. Netrin-1, whose expression is induced in macrophages by the saturated fatty acid palmitate, acts via its receptor Unc5b to block their migration. In a mouse model of diet-induced obesity, we show that adipose tissue macrophages exhibit reduced migratory capacity, which can be restored by blocking netrin-1. Furthermore, hematopoietic deletion of Ntn1 facilitates adipose tissue macrophage emigration, reduces inflammation and improves insulin sensitivity. Collectively, these findings identify netrin-1 as a macrophage retention signal in adipose tissue during obesity that promotes chronic inflammation and insulin resistance.

Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice

Plasma high‐density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR‐33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short‐term anti‐miR‐33 treatment has been previously studied, the long‐term effect of miR‐33 antagonism in vivo remains to be elucidated. Here, we show that long‐term therapeutic silencing of miR‐33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high‐fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR‐33 increases the expression of genes involved in fatty acid synthesis such as acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR‐33 antisense oligonucleotides. We also report that anti‐miR‐33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP‐responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR‐33 when mice are fed a high‐fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR‐33 in non‐human primates before we can translate this therapy to humans.

Control of Cholesterol Metabolism and Plasma High-Density Lipoprotein Levels by microRNA-144Novelty and Significance

Rationale: Foam cell formation because of excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate–binding cassette (ABC) transporters, including adenosine triphosphate–binding cassette transporter A1 (ABCA1) and adenosine triphosphate–binding cassette transporter G1 (ABCG1). ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters. Objective: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists. Methods and Results: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed an miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to apolipoproteinA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 seems to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver. Conclusions: miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression and modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease.

Neuroimmune Guidance Cue Semaphorin 3E Is Expressed in Atherosclerotic Plaques and Regulates Macrophage Retention

Objective—The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques. Approach and Results—Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe–/– mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque. Conclusions—Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.

S-Nitroso-N-Acetylcysteine (SNAC) Prevents Myocardial Alterations in Hypercholesterolemic LDL Receptor Knockout Mice by Antiinflammatory Action

We investigated the ability of S-nitroso-N-acetylcyseine (SNAC) to prevent structural and functional myocardial alterations in hypercholesterolemic mice. C57BL6 wild-type (WT) and LDL-R−/− male mice (S) were fed a standard diet for 15 days. LDL-R−/− mice (S) showed an 11% increase in blood pressure, 62% decrease in left atrial contractility, and lower CD40L and eNOS expression relative to WT. LDL-R−/− mice fed an atherogenic diet for 15 days (Chol) showed significant increased left ventricular mass compared to S, which was characterized by: (1) 1.25-fold increase in the LV weight/body weight ratio and cardiomyocyte diameter; (2) enhanced expression of the NOS isoforms, CD40L, and collagen amount; and (3) no alteration in the atrial contractile performance. Administration of SNAC to Chol mice (Chol + SNAC) (0.51 μmol/kg/day for 15 day, IP) prevented increased left ventricular mass, collagen deposit, NOS isoforms, and CD40L overexpression, but it had no effect on the increased blood pressure or atrial basal hypocontractility. Deletion of the LDL receptor gene in mice resulted in hypertension and a marked left atrial contractile deficit, which may be related to eNOS underexpression. Our data show that SNAC treatment has an antiinflammatory action that might contribute to prevention of structural and functional myocardial alterations in atherosclerotic mice independently of changes in blood pressure.

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.

A Regulator of Secretory Vesicle Size, Kelch-Like Protein 12, Facilitates the Secretion of Apolipoprotein B100 and Very-Low-Density Lipoproteins—Brief Report

Objective—One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined. Approach and Results—Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII–dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER. Conclusions—KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

Genetic Evidence Supports a Major Role for Akt1 in VSMCs During Atherogenesis

RATIONALE Coronary artery disease, the direct result of atherosclerosis, is the most common cause of death in Western societies. Vascular smooth muscle cell (VSMC) apoptosis occurs during the progression of atherosclerosis and in advanced lesions and promotes plaque necrosis, a common feature of high-risk/vulnerable atherosclerotic plaques. Akt1, a serine/threonine protein kinase, regulates several key endothelial cell and VSMC functions including cell growth, migration, survival, and vascular tone. Although global deficiency of Akt1 results in impaired angiogenesis and massive atherosclerosis, the specific contribution of VSMC Akt1 remains poorly characterized. OBJECTIVE To investigate the contribution of VSMC Akt1 during atherogenesis and in established atherosclerotic plaques. METHODS AND RESULTS We generated 2 mouse models in which Akt1 expression can be suppressed specifically in VSCMs before (Apoe(-/-)Akt1(fl/fl)Sm22α(CRE)) and after (Apoe(-/-)Akt1(fl/fl)SM-MHC-CreER(T2E)) the formation of atherosclerotic plaques. This approach allows us to interrogate the role of Akt1 during the initial and late steps of atherogenesis. The absence of Akt1 in VSMCs during the progression of atherosclerosis results in larger atherosclerotic plaques characterized by bigger necrotic core areas, enhanced VSMC apoptosis, and reduced fibrous cap and collagen content. In contrast, VSMC Akt1 inhibition in established atherosclerotic plaques does not influence lesion size but markedly reduces the relative fibrous cap area in plaques and increases VSMC apoptosis. CONCLUSIONS Akt1 expression in VSMCs influences early and late stages of atherosclerosis. The absence of Akt1 in VSMCs induces features of plaque vulnerability including fibrous cap thinning and extensive necrotic core areas. These observations suggest that interventions enhancing Akt1 expression specifically in VSMCs may lessen plaque progression.