Kasey C. Vickers

Intercellular Transport of MicroRNAs

Extracellular microRNAs (miRNA) are present in most biological fluids, relatively stable, and hold great potential for disease biomarkers and novel therapeutics. Circulating miRNAs are transported by membrane-derived vesicles (exosomes and microparticles), lipoproteins, and other ribonucleoprotein complexes. Evidence suggests that miRNAs are selectively exported from cells with distinct signatures that have been found to be altered in many pathophysiologies, including cardiovascular disease. Protected from plasma ribonucleases by their carriers, functional miRNAs are delivered to recipient cells by various routes. Transferred miRNAs use cellular machinery to reduce target gene expression and alter cellular phenotype. Similar to soluble factors, miRNAs mediate cell-to-cell communication linking disparate cell types, diverse biological mechanisms, and homeostatic pathways. Although significant advances have been made, miRNA intercellular communication is full of complexities and many questions remain. This review brings into focus what is currently known and outstanding in a novel field of study with applicability to cardiovascular disease.

Lipid-based carriers of microRNAs and intercellular communication.

Purpose of review Extracellular microRNAs (miRNAs) are uniquely stable in plasma, and the levels of specific circulating miRNAs can differ with disease. Extracellular miRNAs are associated with lipid-based carriers and lipid-free proteins. miRNAs can be transferred from cell-to-cell by lipid-based carriers and affect gene expression. This review summarizes recent studies that demonstrate the transfer of miRNA between cells and their potential role in intercellular communication. Recent findings Microvesicles, exosomes, apoptotic bodies, lipoproteins, and large microparticles contain miRNAs. Recent studies have demonstrated that miRNAs are transferred between dendritic cells, hepatocellular carcinoma cells, and adipocytes in lipid-based carriers. miRNAs are also transferred from T cells to antigen-presenting cells, from stem cells to endothelial cells and fibroblasts, from macrophages to breast cancer cells, and from epithelial cells to hepatocytes in lipid-based carriers. The cellular export of miRNAs in lipid-based carriers is regulated by the ceramide pathway, and the delivery of lipid-associated miRNAs to recipient cells is achieved by various routes, including endocytotic uptake, membrane-fusion, and scavenger receptors. Summary Cellular miRNAs are exported in and to lipid-based carriers (vesicles and lipoprotein particles) and transferred to recipient cells with gene expression changes as intercellular communication.

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells.

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223(-/-) mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDLs anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

MicroRNA-223 coordinates cholesterol homeostasis

Significance Results from this study represent a breakthrough in our understanding of posttranscriptional control of cholesterol metabolism and how microRNAs (miRNAs) are at the heart of cholesterol regulatory circuitry and homeostasis. Although cells are adept at maintaining proper cholesterol levels, it was unknown how cells posttranscriptionally coordinate cholesterol uptake, efflux, and synthesis. MicroRNA-223 (miR-223) transcription and expression are maintained by cholesterol, and, as a feedback network, miR-223 inhibits cholesterol biosynthesis and uptake and increases cholesterol efflux. This study clearly demonstrates the extensive role that miRNAs play in coordinating metabolic adaptation to disease and general homeostasis. This work highlights a unique regulatory control point for cholesterol homeostasis and illustrates how important the study of miRNAs is to the greater understanding of dyslipidemia and cardiovascular disease. MicroRNAs (miRNAs) regulate a wide variety of biological processes and contribute to metabolic homeostasis. Here, we demonstrate that microRNA-223 (miR-223), an miRNA previously associated with inflammation, also controls multiple mechanisms associated with cholesterol metabolism. miR-223 promoter activity and mature levels were found to be linked to cellular cholesterol states in hepatoma cells. Moreover, hypercholesterolemia was associated with increased hepatic miR-223 levels in athero-prone mice. miR-223 was found to regulate high-density lipoprotein-cholesterol (HDL-C) uptake, through direct targeting and repression of scavenger receptor BI, and to inhibit cholesterol biosynthesis through the direct repression of sterol enzymes 3-hydroxy-3-methylglutaryl-CoA synthase 1 and methylsterol monooxygenase 1 in humans. Additionally, miR-223 was found to indirectly promote ATP-binding cassette transporter A1 expression (mRNA and protein) through Sp3, thereby enhancing cellular cholesterol efflux. Finally, genetic ablation of miR-223 in mice resulted in increased HDL-C levels and particle size, as well as increased hepatic and plasma total cholesterol levels. In summary, we identified a critical role for miR-223 in systemic cholesterol regulation by coordinated posttranscriptional control of multiple genes in lipoprotein and cholesterol metabolism.

KRAS-dependent sorting of miRNA to exosomes

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC. DOI: http://dx.doi.org/10.7554/eLife.07197.001

microRNAs in the onset and development of cardiovascular disease

Physiological and pathological roles for small non-encoding miRNAs (microRNAs) in the cardiovascular system have recently emerged and are now widely studied. The discovery of widespread functions of miRNAs has increased the complexity of gene-regulatory processes and networks in both the cardiovascular system and cardiovascular diseases. Indeed, it has recently been shown that miRNAs are implicated in the regulation of many of the steps leading to the development of cardiovascular disease. These findings represent novel aspects in miRNA biology and, therefore, our understanding of the role of these miRNAs during the pathogenesis of cardiovascular disease is critical for the development of novel therapies and diagnostic interventions. The present review will focus on understanding how miRNAs are involved in the onset and development of cardiovascular diseases.

MicroRNAs in atherosclerosis and lipoprotein metabolism.

Purpose of reviewMicroRNAs (miRNA) are mediators of post-transcriptional gene expression that likely regulate most biological pathways and networks. The study of miRNAs is a rapidly emerging field; recent findings have revealed a significant role for miRNAs in atherosclerosis and lipoprotein metabolism, which will be described in this review. Recent findingsThe discovery of miRNA gene regulatory mechanisms contributing to endothelial integrity, macrophage inflammatory response to atherogenic lipids, vascular smooth muscle-cell proliferation, and cholesterol synthesis are described. Furthermore, recent evidence suggests that miRNAs may play a role in mediating the beneficial pleiotropic effects observed with statin-based lipid-lowering therapies. New modifications to miRNA mimetics and inhibitors, increasing targeting efficacy and cellular uptake, will likely enable future therapies to exploit miRNA gene regulatory networks. SummaryAt this time, the applicability and full potential of miRNAs in clinical practice is unknown. Nonetheless, recent advances in miRNA delivery and inhibition hold great promise of a tremendous clinical impact in atherosclerosis and cholesterol regulation.

microRNA-29 fine-tunes the expression of key FOXA2-activated lipid metabolism genes and is dysregulated in animal models of insulin resistance and diabetes

MicroRNAs (miRNAs) have emerged as biomarkers of metabolic status, etiological factors in complex disease, and promising drug targets. Recent reports suggest that miRNAs are critical regulators of pathways underlying the pathophysiology of type 2 diabetes. In this study, we demonstrate by deep sequencing and real-time quantitative PCR that hepatic levels of Foxa2 mRNA and miR-29 are elevated in a mouse model of diet-induced insulin resistance. We also show that Foxa2 and miR-29 are significantly upregulated in the livers of Zucker diabetic fatty (fa/fa) rats and that the levels of both returned to normal upon treatment with the insulin-sensitizing agent pioglitazone. We present evidence that miR-29 expression in human hepatoma cells is controlled in part by FOXA2, which is known to play a critical role in hepatic energy homeostasis. Moreover, we demonstrate that miR-29 fine-tunes FOXA2-mediated activation of key lipid metabolism genes, including PPARGC1A, HMGCS2, and ABHD5. These results suggest that miR-29 is an important regulatory factor in normal metabolism and may represent a novel therapeutic target in type 2 diabetes and related metabolic syndromes.

Prenatal Environmental Tobacco Smoke Exposure Promotes Adult Atherogenesis and Mitochondrial Damage in Apolipoprotein E−/− Mice Fed a Chow Diet

Background—Environmental tobacco smoke (ETS) exposure is recognized as a cardiovascular disease risk factor; however, the impact of prenatal ETS exposure on adult atherogenesis has not been examined. We hypothesized that in utero ETS exposure promotes adult atherosclerotic lesion formation and mitochondrial damage. Methods and Results—Atherosclerotic lesion formation, mitochondrial DNA damage, antioxidant activity, and oxidant load were determined in cardiovascular tissues from adult apolipoprotein E−/− mice exposed to either filtered air or ETS in utero and fed a standard chow diet (4.5% fat) from weaning until euthanasia. All parameters were significantly altered in male mice exposed in utero to ETS. Conclusions—These data support the hypothesis that prenatal ETS exposure is sufficient to promote adult cardiovascular disease development.

HDL and cholesterol: life after the divorce?

For decades, HDL and HDL-cholesterol (HDL-C) levels were viewed as synonymous, and modulation of HDL-C levels by drug therapy held great promise for the prevention and treatment of cardiovascular disease. Nevertheless, recent failures of drugs that raise HDL-C to reduce cardiovascular risk and the now greater understanding of the complexity of HDL composition and biology have prompted researchers in the field to redefine HDL. As such, the focus of HDL has now started to shift away from a cholesterol-centric view toward HDL particle number, subclasses, and other alternative metrics of HDL. Many of the recently discovered functions of HDL are, in fact, not strictly conferred by its ability to promote cholesterol flux but by the other molecules it transports, including a diverse set of proteins, small RNAs, hormones, carotenoids, vitamins, and bioactive lipids. Based on HDLs ability to interact with almost all cells and transport and deliver fat-soluble cargo, HDL has the remarkable capacity to affect a wide variety of endocrine-like systems. In this review, we characterize HDLs unique cargo and address the functional relevance and consequences of HDL transport and delivery of noncholesterol molecules to recipient cells and tissues.