Katey J. Rayner

Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention. MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3–5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels and providing protection against atherosclerosis; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.

miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.

The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

microRNAs and cholesterol metabolism

Cholesterol metabolism is tightly regulated at the cellular level. In addition to classic transcriptional regulation of cholesterol metabolism (e.g. by SREBP and LXR), members of a class of non-coding RNAs termed microRNAs (miRNAs) have recently been identified to be potent post-transcriptional regulators of lipid metabolism genes, including cholesterol homeostasis. We and others have recently shown that miR-33 regulates cholesterol efflux and HDL biogenesis by downregulating the expression of the ABC transporters, ABCA1 and ABCG1. In addition to miR-33, miR-122 and miR-370 have been shown to play important roles in regulating cholesterol and fatty acid metabolism. These new data suggest important roles of microRNAs in the epigenetic regulation of cholesterol metabolism and have opened new avenues for the treatment of dyslipidemias.

MicroRNAs regulating lipid metabolism in atherogenesis

MicroRNAs have emerged as important post-transcriptional regulators of lipid metabolism, and represent a new class of targets for therapeutic intervention. Recently, microRNA-33a and b (miR-33a/b) were discovered as key regulators of metabolic programs including cholesterol and fatty acid homeostasis. These intronic microRNAs are embedded in the sterol response element binding protein genes, SREBF2 and SREBF1, which code for transcription factors that coordinate cholesterol and fatty acid synthesis. By repressing a variety of genes involved in cholesterol export and fatty acid oxidation, including ABCA1, CROT, CPT1, HADHB and PRKAA1, miR-33a/b act in concert with their host genes to boost cellular sterol levels. Recent work in animal models has shown that inhibition of these small non-coding RNAs has potent effects on lipoprotein metabolism, including increasing plasma high-density lipoprotein (HDL) and reducing very low density lipoprotein (VLDL) triglycerides. Furthermore, other microRNAs are being discovered that also target the ABCA1 pathway, including miR-758, suggesting that miRNAs may work cooperatively to regulate this pathway. These exciting findings support the development of microRNA antagonists as potential therapeutics for the treatment of dyslipidaemia, atherosclerosis and related metabolic diseases.

The Plaque “Micro” Environment: microRNAs Control the Risk and the Development of Atherosclerosis

While the discovery of microRNAs has exponentially expanded our understanding of the regulatory mechanisms governing gene networks in many biological processes, the study of these tiny RNA powerhouses in cardiovascular disease is in its infancy. To date, there have been over 1200 human microRNAs identified, and they are estimated to affect the expression of over half of the protein-coding portion of the human genome. In this review, we will discuss miRNAs that are integral players in processes affecting risk factors for CVD, as well as miRNAs that act at the level of the vessel wall to affect atherogenesis. We will discuss how microRNAs are not only advancing the field of cardiovascular biology, but how some miRNAs are at the forefront of drug development and may be soon advancing into the clinic.