Faculty Bibliography

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.

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

OBJECTIVE: The purpose of this study was to determine whether myeloid differentiation factor 88 (MyD88) and its related Toll-like receptors (TLRs) 2 and 4 contributed to the development of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and atherosclerosis. METHODS AND RESULTS: AngII was infused into either apoE(-/-) or LDL receptor (LDLR)(-/-) male mice that were either MyD88(+/+) or (-/-). MyD88 deficiency profoundly reduced AngII-induced AAAs and atherosclerosis in both strains. To define whether deficiency of specific TLRs had similar effects, AngII was infused into LDLR(-/-) mice that were also deficient in either TLR2 or TLR4. TLR2 deficiency had no effect on AAA development but inhibited atherosclerosis. In contrast, TLR4 deficiency attenuated both AAAs and atherosclerosis. To resolve whether MyD88 and TLR4 exerted their effects through cells of hematopoietic lineage, LDLR(-/-) mice were lethally irradiated and repopulated with bone marrow-derived cells from either MyD88 or TLR4 strains. MyD88 deficiency in bone marrow-derived cells profoundly reduced both AngII-induced AAAs and atherosclerosis. However, TLR4 deficiency in bone marrow-derived cells had no effect on either pathology. CONCLUSIONS: These studies demonstrate that MyD88 deficiency in leukocytes profoundly reduces AngII-induced AAAs and atherosclerosis via mechanisms independent of either TLR2 or TLR4

Although the roles of the sterol response element binding protein-1 (SREBP1) and SREBP2 transcription factors in regulating fatty acid and cholesterol synthesis and uptake have been known for some time, it was recently discovered that 2 related microRNAs (miRs), miR-33a and miR-33b, are embedded in these genes. Studies indicate that miR-33a and miR-33b act with their host genes, Srebp2 and Srebp1, respectively, to reciprocally regulate cholesterol homeostasis and fatty acid metabolism in a negative feedback loop. miR-33 has been shown to posttranscriptionally repress key genes involved in cellular cholesterol export and high-density lipoprotein metabolism (Abca1, Abcg1, Npc1), fatty acid oxidation (Crot, Cpt1a, Hadhb, Ampk), and glucose metabolism (Sirt6, Irs2). Delivery of inhibitors of miR-33 in vitro and in vivo relieves repression of these genes, resulting in upregulation of the associated metabolic pathways. In mouse models, miR-33 antagonism has proven to be an effective strategy for increasing plasma high-density lipoprotein cholesterol and fatty acid oxidation and protecting from atherosclerosis. These exciting findings have opened up promising new avenues for the development of therapeutics to treat dyslipidemia and other metabolic disorders

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

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid beta (Abeta) in the brain. Abeta binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Abeta binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Abeta binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Abeta to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Abeta concentration, time required for Abeta binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Abeta binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Abeta to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 mum, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Abeta to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Abeta to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Abeta-CD36 interactions

MicroRNAs (miRNAs) represent an elegant mechanism of posttranscriptional control of gene expression that serves to fine-tune biological processes. These tiny noncoding RNAs (20-22 nucleotide) bind to the 3' untranslated region of mRNAs, thereby repressing gene expression. Recent advances in the understanding of lipid metabolism have revealed that miRNAs, particularly miR-122 and miR-33, play major roles in regulating cholesterol and fatty acid homeostasis. miR-122, the most abundant miRNA in the liver, appears to maintain the hepatic cell phenotype, and its inhibition decreases total serum cholesterol. miR-33, an intronic miRNA located with the sterol response element-binding protein (SREBP)-2 gene, regulates cholesterol efflux, fatty acid beta oxidation, and high-density lipoprotein metabolism. These findings have highlighted the complexity of lipid homeostasis and the important role that miRNAs play in these processes, potentially opening new avenues for the treatment of dyslipidemias

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr-/- mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease

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-alpha. 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