Carlos Fernandez-Hernando, Ph.D.

Two-tiered Approach Identifies a Network of Cancer and Liver Disease-related Genes Regulated by miR-122
Boutz, Daniel R; Collins, Patrick J; Suresh, Uthra; Lu, Mingzhu; Ramirez, Cristina M; Fernandez-Hernando, Carlos; Huang, Yufei; de Sousa Abreu, Raquel; Le, Shu-Yun; Shapiro, Bruce A; Liu, Angela M; Luk, John M; Force Aldred, Shelley; Trinklein, Nathan D; Marcotte, Edward M; Penalva, Luiz O F
2011 May 20;286(20):18066-18078, Journal of biological chemistry
MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function
— id: 134201, year: 2011, vol: 286, page: 18066, stat: Journal Article,

MicroRNA-16 and MicroRNA-424 Regulate Cell-Autonomous Angiogenic Functions in Endothelial Cells via Targeting Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1
Chamorro-Jorganes, Aranzazu; Araldi, Elisa; Penalva, Luiz O F; Sandhu, Devraj; Fernandez-Hernando, Carlos; Suarez, Yajaira
2011 Nov;31(11):2595-2606, Arteriosclerosis, thrombosis, & vascular biology
OBJECTIVE-: MicroRNAs play key roles in modulating a variety of cellular processes by posttranscriptional regulation of their target genes. Vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and fibroblast growth factor receptor-1 (FGFR1) were identified by bioinformatic approaches and subsequently validated as targets of microRNA (miR)-16 and miR-424 in endothelial cells (ECs). METHODS AND RESULTS-: Mimetics of these microRNAs reduced VEGF, VEGFR2, and FGFR1 expression, whereas specific antagonists enhanced their expression. Expression of mature miR-16 and miR-424 was upregulated on VEGF or basic fibroblast growth factor (bFGF) treatment. This upregulation was accompanied by a parallel increase in primary transcript (pri-miR)-16-1 and pri-miR-16-2 but not in pri-miR-424 levels, indicating a VEGF/bFGF-dependent transcriptional and posttranscriptional regulation of miR-16 and miR-424, respectively. Reduced expression of VEGFR2 and FGFR1 by miR-16 or miR-424 overexpression regulated VEGF and bFGF signaling through these receptors, thereby affecting the activity of downstream components of the pathways. Functionally, miR-16 or miR-424 overexpression reduced proliferation, migration, and cord formation of ECs in vitro, and lentiviral overexpression of miR-16 reduced the ability of ECs to form blood vessels in vivo. CONCLUSION-: We conclude that these miRNAs fine-tune the expression of selected endothelial angiogenic mediators in response to these growth factors. Altogether, these findings suggest that miR-16 and miR-424 play important roles in regulating cell-intrinsic angiogenic activity of ECs
— id: 139472, year: 2011, vol: 31, page: 2595, stat: Journal Article,

miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling
Davalos, Alberto; Goedeke, Leigh; Smibert, Peter; Ramirez, Cristina M; Warrier, Nikhil P; Andreo, Ursula; Cirera-Salinas, Daniel; Rayner, Katey; Suresh, Uthra; Pastor-Pareja, Jose Carlos; Esplugues, Enric; Fisher, Edward A; Penalva, Luiz O F; Moore, Kathryn J; Suarez, Yajaira; Lai, Eric C; Fernandez-Hernando, Carlos
2011 May 31;108(22):9232-9237, Proceedings of the National Academy of Sciences of the United States of America
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
— id: 133351, year: 2011, vol: 108, page: 9232, stat: Journal Article,

Statins Promote the Regression of Atherosclerosis via Activation of the CCR7-Dependent Emigration Pathway in Macrophages
Feig, Jonathan E; Shang, Yueting; Rotllan, Noemi; Vengrenyuk, Yuliya; Wu, Chaowei; Shamir, Raanan; Torra, Ines Pineda; Fernandez-Hernando, Carlos; Fisher, Edward A; Garabedian, Michael J
2011 ;6(12):e28534-e28534, PLoS ONE
HMG-CoA reductase inhibitors (statins) decrease atherosclerosis by lowering low-density-lipoprotein cholesterol. Statins are also thought to have additional anti-atherogenic properties, yet defining these non-conventional modes of statin action remains incomplete. We have previously developed a novel mouse transplant model of atherosclerosis regression in which aortic segments from diseased donors are placed into normolipidemic recipients. With this model, we demonstrated the rapid loss of CD68+ cells (mainly macrophages) in plaques through the induction of a chemokine receptor CCR7-dependent emigration process. Because the human and mouse CCR7 promoter contain Sterol Response Elements (SREs), we hypothesized that Sterol Regulatory Element Binding Proteins (SREBPs) are involved in increasing CCR7 expression and through this mechanism, statins would promote CD68+ cell emigration from plaques. We examined whether statin activation of the SREBP pathway in vivo would induce CCR7 expression and promote macrophage emigration from plaques. We found that western diet-fed apoE(-/-) mice treated with either atorvastatin or rosuvastatin led to a substantial reduction in the CD68+ cell content in the plaques despite continued hyperlipidemia. We also observed a significant increase in CCR7 mRNA in CD68+ cells from both the atorvastatin and rosuvastatin treated mice associated with emigration of CD68+ cells from plaques. Importantly, CCR7(-/-)/apoE(-/-) double knockout mice failed to display a reduction in CD68+ cell content upon statin treatment. Statins also affected the recruitment of transcriptional regulatory proteins and the organization of the chromatin at the CCR7 promoter to increase the transcriptional activity. Statins promote the beneficial remodeling of plaques in diseased mouse arteries through the stimulation of the CCR7 emigration pathway in macrophages. Therefore, statins may exhibit some of their clinical benefits by not only retarding the progression of atherosclerosis, but also accelerating its regression
— id: 146266, year: 2011, vol: 6, page: e28534, stat: Journal Article,

MicroRNA Modulation of Cholesterol Homeostasis
Fernandez-Hernando, Carlos; Moore, Kathryn J
2011 Nov;31(11):2378-2382, Arteriosclerosis, thrombosis, & vascular biology
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
— id: 139482, year: 2011, vol: 31, page: 2378, stat: Journal Article,

MicroRNAs in lipid metabolism
Fernandez-Hernando, Carlos; Suarez, Yajaira; Rayner, Katey J; Moore, Kathryn J
2011 Apr;22(2):86-92, Current opinion in lipidology
PURPOSE OF REVIEW: Although the role for microRNAs (miRNAs) in regulating multiple physiological processes including apoptosis, cell differentiation, and cancer is well established, the importance of these tiny RNAs in regulating lipid metabolism has only recently been uncovered. This review summarizes the evidence for a critical role of miRNAs in regulating lipid metabolism. RECENT FINDINGS: Lipid 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 noncoding RNAs termed miRNAs have now been identified to be potent post-transcriptional regulators of lipid metabolism genes involved in cholesterol homeostasis and fatty acid oxidation. Several reports 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, miR-33 also inhibits the translation of several transcripts encoding proteins involved in fatty acid beta-oxidation including CPT1a, CROT, and HADHB, thereby reducing fatty acid degradation. Other miRNAs including miR-122, miR-370, miR-335, and miR-378/378*, miR-27 and miR-125a-5p have been implicated in regulating cholesterol homeostasis, fatty acid metabolism and lipogenesis. SUMMARY: Recent advances in the understanding of the regulation of lipid metabolism indicate that miRNAs play major roles in regulating cholesterol and fatty acid metabolism. These new findings may open new avenues for the treatment of dyslipidemias
— id: 128788, year: 2011, vol: 22, page: 86, stat: Journal Article,

The role of microRNAs in cholesterol efflux and hepatic lipid metabolism
Moore, Kathryn J; Rayner, Katey J; Suarez, Yajaira; Fernandez-Hernando, Carlos
2011 Aug 21;31:49-63, Annual review of nutrition
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
— id: 138339, year: 2011, vol: 31, page: 49, stat: Journal Article,

MicroRNA-758 Regulates Cholesterol Efflux Through Posttranscriptional Repression of ATP-Binding Cassette Transporter A1
Ramirez, Cristina M; Davalos, Alberto; Goedeke, Leigh; Salerno, Alessandro G; Warrier, Nikhil; Cirera-Salinas, Daniel; Suarez, Yajaira; Fernandez-Hernando, Carlos
2011 Nov;31(11):2707-2714, Arteriosclerosis, thrombosis, & vascular biology
OBJECTIVE-: The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of macrophage cholesterol efflux and protects cells from excess intracellular cholesterol accumulation; however, the mechanism involved in posttranscriptional regulation of ABCA1 is poorly understood. We previously showed that microRNA-33 (miR-33) is 1 regulator. Here, we investigated the potential contribution of other microRNAs (miRNAs) to posttranscriptional regulation of ABCA1 and macrophage cholesterol efflux. METHODS AND RESULTS-: We performed a bioinformatic analysis for identifying miRNA target prediction sites in ABCA1 gene and an unbiased genome-wide screen to identify miRNAs modulated by cholesterol excess in mouse peritoneal macrophages. Quantitative real-time reverse transcription-polymerase chain reaction confirmed that miR-758 is repressed in cholesterol-loaded macrophages. Under physiological conditions, high dietary fat excess in mice repressed miR-758 both in peritoneal macrophages and, to a lesser extent, in the liver. In mouse and human cells in vitro, miR-758 repressed the expression of ABCA1, and conversely, the inhibition of this miRNA by using anti-miR-758 increased ABCA1 expression. In mouse cells, miR-758 reduced cellular cholesterol efflux to apolipoprotein A1 (apoA1), and anti-miR-758 increased it. miR-758 directly targets the 3'-untranslated region of Abca1 as assessed by 3'-untranslated region luciferase reporter assays. Interestingly, miR-758 is highly expressed in the brain, where it also targets several genes involved in neurological functions, including Slc38a1, Ntm, Epha7, and Mytl1. CONCLUSION-: We identified miR-758 as a novel miRNA that posttranscriptionally controls ABCA1 levels in different cells and regulates macrophage cellular cholesterol efflux to apoA1, opening new avenues to increase apoA1 and raise high-density lipoprotein levels
— id: 139473, year: 2011, vol: 31, page: 2707, stat: Journal Article,

"Micromanaging" metabolic syndrome
Ramirez, Cristina M; Goedeke, Leigh; Fernandez-Hernando, Carlos
2011 Oct 1;10(19):3249-3252, Cell cycle
Metabolic diseases are characterized by the failure of regulatory genes or enzymes to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators of metabolic homeostasis, recent discoveries have shown the remarkable role of small non-coding RNAs (microRNAs) in the post-transcriptional regulation of a number of genes, and their involvement in many pathological states, such as diabetes, atherosclerosis and cancer. Of note is microRNA-33 (miR-33), an intronic microRNA (miRNA) located within the sterol regulatory element-binding protein (SREBP) genes, one of the master regulators of cholesterol and fatty acid metabolism. We have recently shown that miR-33 regulates cholesterol efflux and high-density lipoprotein (HDL) formation, as well as fatty acid oxidation and insulin signaling. These results describe a model in which miR-33 works in concert with its host genes to ensure that the cell's metabolic state is balanced, thus highlighting the clinical potential of miRNAs as novel therapeutic targets for treating cardiometabolic diseases
— id: 139919, year: 2011, vol: 10, page: 3249, stat: Journal Article,

Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides
Rayner, Katey J; Esau, Christine C; Hussain, Farah N; McDaniel, Allison L; Marshall, Stephanie M; van Gils, Janine M; Ray, Tathagat D; Sheedy, Frederick J; Goedeke, Leigh; Liu, Xueqing; Khatsenko, Oleg G; Kaimal, Vivek; Lees, Cynthia J; Fernandez-Hernando, Carlos; Fisher, Edward A; Temel, Ryan E; Moore, Kathryn J
2011 Oct 20;478(7369):404-407, Nature
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
— id: 139483, year: 2011, vol: 478, page: 404, stat: Journal Article,

Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis
Rayner, Katey J; Sheedy, Frederick J; Esau, Christine C; Hussain, Farah N; Temel, Ryan E; Parathath, Saj; van Gils, Janine M; Rayner, Alistair J; Chang, Aaron N; Suarez, Yajaira; Fernandez-Hernando, Carlos; Fisher, Edward A; Moore, Kathryn J
2011 Jul 1;121(7):2921-2931, Journal of clinical investigation
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
— id: 136939, year: 2011, vol: 121, page: 2921, stat: Journal Article,

The PLIN4 Variant rs8887 Modulates Obesity Related Phenotypes in Humans through Creation of a Novel miR-522 Seed Site
Richardson, Kris; Louie-Gao, Qiong; Arnett, Donna K; Parnell, Laurence D; Lai, Chao-Qiang; Davalos, Alberto; Fox, Caroline S; Demissie, Serkalem; Cupples, L Adrienne; Fernandez-Hernando, Carlos; Ordovas, Jose M
2011 ;6(4):e17944-e17944, PLoS ONE
PLIN4 is a member of the PAT family of lipid storage droplet (LSD) proteins. Associations between seven single nucleotide polymorphisms (SNPs) at human PLIN4 with obesity related phenotypes were investigated using meta-analysis followed by a determination if these phenotypes are modulated by interactions between PLIN4 SNPs and dietary PUFA. Samples consisted of subjects from two populations of European ancestry. We demonstrated association of rs8887 with anthropometrics. Meta-analysis demonstrated significant interactions between the rs8887 minor allele with PUFA n3 modulating anthropometrics. rs884164 showed interaction with both n3 and n6 PUFA modulating anthropometric and lipid phenotypes. In silico analysis of the PLIN4 3'UTR sequence surrounding the rs8887 minor A allele predicted a seed site for the human microRNA-522 (miR-522), suggesting a functional mechanism. Our data showed that a PLIN4 3'UTR luciferase reporter carrying the A allele of rs8887 was reduced in response to miR-522 mimics compared to the G allele. These results suggest variation at the PLIN4 locus, and its interaction with PUFA as a modulator of obesity related phenotypes, acts in part through creation of a miR-522 regulatory site
— id: 133425, year: 2011, vol: 6, page: e17944, stat: Journal Article,

Endothelial-specific overexpression of caveolin-1 accelerates atherosclerosis in apolipoprotein E-deficient mice
Fernandez-Hernando, Carlos; Yu, Jun; Davalos, Alberto; Prendergast, Jay; Sessa, William C
2010 Aug;177(2):998-1003, American journal of pathology
Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Genetic ablation of Cav-1 on an apolipoprotein E knockout background inhibits the progression of atherosclerosis, whereas re-expression of Cav-1 in the endothelium promotes lesion expansion. Although Cav-1-null mice are useful to delineate the importance of caveolae in atherosclerosis, there are additional problems that are difficult to dissect because loss of Cav-1 abolishes both the caveolae organelle as well as the Cav-1-mediated signaling pathways. To study how Cav-1 influences the progression of atherosclerosis in mice with caveolae, we generated a transgenic mouse that overexpresses Cav-1 in the endothelial cells in an apolipoprotein E-deficient background. We found that endothelial-specific overexpression of Cav-1 enhanced the progression of atherosclerosis in mice. Mechanistically, overexpression of Cav-1 reduced endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1 in vivo
— id: 133802, year: 2010, vol: 177, page: 998, stat: Journal Article,

microRNAs and cholesterol metabolism
Moore, Kathryn J; Rayner, Katey J; Suarez, Yajaira; Fernandez-Hernando, Carlos
2010 Dec;21(12):699-706, Trends in endocrinology & 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
— id: 138240, year: 2010, vol: 21, page: 699, stat: Journal Article,

Mir-33 Coordinates Genes Regulating Cholesterol Homeostasis
Rayner, Katey J.; Suarez, Yajaira; Davalos, Alberto; Parathath, Saj; Fitzgerald, Michael L.; Tamehiro, Norimasa; Fisher, Edward A.; Moore, Kathryn J.; Fernandez-Hernando, Carlos
2010 NOV ;30(11):E191-E191, Arteriosclerosis, thrombosis, & vascular biology
— id: 117305, year: 2010, vol: 30, page: E191, stat: Journal Article,

MiR-33 contributes to the regulation of cholesterol homeostasis
Rayner, Katey J; Suarez, Yajaira; Davalos, Alberto; Parathath, Saj; Fitzgerald, Michael L; Tamehiro, Norimasa; Fisher, Edward A; Moore, Kathryn J; Fernandez-Hernando, Carlos
2010 Jun 18;328(5985):1570-1573, Science
Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element-binding factor-2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate-binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux
— id: 138135, year: 2010, vol: 328, page: 1570, stat: Journal Article,

Endothelial caveolin-1 regulates pathologic angiogenesis in a mouse model of colitis
Chidlow, John H Jr; Greer, Joshua J M; Anthoni, Christoph; Bernatchez, Pascal; Fernandez-Hernando, Carlos; Bruce, Megan; Abdelbaqi, Maisoun; Shukla, Deepti; Granger, D Neil; Sessa, William C; Kevil, Christopher G
2009 Feb;136(2):575-84.e2, Gastroenterology
BACKGROUND & AIMS: Increased vascular density has been associated with progression of human inflammatory bowel diseases (IBDs) and animal models of colitis. Pathologic angiogenesis in chronically inflamed tissues is mediated by several factors that are regulated at specialized lipid rafts known as caveolae. Caveolin-1 (Cav-1), the major structural protein of caveolae in endothelial cells, is involved in the regulation of angiogenesis, so we investigated its role in experimental colitis. METHODS: Colitis was induced by administration of dextran sodium sulfate to wild-type and Cav-1(-/-) mice, as well as Cav-1(-/-) mice that overexpress Cav-1 only in the endothelium. Colon tissues were analyzed by histologic analyses. Leukocyte recruitment was analyzed by intravital microscopy; angiogenesis was evaluated by immunohistochemistry and in vivo disk assays. RESULTS: Cav-1 protein levels increased after the induction of colitis in wild-type mice. In Cav-1(-/-) mice or mice given a Cav-1 inhibitory peptide, the colitis histopathology scores, vascular densities, and levels of inflammatory infiltrates decreased significantly compared with controls. Lower levels of leukocyte and platelet rolling and adhesion colitis also were observed in Cav-1(-/-) mice and mice given a Cav-1 inhibitory peptide, compared with controls. Cav-1(-/-) mice that received transplants of wild-type bone marrow had a lower colitis score than wild-type mice. Data from mice that overexpress Cav-1 only in the endothelium indicated that endothelial Cav-1 is the critical regulator of colitis. Genetic deletion or pharmacologic inhibition of endothelial Cav-1 also significantly decreased vascular densities and angiogenesis scores, compared with controls. CONCLUSIONS: Endothelial Cav-1 mediates angiogenesis in experimental colitis. Modulation of Cav-1 could provide a novel therapeutic target for IBD
— id: 103229, year: 2009, vol: 136, page: 575, stat: Journal Article,

Akt1 is critical for acute inflammation and histamine-mediated vascular leakage
Di Lorenzo, Annarita; Fernandez-Hernando, Carlos; Cirino, Giuseppe; Sessa, William C
2009 Aug 25;106(34):14552-14557, Proceedings of the National Academy of Sciences of the United States of America
Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. Akt1(-/-) mice showed a markedly reduced edema versus Akt2(-/-) and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in Akt1(-/-) versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and leukocyte extravasation
— id: 103239, year: 2009, vol: 106, page: 14552, stat: Journal Article,

Absence of Akt1 Reduces Vascular Smooth Muscle Cell Migration and Survival and Induces Features of Plaque Vulnerability and Cardiac Dysfunction During Atherosclerosis
Fernandez-Hernando, Carlos; Jozsef, Levente; Jenkins, Deborah; Di Lorenzo, Annarita; Sessa, William C
2009 Dec;29(12):2033-2040, Arteriosclerosis, thrombosis, & vascular biology
OBJECTIVE: Deletion of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Vascular smooth muscle cells (VSMCs) are an important component of atherosclerotic plaques, responsible for promoting plaque stability in advanced lesions. Fibrous caps of unstable plaques contain less collagen and ECM components and fewer VSMCs than caps from stable lesions. Here, we investigated the role of Akt1 in VSMC proliferation, migration, and oxidative stress-induced apoptosis. In addition, we also characterized the atherosclerotic plaque morphology and cardiac function in an atherosclerosis-prone mouse model deficient in Akt1. METHODS AND RESULTS: Absence of Akt1 reduces VSMC proliferation and migration. Mechanistically, the proliferation and migratory phenotype found in Akt1-null VSMCs was linked to reduced Rac-1 activity and MMP-2 secretion. Serum starvation and stress-induced apoptosis was enhanced in Akt1 null VSMCs as determined by flow cytometry using Annexin V/PI staining. Immunohistochemical analysis of atherosclerotic plaques from Akt1(-)(/)(-ApoE)(-)(/)(-) mice showed a dramatic increase in plaque vulnerability characteristics such as enlarged necrotic core and reduced fibrous cap and collagen content. Finally, we show evidence of myocardial infarcts and cardiac dysfunction in Akt1(-)(/)(-ApoE)(-)(/)(-) mice analyzed by immunohistochemistry and echocardiography, respectively. CONCLUSIONS: Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress-induced apoptosis. Absence of Akt1 induces features of plaque vulnerability and cardiac dysfunction in a mouse model of atherosclerosis
— id: 103243, year: 2009, vol: 29, page: 2033, stat: Journal Article,

Genetic evidence supporting a critical role of endothelial caveolin-1 during the progression of atherosclerosis
Fernandez-Hernando, Carlos; Yu, Jun; Suarez, Yajaira; Rahner, Christoph; Davalos, Alberto; Lasuncion, Miguel A; Sessa, William C
2009 Jul;10(1):48-54, Cell metabolism
The accumulation of LDL-derived cholesterol in the artery wall is the initiating event that causes atherosclerosis. However, the mechanisms that lead to the initiation of atherosclerosis are still poorly understood. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in promoting atherogenesis. Mice were generated lacking Cav-1 and apoE but expressing endothelial-specific Cav-1 in the double knockout background. Genetic ablation of Cav-1 on an apoE knockout background inhibits the progression of atherosclerosis, while re-expression of Cav-1 in the endothelium promotes lesion expansion. Mechanistically, the loss of Cav-1 reduces LDL infiltration into the artery wall, promotes nitric oxide production, and reduces the expression of leukocyte adhesion molecules, effects completely reversed in transgenic mice. In summary, this unique model provides physiological evidence supporting the important role of endothelial Cav-1 expression in regulating the entry of LDL into the vessel wall and the initiation of atherosclerosis
— id: 103238, year: 2009, vol: 10, page: 48, stat: Journal Article,

Nogo-B receptor stabilizes Niemann-Pick type C2 protein and regulates intracellular cholesterol trafficking
Harrison, Kenneth D; Miao, Robert Qing; Fernandez-Hernando, Carlos; Suarez, Yajaira; Davalos, Alberto; Sessa, William C
2009 Sep;10(3):208-218, Cell metabolism
The Nogo-B receptor (NgBR) is a recently identified receptor for the N terminus of reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast two-hybrid screen for interacting proteins, using the C-terminal domain as bait, and identified Niemann-Pick type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR, and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER) and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR lead to a decrease in NPC2 levels, increased intracellular cholesterol accumulation, and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking
— id: 103241, year: 2009, vol: 10, page: 208, stat: Journal Article,

Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-Delta24-reductase-deficient cell line
Rodriguez-Acebes, Sara; de la Cueva, Paloma; Fernandez-Hernando, Carlos; Ferruelo, Antonio J; Lasuncion, Miguel A; Rawson, Robert B; Martinez-Botas, Javier; Gomez-Coronado, Diego
2009 Jun 1;420(2):305-315, Biochemical journal
Cholesterol homoeostasis is critical for cell viability and proliferation. The SREBP (sterol regulatory element-binding protein) pathway is crucial for the maintenance of cholesterol homoeostasis. This pathway is controlled by cholesterol and cholesterol-derived oxysterols. J774 cells cannot convert desmosterol into cholesterol, a defect resulting from the absence of mRNA for sterol-Delta24-reductase. Using J774 cells, we addressed the capacity of desmosterol to replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway. J774 cells were able to grow indefinitely after the virtually total replacement of cholesterol by desmosterol (J774-D cells). Inhibition of sterol biosynthesis with lovastatin suppressed J774-D cell proliferation. Desmosterol prevented this effect, but its analogue, cholest-5,22-trans-dien-3beta-ol, did not. Addition of desmosterol inhibited processing of SREBP-1 and -2 and also reduced the expression of SREBP-targeted genes. As occurs in cholesterol-containing cells, 25-hydroxycholesterol was more potent than desmosterol or cholesterol in suppressing these processes. Moreover, desmosterol addition enhanced the expression of Abca1 and Srebf1c, two LXR (liver X receptor)-targeted genes. To test the ability of endogenously produced desmosterol to regulate gene expression, J774-D cells were pretreated with lovastatin to inhibit sterol biosynthesis. After removal of the inhibitor the expression of SREBP-targeted genes decreased and that of an LXR-targeted gene increased, reaching control levels. Our results demonstrate that the virtually complete replacement of cholesterol by desmosterol is compatible with cell growth and the functioning of the SREBP pathway. In these cells, desmosterol suppresses SREBP processing and targeted gene expression, and it is especially effective activating LXR-targeted genes
— id: 103234, year: 2009, vol: 420, page: 305, stat: Journal Article,

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair
Yu, Jun; Fernandez-Hernando, Carlos; Suarez, Yajaira; Schleicher, Michael; Hao, Zhengrong; Wright, Paulette L; DiLorenzo, Annarita; Kyriakides, Themis R; Sessa, William C
2009 Oct 13;106(41):17511-17516, Proceedings of the National Academy of Sciences of the United States of America
Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control
— id: 103244, year: 2009, vol: 106, page: 17511, stat: Journal Article,

Dose-dependent dual effects of cholesterol and desmosterol on J774 macrophage proliferation
Rodriguez-Acebes, Sara; de la Cueva, Paloma; Ferruelo, Antonio J; Fernandez-Hernando, Carlos; Lasuncion, Miguel A; Martinez-Botas, Javier; Gomez-Coronado, Diego
2008 Dec 12;377(2):484-488, Biochemical & biophysical research communications
We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 microM lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition of mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation
— id: 103225, year: 2008, vol: 377, page: 484, stat: Journal Article,

Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence
Schleicher, Michael; Shepherd, Benjamin R; Suarez, Yajaira; Fernandez-Hernando, Carlos; Yu, Jun; Pan, Yong; Acevedo, Lisette M; Shadel, Gerald S; Sessa, William C
2008 Jan 14;180(1):101-112, Journal of cell biology
Prohibitin 1 (PHB1) is a highly conserved protein that is mainly localized to the inner mitochondrial membrane and has been implicated in regulating mitochondrial function in yeast. Because mitochondria are emerging as an important regulator of vascular homeostasis, we examined PHB1 function in endothelial cells. PHB1 is highly expressed in the vascular system and knockdown of PHB1 in endothelial cells increases mitochondrial production of reactive oxygen species via inhibition of complex I, which results in cellular senescence. As a direct consequence, both Akt and Rac1 are hyperactivated, leading to cytoskeletal rearrangements and decreased endothelial cell motility, e.g., migration and tube formation. This is also reflected in an in vivo angiogenesis assay, where silencing of PHB1 blocks the formation of functional blood vessels. Collectively, our results provide evidence that PHB1 is important for mitochondrial function and prevents reactive oxygen species-induced senescence and thereby maintains the angiogenic capacity of endothelial cells
— id: 103213, year: 2008, vol: 180, page: 101, stat: Journal Article,

Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis
Suarez, Yajaira; Fernandez-Hernando, Carlos; Yu, Jun; Gerber, Scott A; Harrison, Kenneth D; Pober, Jordan S; Iruela-Arispe, M Luisa; Merkenschlager, Matthias; Sessa, William C
2008 Sep 16;105(37):14082-14087, Proceedings of the National Academy of Sciences of the United States of America
Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the up-regulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response
— id: 103223, year: 2008, vol: 105, page: 14082, stat: Journal Article,

Phospholipase C beta3 deficiency leads to macrophage hypersensitivity to apoptotic induction and reduction of atherosclerosis in mice
Wang, Zhenglong; Liu, Bei; Wang, Ping; Dong, Xuemei; Fernandez-Hernando, Carlos; Li, Zhong; Hla, Timothy; Li, Zihai; Claffey, Kevin; Smith, Jonathan D; Wu, Dianqing
2008 Jan;118(1):195-204, Journal of clinical investigation
Atherosclerosis is an inflammatory disease that is associated with monocyte recruitment and subsequent differentiation into lipid-laden macrophages at sites of arterial lesions, leading to the development of atherosclerotic plaques. PLC is a key member of signaling pathways initiated by G protein-coupled ligands in macrophages. However, the role of this enzyme in the regulation of macrophage function is not known. Here, we studied macrophages from mice lacking PLC beta2, PLC beta3, or both PLC isoforms and found that PLC beta3 is the major functional PLC beta isoform in murine macrophages. Although PLC beta3 deficiency did not affect macrophage migration, adhesion, or phagocytosis, it resulted in macrophage hypersensitivity to multiple inducers of apoptosis. PLC beta3 appeared to regulate this sensitivity via PKC-dependent upregulation of Bcl-XL. The significance of PLC beta signaling in vivo was examined using the apoE-deficient mouse model of atherosclerosis. Mice lacking both PLC beta3 and apoE exhibited fewer total macrophages and increased macrophage apoptosis in atherosclerotic lesions, as well as reduced atherosclerotic lesion size when compared with mice lacking only apoE. These results demonstrate what we believe to be a novel role for PLC activity in promoting macrophage survival in atherosclerotic plaques and identify PLC beta3 as a potential target for treatment of atherosclerosis
— id: 103212, year: 2008, vol: 118, page: 195, stat: Journal Article,

Myoferlin regulates vascular endothelial growth factor receptor-2 stability and function
Bernatchez, Pascal N; Acevedo, Lisette; Fernandez-Hernando, Carlos; Murata, Takahisa; Chalouni, Cecile; Kim, Jiae; Erdjument-Bromage, Hediye; Shah, Vijay; Gratton, Jean-Philippe; McNally, Elizabeth M; Tempst, Paul; Sessa, William C
2007 Oct 19;282(42):30745-30753, Journal of biological chemistry
Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling
— id: 103209, year: 2007, vol: 282, page: 30745, stat: Journal Article,

Loss of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease
Fernandez-Hernando, Carlos; Ackah, Eric; Yu, Jun; Suarez, Yajaira; Murata, Takahisa; Iwakiri, Yasuko; Prendergast, Jay; Miao, Robert Q; Birnbaum, Morris J; Sessa, William C
2007 Dec;6(6):446-457, Cell metabolism
The Akt signaling pathway controls several cellular functions in the cardiovascular system; however, its role in atherogenesis is unknown. Here, we show that the genetic ablation of Akt1 on an apolipoprotein E knockout background (ApoE(-/-)Akt1(-/-)) increases aortic lesion expansion and promotes coronary atherosclerosis. Mechanistically, lesion formation is due to the enhanced expression of proinflammatory genes and endothelial cell and macrophage apoptosis. Bone marrow transfer experiments showing that macrophages from ApoE(-/-)Akt1(-/-) donors were not sufficient to worsen atherogenesis when transferred to ApoE(-/-) recipients suggest that lesion expansion in the ApoE(-/-)Akt1(-/-) strain might be of vascular origin. In the vessel wall, the loss of Akt1 increases inflammatory mediators and reduces eNOS phosphorylation, suggesting that Akt1 exerts vascular protection against atherogenesis. The presence of coronary lesions in ApoE(-/-)Akt1(-/-) mice provides a new model for studying the mechanisms of acute coronary syndrome in humans
— id: 103211, year: 2007, vol: 6, page: 446, stat: Journal Article,

Cholesterol starvation induces differentiation of human leukemia HL-60 cells
Sanchez-Martin, Carolina C; Davalos, Alberto; Martin-Sanchez, Covadonga; de la Pena, Gema; Fernandez-Hernando, Carlos; Lasuncion, Miguel A
2007 Apr 1;67(7):3379-3386, Cancer research
Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G(2) phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-alpha demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D(3), which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal-regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Delta(7)-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy
— id: 103203, year: 2007, vol: 67, page: 3379, stat: Journal Article,

Dicer dependent microRNAs regulate gene expression and functions in human endothelial cells
Suarez, Yajaira; Fernandez-Hernando, Carlos; Pober, Jordan S; Sessa, William C
2007 Apr 27;100(8):1164-1173, Circulation research
Dicer is a key enzyme involved in the maturation of microRNAS (miRNAs). miRNAs have been shown to be regulators of gene expression participating in the control of a wide range of physiological pathways. To assess the role of Dicer and consequently the importance of miRNAs in the biology and functions of human endothelial cells (EC) during angiogenesis, we globally reduced miRNAs in ECs by specific silencing Dicer using siRNA and examined the effects on EC phenotypes in vitro. The knockdown of Dicer in ECs altered the expression (mRNA and/or protein) of several key regulators of endothelial biology and angiogenesis, such as TEK/Tie-2, KDR/VEGFR2, Tie-1, endothelial nitric oxide synthase and IL-8. Although, Dicer knockdown increased activation of the endothelial nitric oxide synthase pathway it reduced proliferation and cord formation of EC in vitro. The miRNA expression profile of EC revealed 25 highly expressed miRNAs in human EC and using miRNA mimicry, miR-222/221 regulates endothelial nitric oxide synthase protein levels after Dicer silencing. Collectively, these results indicate that maintenance and regulation of endogenous miRNA levels via Dicer mediated processing is critical for EC gene expression and functions in vitro
— id: 103202, year: 2007, vol: 100, page: 1164, stat: Journal Article,

Red grape juice polyphenols alter cholesterol homeostasis and increase LDL-receptor activity in human cells in vitro
Davalos, Alberto; Fernandez-Hernando, Carlos; Cerrato, Francisca; Martinez-Botas, Javier; Gomez-Coronado, Diego; Gomez-Cordoves, Carmen; Lasuncion, Miguel A
2006 Jul;136(7):1766-1773, Journal of nutrition
Red grape juice (RGJ) polyphenols have been shown to reduce circulating levels of LDL cholesterol and to increase LDL receptor activity. To explore the effect of RGJ-derived polyphenols on intracellular cholesterol homeostasis, human hepatocarcinoma HepG2 and promyelocytic HL-60 cell lines were incubated in serum-free medium, with or without LDL, in the presence or absence of RGJ. In the presence of LDL, RGJ increased both the activity and cell surface expression of the LDL receptor, and increased the cell total cholesterol content. In cells exposed to LDL, RGJ also increased levels of the active form of sterol regulatory element-binding protein-1 and mRNA expression of the LDL receptor and hydroxymethylglutaryl-CoA reductase. In contrast, RGJ caused a marked reduction in the expression of CYP7A1, apolipoprotein B, ABCA1, and ABCG5. Experiments using the acyl-CoA cholesterol acyltransferase inhibitor S-58035 indicated that no measurable free cholesterol from endocytosed LDL reaches the endoplasmic reticulum in cells treated with RGJ. Finally, fluorescence microscopy revealed that in RGJ-treated cells, DiI-labeled LDL did not colocalize with CD63, a protein localized at steady state in the internal vesicles of late endosomes. These results indicate that RGJ polyphenols disrupt or delay LDL trafficking through the endocytic pathway, thus preventing LDL cholesterol from exerting regulatory effects on intracellular lipid homeostasis
— id: 103193, year: 2006, vol: 136, page: 1766, stat: Journal Article,

Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase
Fernandez-Hernando, Carlos; Fukata, Masaki; Bernatchez, Pascal N; Fukata, Yuko; Lin, Michelle I; Bredt, David S; Sessa, William C
2006 Jul 31;174(3):369-377, Journal of cell biology
Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function
— id: 103195, year: 2006, vol: 174, page: 369, stat: Journal Article,

Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression
Fernandez, Carlos; Martin, Miguel; Gomez-Coronado, Diego; Lasuncion, Miguel A
2005 May;46(5):920-929, Journal of lipid research
Cholesterol is a major lipid component of the plasma membrane in animal cells. In addition to its structural requirement, cholesterol is essential in cell proliferation and other cell processes. The aim of the present study was to elucidate the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression, compared with other sterols of the cholesterol biosynthesis pathway. Human promyelocytic HL-60 cells were cultured in cholesterol-free medium and treated with different distal inhibitors of cholesterol biosynthesis (zaragozic acid, SKF 104976, SR 31747, BM 15766, and AY 9944), which allow the synthesis of isoprenoid derivatives and different sets of sterol intermediates, but not cholesterol. The results showed that only the inhibition of sterol Delta7-reductase was compatible with cell proliferation. Blocking cholesterol biosynthesis upstream of this enzyme resulted in the inhibition of cell proliferation and cell cycle arrest selectively in G2/M phase
— id: 104335, year: 2005, vol: 46, page: 920, stat: Journal Article,

Lovastatin-induced PC-12 cell differentiation is associated with RhoA/RhoA kinase pathway inactivation
Fernandez-Hernando, Carlos; Suarez, Yajaira; Lasuncion, Miguel A
2005 Aug;29(4):591-602, Molecular & cellular neurosciences
Relatively little is known about the relationship between cellular lipid composition and the ability of neuroblasts to elaborate axonal and dendritic processes. We have studied the role of cholesterol and non-sterol isoprenoids during neurite outgrowth in PC-12 cells using inhibitors of cholesterol biosynthesis that act at different points in the biosynthetic pathway. We provide evidence that inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leads to extensive, sterol-dependent neurite outgrowth, via a mechanism that is independent of the requirement for sterols during proliferation. This effect is prevented by non-sterol mevalonate derivatives, suggesting the involvement of protein prenylation in the regulation of neurite outgrowth. Furthermore, we show that lovastatin inhibits both RhoA activation and Cofilin phosphorylation, while geranylgeraniol reverses these effects. Finally, the effect of geranylgeraniol on neurite outgrowth is prevented by Y-27632, an inhibitor of RhoA kinase. Taken together, our results suggest that inhibition of geranylgeraniol synthesis causes sterol-dependent neurite outgrowth in a process that is mediated by inhibition of RhoA signaling
— id: 103186, year: 2005, vol: 29, page: 591, stat: Journal Article,

Sterol stringency of proliferation and cell cycle progression in human cells
Suarez, Yajaira; Fernandez, Carlos; Ledo, Beatriz; Martin, Miguel; Gomez-Coronado, Diego; Lasuncion, Miguel A
2005 May 15;1734(2):203-213, Biochimica & biophysica acta
Cholesterol is a major component of the plasma membrane in mammalian cells, where it acts as a modulator of bulk physical state and integrity. In addition to its structural role, cholesterol is essential for proliferation and other cell processes. The present study was undertaken to explore the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression. Comparisons were made between cholesterol and other sterol analogs that differ from cholesterol in three specific elements: the presence of a Delta5 double bond in ring B, the hydroxyl group at C-3, and the presence of an aliphatic side chain. The human leukemia cells HL-60 and MOLT-4 were cultured in cholesterol-free medium and treated with different sterols in the presence or absence of SKF 104976, a competitive inhibitor of lanosterol 14alpha-demethylase that allows the synthesis of isoprenoid derivatives but not cholesterol. Our results show that the beta-hydroxyl group at C-3 and the unsaturated bond at Delta5 are necessary for cell proliferation and cell cycle progression. The sterol analog 5alpha-cholestan-3beta-ol (dihydrocholesterol), which is saturated at Delta5 and has an A/B ring junction in the trans configuration, was also able to support cell growth. However, 5beta-cholestan-3beta-ol and 5beta-cholestan-3alpha-ol, both of which have an A/B ring junction in the cis configuration, were totally ineffective in supporting cell growth. Indeed, they produced an inhibition of cell proliferation and arrested the cell cycle specifically in the G2/M phase. These effects of 5beta-cholestanols were abrogated by cholesterol in a concentration-dependent manner. Moreover, 5beta-cholestanols potently inhibited cholesterol biosynthesis and transcription driven by the sterol response element. In addition to providing a description of the structural features of sterols associated with their supporting action on cell proliferation in mammalian cells, the present results demonstrate that selected cholesterol analogs may act as cytostatic agents, interrupting cell cycle progression specifically in the G2/M phase
— id: 103953, year: 2005, vol: 1734, page: 203, stat: Journal Article,

Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation
Fernandez, Carlos; Lobo Md, Maria del Val T; Gomez-Coronado, Diego; Lasuncion, Miguel A
2004 Oct 15;300(1):109-120, Experimental cell research
As an essential component of mammalian cell membranes, cells require cholesterol for proliferation, which is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-CoA. In addition to cholesterol, other non-sterol mevalonate derivatives are necessary for DNA synthesis, such as the phosphorylated forms of isopentane, farnesol, geranylgeraniol, and dolichol. The aim of the present study was to elucidate the role of cholesterol in mitosis. For this, human leukemia cells (HL-60) were incubated in a cholesterol-free medium and treated with SKF 104976, which inhibits cholesterol biosynthesis by blocking sterol 14alpha-demethylase, and the expression of relevant cyclins in the different phases of the cell cycle was analyzed by flow cytometry. Prolonged cholesterol starvation induced the inhibition of cytokinesis and the formation of polyploid cells, which were multinucleated and had mitotic aberrations. Supplementing the medium with cholesterol completely abolished these effects, demonstrating they were specifically due to cholesterol deficiency. This is the first evidence that cholesterol is essential for mitosis completion and that, in the absence of cholesterol, the cells fail to undergo cytokinesis, entered G1 phase at higher DNA ploidy (tetraploidy), and then progressed through S (rereplication) into G2, generating polyploid cells
— id: 104340, year: 2004, vol: 300, page: 109, stat: Journal Article,

Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study
Ruiz-Llorente, Lidia; Ortega-Gutierrez, Silvia; Viso, Alma; Sanchez, Maria G; Sanchez, Ana M; Fernandez, Carlos; Ramos, Jose A; Hillard, Cecilia; Lasuncion, Miguel A; Lopez-Rodriguez, Maria L; Diaz-Laviada, Ines
2004 Feb;141(3):457-467, British journal of pharmacology
The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7+/-0.2 microm and Vmax=3.3+/-0.3 pmol min-1 (10(6) cells)-1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1-8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3+/-0.5 microM, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator.British Journal of Pharmacology (2004) 141, 457-467. doi:10.1038/sj.bjp.0705628
— id: 104341, year: 2004, vol: 141, page: 457, stat: Journal Article,

Synergistic upregulation of low-density lipoprotein receptor activity by tamoxifen and lovastatin
Suarez, Yajaira; Fernandez, Carlos; Gomez-Coronado, Diego; Ferruelo, Antonio J; Davalos, Alberto; Martinez-Botas, Javier; Lasuncion, Miguel A
2004 Nov 1;64(2):346-355, Cardiovascular research
OBJECTIVE: To study the mechanism involved in the cholesterol-lowering activity of tamoxifen, an estrogen receptor (ER) modulator widely used in breast cancer therapy. METHODS AND RESULTS: We used MOLT-4 cells, which do not express estrogen receptors and require important amounts of cholesterol for proliferation. We firstly confirmed that tamoxifen reduced cholesterol biosynthesis by inhibiting sterol Delta(8,7)-isomerase and Delta(24)-reductase activities, which resulted in the accumulation of zymosterol. In cells incubated in the presence of low-density lipoprotein (LDL) (120 microg cholesterol/ml), tamoxifen stimulated LDL receptor activity and expression in a dose-dependent manner, as determined by 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-labeled LDL uptake, LDL receptor expression on the cell surface and LDL receptor mRNA levels. Furthermore, tamoxifen, but not lovastatin, inhibited the egress of LDL-derived cholesterol from lysosomes, as ascertained by filipin staining in both MOLT-4 and HepG2 cells. When studied in combination, especially at relatively high LDL concentrations in the medium, tamoxifen and lovastatin stimulated LDL receptor activity synergistically, which is attributed to the different mechanism of action these drugs exhibit. CONCLUSIONS: The present study demonstrates the stimulation of the LDL receptor by tamoxifen. These results explain the long-known hypolipidemic effect of tamoxifen and support its use, or that of other intracellular cholesterol trafficking inhibitors, in combination with statins for the reduction of plasma LDL cholesterol levels
— id: 103932, year: 2004, vol: 64, page: 346, stat: Journal Article,

Efecto de los fitosteroles sobre la biosintesis de colesterol y la proliferacion en celulas humanas
Fernandez C; Martin M; Gomez-Coronado D; Lasuncion MA
2003 ;15(5):175-183, Clinica e investigacion en arteriosclerosis
Introduccion y objetivos. Se sabe que el consumo de fitosteroles reduce la concentracion de colesterol en plasma, debido a la interferencia de estos compuestos en la absorcion intestinal de colesterol. Aunque en pequena proporcion, los fitosteroles de la dieta tambien se asimilan, y su concentracion en el plasma es mil veces inferior a la del colesterol. Dada su analogia estructural con el colesterol, nos planteamos determinar el efecto de distintos fitosteroles sobre la biosintesis de colesterol en celulas humanas y su repercusion sobre la proliferacion celular. Metodo. Las celulas de la linea promielocitica humana HL-60 se cultivaron en un medio libre de colesterol (DCCM-l) en presencia de los diferentes esteroles en estudio y de [14C]-acetato como precursor para la biosintesis de colesterol. Al cabo de 8 h de incubacion se lisaron las celulas y se extrajeron los lipidos no saponificables, que posteriormente fueron analizados mediante cromatografia liquida de alta resolucion (HPLC). La proliferacion celular se analizo determinando la incorporacion de [3H]-timidina al ADN y por recuento de las celulas. Resultados. Los fitosteroles insaturados en el C22 de la cadena lateral-estigmasterol, brasicasterol y ergosterol- inhibieron la biosintesis de colesterol en celulas HL-60 en concentraciones fisiologicas, de 1 µg/ml, mientras que los otros fitosteroles-ß-sitosterol y campesterol- fueron inactivos incluso en concentraciones de 30 µg/ml. El 5, 22-colestadien-3ß-ol, un esterol no natural que contiene tambien un doble enlace en C22, inhibio la incorporacion de [14C]-acetato a colesterol mucho mas intensamente que los anteriores. El descenso de 14C-colesterol se acompano de un aumento de la radiactividad en desmosterol y tambien, aunque menos intensamente, en 5, 7, 24-colestatrien-3ß-ol. A pesar de estos cambios en la composicion celular de esteroles, los fitosteroles no afectaron la viabilidad ni la proliferacion de las celulas HL-60, lo que sugiere que los esteroles intermediarios pueden suplir al colesterol en sus funciones en la division celular. Conclusiones. Los fitosteroles con doble enlace en C22 inhiben la biosintesis de colesterol a valor de la esterol delta24-reductasa en concentraciones que pueden considerarse fisiologicas, por lo que esta accion puede considerarse un mecanismo adicional para explicar los efectos hipolipemiantes de estos compuestos
— id: 104343, year: 2003, vol: 15, page: 175, stat: Journal Article,

Inhibition of cholesterol biosynthesis by Delta22-unsaturated phytosterols via competitive inhibition of sterol Delta24-reductase in mammalian cells
Fernandez, Carlos; Suarez, Yajaira; Ferruelo, Antonio J; Gomez-Coronado, Diego; Lasuncion, Miguel A
2002 Aug 15;366(Pt 1):109-119, Biochemical journal
Dietary phytosterols are cholesterol-lowering agents that interfere with the intestinal absorption of cholesterol. In the present study, we have studied their effects on cholesterol biosynthesis in human cells, particularly in the sterol-conversion pathway. For this, both Caco-2 (intestinal mucosa) and HL-60 (promyelocytic) human cell lines were incubated with [(14)C]acetate, and the incorporation of radioactivity into sterols was determined using HPLC and radioactivity detection online. Sterols containing a double bond at C-22 in the side chain (stigmasterol, brassicasterol and ergosterol) dramatically inhibited the activity of sterol Delta(24)-reductase, as indicated by the decrease in radioactivity incorporation into cholesterol and the accumulation of its precursors (mainly desmosterol). Phytosterols with the saturated side chain (beta-sitosterol and campesterol) were inactive in this regard. The inhibition of sterol (24)-reductase was confirmed in rat liver microsomes by using (14)C-labelled desmosterol as the substrate. The (22)-unsaturated phytosterols acted as competitive inhibitors of sterol (24)-reductase, with K(i) values (41.1, 42.7 and 36.8 microM for stigmasterol, brassicasterol and ergosterol respectively) similar to the estimated K(m) for desmosterol (26.3 microM). The sterol 5,22-cholestedien-3beta-ol, an unusual desmosterol isomer that lacks the alkyl groups characteristic of phytosterols, acted as a much stronger inhibitor of (24)-reductase (K(i)=3.34 microM). The usually low intracellular concentrations of the physiological substrates of (24)-reductase explains the strong inhibition of cholesterol biosynthesis that these compounds exert in cells. Given that inhibition of sterol (24)-reductase was achieved at physiologically relevant concentrations, it may represent an additional mechanism for the cholesterol-lowering action of phytosterols, and opens up the possibility of using certain (22)-unsaturated sterols as effective hypocholesterolaemic agents
— id: 103883, year: 2002, vol: 366, page: 109, stat: Journal Article,

Differential effects of ergosterol and cholesterol on Cdk1 activation and SRE-driven transcription
Suarez, Yajaira; Fernandez, Carlos; Ledo, Beatriz; Ferruelo, Antonio J; Martin, Miguel; Vega, Miguel A; Gomez-Coronado, Diego; Lasuncion, Miguel A
2002 Mar;269(6):1761-1771, European journal of biochemistry
Cholesterol is essential for cell growth and division, but whether this is just a consequence of its use in membrane formation or whether it also elicits regulatory actions in cell cycle machinery remains to be established. Here, we report on the specificity of this action of cholesterol in human cells by comparing its effects with those of ergosterol, a yeast sterol structurally similar to cholesterol. Inhibition of cholesterol synthesis by means of SKF 104976 in cells incubated in a cholesterol-free medium resulted in cell proliferation inhibition and cell cycle arrest at G2/M phase. These effects were abrogated by cholesterol added to the medium but not by ergosterol, despite that the latter was used by human cells and exerted similar homeostatic actions, as the regulation of the transcription of an SRE-driven gene construct. In contrast to cholesterol, ergosterol was unable to induce cyclin B1 expression, to activate Cdk1 and to resume cell cycle in cells previously arrested at G2. This lack of effect was not due to cytotoxicity, as cells exposed to ergosterol remained viable and, upon supplementing with UCN-01, an activator of Cdk1, they progressed through mitosis. However, in the presence of suboptimal concentrations of cholesterol, ergosterol exerted synergistic effects on cell proliferation. This is interpreted on the basis of the differential action of these sterols, ergosterol contributing to cell membrane formation and cholesterol being required for Cdk1 activation. In summary, the action of cholesterol on G2 traversal is highly specific and exerted through a mechanism different to that used for cholesterol homeostasis, reinforcing the concept that cholesterol is a specific regulator of cell cycle progression in human cells
— id: 103870, year: 2002, vol: 269, page: 1761, stat: Journal Article,

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives
Martinez-Botas, J; Ferruelo, A J; Suarez, Y; Fernandez, C; Gomez-Coronado, D; Lasuncion, M A
2001 Jun 29;1532(3):185-194, Biochimica & biophysica acta
The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling
— id: 104342, year: 2001, vol: 1532, page: 185, stat: Journal Article,