Publications by authors named "Michihisa Umetani"

33 Publications

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

Angew Chem Int Ed Engl 2021 Mar 8. Epub 2021 Mar 8.

Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, 77030, USA.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K ≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC ≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202100345DOI Listing
March 2021

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

FASEB J 2021 Jan;35(1):e21262

Department of Future Basic Medicine, Nara Medical University, Nara, Japan.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202002077RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771643PMC
January 2021

Obesity and Cancer: 27-Hydroxycholesterol, the Missing Link.

Int J Mol Sci 2020 Jul 8;21(14). Epub 2020 Jul 8.

Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5056, USA.

Obesity is currently affecting more than 40% of the Americans, and if it progresses with this rate, soon one out of two Americans will be obese. Obesity is an important risk factor for several disorders including cardiovascular disease, the first cause of death in the United States. Cancer follows as the second deadliest disease, and a link between obesity and cancer has been suggested. However, it is very hard to establish an exact connection between obesity and cancers due to the multifactorial nature of obesity. Hypercholesterolemia is a comorbidity of obesity and also linked to several cancers. Recently a cholesterol metabolite 27-hydroxycholesterol (27HC) was found to be an endogenous selective estrogen receptor modulator (SERM), which opened new doors toward several interesting studies on the role of this molecule in biological disorders. It is speculated that 27HC might be the missing link in the obesity and cancer chain. Here, we explored the effects of 27-hydroxycholesterol on obesity and cancers with a focus on the SERM capacity of 27HC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21144822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404106PMC
July 2020

Vascular Protection by Exercise in Obesity: Inflammasome-associated Mechanisms.

Med Sci Sports Exerc 2020 12;52(12):2538-2545

Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, Houston, TX.

Purpose: The nodlike receptor family pyrin domain containing 3 (NLRP3) inflammasome is a critical player in vascular pathology as it regulates caspase-1-mediated interleukin (IL)-1β processing. Physical activity ameliorates obesity-induced inflammation and vascular dysfunction, but the mechanisms responsible for these positive changes are incompletely understood. Here, the protective effect of physical activity on the inflammasome-associated vascular dysfunction in obesity and its putative mechanisms were investigated.

Methods: Mice were fed a control low-fat diet (LFD) or a high-fat diet (HFD; 45% of calories from fat) and provided with running wheel access (LF-RUN or HF-RUN) or denied wheel access for our sedentary condition (LF-SED or HF-SED). The NLRP3 inflammasome-associated pathway, including NLRP3, caspase-1, and IL-1β, in mice aorta was examined by RT-qPCR and FLICA and DAB staining. The protein expression of zonula occluden-1 (ZO-1), ZO-2, adiponectin (APN), and adiponectin receptor 1 (AdipoR1) in aortic endothelial cells was determined by immunofluorescence double staining. Intracellular reactive oxidative stress and nitric oxide (NO) production were monitored with fluorescence probes, dihydroethidium, and diaminofluorecein.

Results: HFD increased caspase-1 and IL-1β at mRNA and protein levels in endothelial cells of the aorta, and this was attenuated by voluntary running. HFD decreased ZO-1 and ZO-2 expression and reduced APN and AdipoR1 signaling; these were restored by running. The elevated intracellular superoxide (O2) production observed in HF-SED was ameliorated in HF-RUN. Finally, HF-RUN improved NO production in the aorta compared with HF-SED.

Conclusions: Our findings suggest that voluntary running ameliorates mechanisms associated with vascular dysfunction by suppressing NLRP3 inflammasome, improving NO production, and reducing oxidative stress. Such benefits of physical activity may be, at least in part, associated with APN-AdipoR1 signaling and tight junction protein expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1249/MSS.0000000000002419DOI Listing
December 2020

Gene expression dynamic analysis reveals co-activation of Sonic Hedgehog and epidermal growth factor followed by dynamic silencing.

Oncotarget 2020 Apr 14;11(15):1358-1372. Epub 2020 Apr 14.

Department of Biostatistics and Data Science, School of Public Heath, University of Texas Health Science Center at Houston, Houston, TX, USA.

Aberrant activation of the Sonic Hedgehog (SHH) gene is observed in various cancers. Previous studies have shown a "cross-talk" effect between the canonical Hedgehog signaling pathway and the Epidermal Growth Factor (EGF) pathway when SHH is active in the presence of EGF. However, the precise mechanism of the cross-talk effect on the entire gene population has not been investigated. Here, we re-analyzed publicly available data to study how SHH and EGF cooperate to affect the dynamic activity of the gene population. We used genome dynamic analysis to explore the expression profiles under different conditions in a human medulloblastoma cell line. Ordinary differential equations, equipped with solid statistical and computational tools, were exploited to extract the information hidden in the dynamic behavior of the gene population. Our results revealed that EGF stimulation plays a dominant role, overshadowing most of the SHH effects. We also identified cross-talk genes that exhibited expression profiles dissimilar to that seen under SHH or EGF stimulation alone. These unique cross-talk patterns were validated in a cell culture model. These cross-talk genes identified here may serve as valuable markers to study or test for EGF co-stimulatory effects in an SHH+ environment. Furthermore, these cross-talk genes may play roles in cancer progression, thus they may be further explored as cancer treatment targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.27547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170495PMC
April 2020

Editorial: Cholesterol and Oxysterols as Signal Molecules in Human Pathophysiology and Cancer: Implications for New Therapeutic Strategies.

Front Endocrinol (Lausanne) 2019 23;10:732. Epub 2019 Oct 23.

Department of Pharmacy and Health and Nutritional Science, University of Calabria, Cosenza, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fendo.2019.00732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819365PMC
October 2019

Contribution of transcription factor EB to adipoRon-induced inhibition of arterial smooth muscle cell proliferation and migration.

Am J Physiol Cell Physiol 2019 11 4;317(5):C1034-C1047. Epub 2019 Sep 4.

Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas.

Abnormal vascular smooth muscle cell (SMC) dedifferentiation with increased proliferation and migration during pathological vascular remodeling is associated with vascular disorders, such as atherosclerosis and in-stent restenosis. AdipoRon, a selective agonist of adiponectin receptor, has been shown to protect against vascular remodeling by preventing SMC dedifferentiation. However, the molecular mechanisms that mediate adipoRon-induced SMC differentiation are not well understood. The present study aimed to elucidate the role of transcription factor EB (TFEB), a master regulator of autophagy, in mediating adipoRon's effect on SMCs. In cultured arterial SMCs, adipoRon dose-dependently increased TFEB activation, which is accompanied by upregulated transcription of genes involved in autophagy pathway and enhanced autophagic flux. In parallel, adipoRon suppressed serum-induced cell proliferation and caused cell cycle arrest. Moreover, adipoRon inhibited SMC migration as characterized by wound-healing retardation, F-actin reorganization, and matrix metalloproteinase-9 downregulation. These inhibitory effects of adipoRon on proliferation and migration were attenuated by TFEB gene silencing. Mechanistically, activation of TFEB by adipoRon is dependent on intracellular calcium, but it is not associated with changes in AMPK, ERK1/2, Akt, or molecular target of rapamycin complex 1 activation. Using ex vivo aortic explants, we demonstrated that adipoRon inhibited sprouts that had outgrown from aortic rings, whereas lentiviral TFEB shRNA transduction significantly reversed this effect of adipoRon on aortic rings. Taken together, our results indicate that adipoRon activates TFEB signaling that helps maintain the quiescent and differentiated status of arterial SMCs, preventing abnormal SMC dedifferentiation. This study provides novel mechanistic insights into understanding the therapeutic effects of adipoRon on TFEB signaling and pathological vascular remodeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00294.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879882PMC
November 2019

27-Hydroxycholesterol Promotes Adiposity and Mimics Adipogenic Diet-Induced Inflammatory Signaling.

Endocrinology 2019 10;160(10):2485-2494

Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, Texas.

27-Hydroxycholesterol (27HC) is an abundant cholesterol metabolite and has detrimental effects on the cardiovascular system, whereas its impact on adiposity is not well known. In this study, we found that elevations in 27HC cause increased body weight gain in mice fed a high-fat/high-cholesterol diet in an estrogen receptor α-dependent manner. Regardless of diet type, body fat mass was increased by 27HC without changes in food intake or fat absorption. 27HC did not alter energy expenditure in mice fed a normal chow diet and increased visceral white adipose mass by inducing hyperplasia but not hypertrophy. Although 27HC did not augment adipocyte terminal differentiation, it increased the adipose cell population that differentiates to mature adipocytes. RNA sequencing analysis revealed that 27HC treatment of mice fed a normal chow diet induces inflammatory gene sets similar to those seen after high-fat/high-cholesterol diet feeding, whereas there was no overlap in inflammatory gene expression among any other 27HC administration/diet change combination. Histological analysis showed that 27HC treatment increased the number of total and M1-type macrophages in white adipose tissues. Thus, 27HC promotes adiposity by directly affecting white adipose tissues and by increasing adipose inflammatory responses. Lowering serum 27HC levels may lead to an approach targeting cholesterol to prevent diet-induced obesity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2019-00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760292PMC
October 2019

Tricyclic antidepressant amitriptyline inhibits autophagic flux and prevents tube formation in vascular endothelial cells.

Basic Clin Pharmacol Toxicol 2019 Apr 15;124(4):370-384. Epub 2018 Nov 15.

Department of Pharmacological & Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas.

Amitriptyline is a tricyclic antidepressant and an inhibitor of lysosomal acid sphingomyelinase (ASM). Amitriptyline is well known for its cardiovascular side effects and toxicity in psychiatric patients. However, the mechanisms underlying the cardiovascular side effects of amitriptyline remain largely undefined. This study aimed to determine the effects of amitriptyline on angiogenic capability of vascular endothelial cells in physiological settings and identify its mechanism of action. The ex vivo aortic ring angiogenesis and in vitro-cultured endothelial cell tube formation assay were used to assess the effects of amitriptyline on endothelial angiogenic capability. It was demonstrated that amitriptyline impaired the angiogenesis of aortic rings, which was similar to that found in aortic rings with haploinsufficiency of the ASM gene. In cultured mouse microvascular endothelial cells (MVECs), amitriptyline impaired the proliferation and tube formation under basal condition, which were accompanied by attenuated angiogenic signalling pathways such as endothelial nitric oxide synthase, Akt and Erk1/2 pathways. Mechanistically, amitriptyline inhibited autophagic flux without affecting autophagosome biogenesis at basal condition. ASM gene silencing or autophagy inhibition mimics the inhibitory effects of amitriptyline on endothelial cell proliferation and tube formation. Collectively, our data suggest that amitriptyline inhibits endothelial cell proliferation and angiogenesis via blockade of ASM-autophagic flux axis. It is implicated that the cardiovascular side effects of amitriptyline may be associated with its inhibitory action on physiological angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bcpt.13146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226027PMC
April 2019

Estrogen Receptor Beta-Mediated Modulation of Lung Cancer Cell Proliferation by 27-Hydroxycholesterol.

Front Endocrinol (Lausanne) 2018 23;9:470. Epub 2018 Aug 23.

Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, United States.

27-hydroxycholesterol (27HC) is an abundant cholesterol metabolite in human circulation and promotes breast cancer cell proliferation. Although lung is one of the organs that contain high levels of 27HC, the role of 27HC in lung is unknown. In this study, we found that 27HC promotes lung cancer cell proliferation in an estrogen receptor β (ERβ)-dependent manner. The expression of 27HC-generating enzyme CYP27A1 is higher in lung cancer cells than in normal lung cells. Treatment with 27HC increased cell proliferation in ERβ-positive lung cancer cells, but not in ERα-positive or ER-negative cells. The effect on cell proliferation is specific to 27HC and another oxysterol, 25-hydroxycholesterol that has a similar oxysterol structure with 27HC. Moreover, among ligands for nuclear receptors tested, only estrogen had the proliferative effect, and the effect by 27HC and estrogen was inhibited by ERβ-specific, but not ERα-specific, inhibitors. In addition, the effect by 27HC was not affected by membrane-bound estrogen receptor GPR30. Interestingly, despite the high expression of CYP27A1, endogenously produced 27HC was not the major contributor of the 27HC-induced cell proliferation. Using kinase inhibitors, we found that the effect by 27HC was mediated by the PI3K-Akt signaling pathway. These results suggest that 27HC promotes lung cancer cell proliferation via ERβ and PI3K-Akt signaling. Thus, lowering 27HC levels may lead to a novel approach for the treatment of lung cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fendo.2018.00470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116707PMC
August 2018

Physical activity protects NLRP3 inflammasome-associated coronary vascular dysfunction in obese mice.

Physiol Rep 2018 06;6(12):e13738

Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, Houston, Texas.

Activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome mediates the release of pro-inflammatory cytokine interleukin (IL)-1β and thereby plays a pivotal role in the inflammatory response in vascular pathology. An active lifestyle has beneficial effects on inflammation-associated vascular dysfunction in obesity. However, it remains unclear how physical activity regulates NLRP3 inflammasome-mediated vascular dysfunction in obesity. Therefore, we explored the protective effect of physical activity on NLRP3 inflammasome-associated vascular dysfunction in mouse hearts, and the potential underlying mechanisms. C57BL/6J male mice were randomly divided into four groups: (1) control low-fat diet (LF-SED), (2) LF diet with free access to a voluntary running wheel (LF-RUN), (3) high-fat diet (HF-SED; 45% of calories from fat), and (4) HF-RUN. We examined NLRP3 inflammasome-related signaling pathways, nitric oxide (NO) signaling, and oxidative stress in coronary arterioles to test effects of HFD and physical activity. Voluntary running reduced NLRP3 inflammasome and its downstream effects, caspase-1 and IL-1β in coronary arteriole endothelium of obese mice in immunofluorescence staining. HF-RUN attenuated HFD-dependent endothelial NO synthase (eNOS) reduction and thus increased NO production compared to HF-SED. HFD elevated intracellular superoxide production in coronary arterioles while voluntary running ameliorated oxidative stress. Our findings provide the first evidence that voluntary running attenuates endothelial NLRP3 inflammasome activation in coronary arterioles of HFD feeding mice. Results further suggest that voluntary running improves obesity-induced vascular dysfunction by preserved NO bioavailability via restored expression of eNOS and reduced oxidative stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14814/phy2.13738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014451PMC
June 2018

27-Hydroxycholesterol induces hematopoietic stem cell mobilization and extramedullary hematopoiesis during pregnancy.

J Clin Invest 2017 Sep 7;127(9):3392-3401. Epub 2017 Aug 7.

Children's Research Institute and.

Extramedullary hematopoiesis (EMH) is induced during pregnancy to support rapid expansion of maternal blood volume. EMH activation requires hematopoietic stem cell (HSC) proliferation and mobilization, processes that depend upon estrogen receptor α (ERα) in HSCs. Here we show that treating mice with estradiol to model estradiol increases during pregnancy induced HSC proliferation in the bone marrow but not HSC mobilization. Treatment with the alternative ERα ligand 27-hydroxycholesterol (27HC) induced ERα-dependent HSC mobilization and EMH but not HSC division in the bone marrow. During pregnancy, 27HC levels increased in hematopoietic stem/progenitor cells as a result of CYP27A1, a cholesterol hydroxylase. Cyp27a1-deficient mice had significantly reduced 27HC levels, HSC mobilization, and EMH during pregnancy but normal bone marrow hematopoiesis and EMH in response to bleeding or G-CSF treatment. Distinct hematopoietic stresses thus induce EMH through different mechanisms. Two different ERα ligands, estradiol and 27HC, work together to promote EMH during pregnancy, revealing a collaboration of hormonal and metabolic mechanisms as well as a physiological function for 27HC in normal mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI94027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669562PMC
September 2017

Nonnuclear Estrogen Receptor Activation Improves Hepatic Steatosis in Female Mice.

Endocrinology 2016 Oct 23;157(10):3731-3741. Epub 2016 Aug 23.

Center for Pulmonary and Vascular Biology (K.L.C., J.B., M.U., J.U., L.H., C.M., P.W.S.), Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, 75390; and Departments of Chemistry (S.H.K., J.A.K.), Food Science and Human Nutrition (Z.M.-E.), and Molecular and Integrative Physiology (B.S.K.), University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.

Estrogens have the potential to afford atheroprotection, to prevent excess adiposity and its metabolic complications including insulin resistance, and to lessen hepatic steatosis. Cellular responses to estrogens occur through gene regulation by nuclear estrogen receptors (ERs), and through signal initiation by plasma membrane-associated ER. Leveraging the potentially favorable cardiometabolic actions of estrogens has been challenging, because their reproductive tract and cancer-promoting effects adversely impact the risk to benefit ratio of the therapy. In previous works, we discovered that an estrogen dendrimer conjugate (EDC) comprised of ethinyl-estradiol (E) molecules linked to a poly(amido)amine dendrimer selectively activates nonnuclear ER, and in mice, EDC does not invoke a uterotrophic response or support ER-positive breast cancer growth. In the present investigation, we employed EDC to determine how selective nonnuclear ER activation impacts atherosclerosis, adiposity, glucose homeostasis, and hepatic steatosis in female mice. In contrast to E, EDC did not blunt atherosclerosis in hypercholesterolemic apoE mice. Also in contrast to E, EDC did not prevent the increase in adiposity caused by Western diet feeding in wild-type mice, and it did not affect Western diet-induced glucose intolerance. However, E and EDC had comparable favorable effect on diet-induced hepatic steatosis, and this was related to down-regulation of fatty acid and triglyceride synthesis genes in the liver. Predictably, only E caused a uterotrophic response. Thus, although nonnuclear ER activation does not prevent atherosclerosis or diet-induced obesity or glucose intolerance, it may provide a potential new strategy to combat hepatic steatosis without impacting the female reproductive tract or increasing cancer risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2015-1629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045504PMC
October 2016

Re-adopting classical nuclear receptors by cholesterol metabolites.

J Steroid Biochem Mol Biol 2016 Mar 10;157:20-6. Epub 2015 Nov 10.

Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, 3517 Cullen Blvd, SERC 545, Houston, TX 77204-5056, USA. Electronic address:

Since the first cloning of the human estrogen receptor (ER) α in 1986 and the subsequent cloning of human ERβ, there has been extensive investigation of the role of estrogen/ER. Estrogens/ER play important roles not only in sexual development and reproduction but also in a variety of other functions in multiple tissues. Selective Estrogen Receptor Modulators (SERMs) are ER lignds that act as agonists or antagonists depending on the target genes and tissues, and until recently, only synthetic SERMs have been recognized. However, the discovery of the first endogenous SERM, 27-hydroxycholesterol (27HC), opened a new dimension of ER action in health and disease. In addition to the identification of 27HC as a SERM, oxysterols have been recently demonstrated as indirect modulators of ER through interaction with the nuclear receptor Liver X Receptor (LXR) β. In this review, the recent progress on these novel roles of oxysterols in ER modulation is summarized.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsbmb.2015.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724260PMC
March 2016

The interaction between metabolism, cancer and cardiovascular disease, connected by 27-hydroxycholesterol.

Clin Lipidol 2014;9(6):617-624

Division of Pulmonary & Vascular Biology, Departments of Pediatrics & Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, USA.

Oxysterols are metabolites of cholesterol that are produced in liver and other peripheral tissues as a means to eliminate cholesterol to bile acid. Recent studies have revealed that the most abundant circulating oxysterol 27-hydroxycholesterol (27HC) is the first identified endogenous selective estrogen receptor modulator. 27HC levels correlate well with that of cholesterol, and also rise progressively with age. 27HC affects estrogen receptor function by the antagonism of estrogen action and also by the direct modulation of the receptor function, and similar to estrogen/estrogen receptors, 27HC has many actions in various tissues. This review article introduces the recent progress in the understanding of the role of 27HC in breast cancer and cardiovascular dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/clp.14.53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306277PMC
January 2014

The cholesterol metabolite 27-hydroxycholesterol promotes atherosclerosis via proinflammatory processes mediated by estrogen receptor alpha.

Cell Metab 2014 Jul 19;20(1):172-82. Epub 2014 Jun 19.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Oxysterols are cholesterol metabolites that serve multiple functions in lipid metabolism, including as liver X receptor (LXR) ligands. 27-hydroxycholesterol (27HC) is an abundant oxysterol metabolized by CYP7B1. How 27HC impacts vascular health is unknown. We show that elevations in 27HC via cyp7b1 deletion promote atherosclerosis in apoe(-/-) mice without altering lipid status; furthermore, estrogen-related atheroprotection is attenuated. In wild-type mice, leukocyte-endothelial cell adhesion is increased by 27HC via estrogen receptor (ER)-dependent processes. In monocytes/macrophages, 27HC upregulates proinflammatory genes and increases adhesion via ERα. In endothelial cells, 27HC is also proadhesive via ERα, and in contrast to estrogen, which blunts NF-κB activation, 27HC stimulates NF-κB activation via Erk1,2 and JNK-dependent IκBα degradation. Whereas 27HC administration to apoe(-/-) mice increases atherosclerosis, apoe(-/-);erα(-/-) are unaffected. Thus, 27HC promotes atherosclerosis via proinflammatory processes mediated by ERα, and it attenuates estrogen-related atheroprotection. Strategies to lower 27HC may complement approaches targeting cholesterol to prevent vascular disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2014.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098728PMC
July 2014

27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology.

Science 2013 Nov;342(6162):1094-8

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27710, USA.

Hypercholesterolemia is a risk factor for estrogen receptor (ER)-positive breast cancers and is associated with a decreased response of tumors to endocrine therapies. Here, we show that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol and an ER and liver X receptor (LXR) ligand, increases ER-dependent growth and LXR-dependent metastasis in mouse models of breast cancer. The effects of cholesterol on tumor pathology required its conversion to 27HC by the cytochrome P450 oxidase CYP27A1 and were attenuated by treatment with CYP27A1 inhibitors. In human breast cancer specimens, CYP27A1 expression levels correlated with tumor grade. In high-grade tumors, both tumor cells and tumor-associated macrophages exhibited high expression levels of the enzyme. Thus, lowering circulating cholesterol levels or interfering with its conversion to 27HC may be a useful strategy to prevent and/or treat breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1241908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899689PMC
November 2013

27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth.

Cell Rep 2013 Nov 7;5(3):637-45. Epub 2013 Nov 7.

Department of Pediatrics, Division of Pulmonary and Vascular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

To date, estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells, and it stimulates cell-autonomous, ER-dependent, and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally modulated, nonaromatized ER ligand that promotes ER+ breast tumor growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2013.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950897PMC
November 2013

LXRβ/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity.

J Clin Invest 2013 Aug 8;123(8):3488-97. Epub 2013 Jul 8.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA.

Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRβ has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRβ to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRβ and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRβ- and ERα-dependent processes. These studies demonstrate that LXRβ has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI66533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726156PMC
August 2013

Coupling of Fcγ receptor I to Fcγ receptor IIb by SRC kinase mediates C-reactive protein impairment of endothelial function.

Circ Res 2011 Oct 22;109(10):1132-40. Epub 2011 Sep 22.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Rationale: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fcγ receptor IIB (FcγRIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5'-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization.

Objective: How CRP activates FcγRIIB in endothelium is not known. We determined the role of Fcγ receptor I (FcγRI) and the basis for coupling of FcγRI to FcγRIIB in endothelium.

Methods And Results: In cultured endothelial cells, FcγRI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via FcγRI. CRP-induced increases in FcγRIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5'-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the γ subunit of FcγRI (FcRγ(-/-)) or FcγRIIB(-/-) mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcRγ(-/-); TG-CRP and FcγRIIB(-/-); TG-CRP mice.

Conclusions: CRP antagonism of eNOS is mediated by the coupling of FcγRI to FcγRIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5'-phosphatase 1, and consistent with this mechanism, both FcγRI and FcγRIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. FcγRI and FcγRIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.111.254573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215097PMC
October 2011

The oxysterol, 27-hydroxycholesterol, links cholesterol metabolism to bone homeostasis through its actions on the estrogen and liver X receptors.

Endocrinology 2011 Dec 20;152(12):4691-705. Epub 2011 Sep 20.

Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, North Carolina 27710, USA.

Osteoporosis and age-related bone loss are important public health concerns. Therefore, there is a high level of interest in the development of medical interventions and lifestyle changes that reduce the incidence of osteoporosis and age-related bone loss. Decreased bone mineral density is associated with high cholesterol, and patients on statins have increased bone mineral densities, strongly implicating cholesterol as a negative regulator of bone homeostasis. In this study, using both molecular and pharmacological approaches, we have been able to demonstrate that the primary cholesterol metabolite, 27-hydroxycholesterol, through its actions on both estrogen receptors and liver X receptors, decreases osteoblast differentiation and enhances osteoclastogenesis, resulting in increased bone resorbtion in mice. Induction of the short heterodimer partner protein by estrogens in osteoblasts can attenuate the liver X receptor-mediated actions of 27-hydroxycholesterol in bone. These data establish a mechanistic link between cholesterol and bone quality, highlight an unexpected target of estrogens in osteoblasts, and define a signaling axis, the therapeutic exploitation of which is likely to yield novel antiosteoporotic drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2011-1298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230052PMC
December 2011

The G protein-coupled bile acid receptor, TGR5, stimulates gallbladder filling.

Mol Endocrinol 2011 Jun 31;25(6):1066-71. Epub 2011 Mar 31.

Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050, USA.

TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5(-/-) mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of fibroblast growth factor 15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2010-0460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100601PMC
June 2011

27-Hydroxycholesterol: the first identified endogenous SERM.

Trends Endocrinol Metab 2011 Apr 23;22(4):130-5. Epub 2011 Feb 23.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA. michihisa.umetani@utsouthwestern

The cholesterol metabolite 27-hydroxycholesterol (27OHC) classically delivers sterols from peripheral tissues to the liver and is a substrate for bile acid synthesis. Recent studies have revealed that 27OHC also binds to and modifies the function of estrogen receptors ERα and ERβ. Experiments in mice lacking the enzyme which synthesizes 27OHC, CYP27A1, or the enzyme which catabolizes 27OHC, CYP7B1, have demonstrated that 27OHC adversely affects estrogen-related cardiovascular protection and bone mineralization. Work in breast cancer cells further indicates that 27OHC alters ER target gene expression to promote cell growth. Therefore, 27OHC is the first identified endogenous selective estrogen receptor modulator (SERM) and could have an important impact upon the cardiovascular system, bone biology, and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tem.2011.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070823PMC
April 2011

Non-nuclear estrogen receptor signaling in the endothelium.

J Biol Chem 2011 Apr 22;286(17):14737-43. Epub 2011 Feb 22.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

In addition to the classical function of estrogen receptors (ER) as transcription factors, evidence continues to accumulate that they mediate non-nuclear processes in numerous cell types, including the endothelium, in which they activate endothelial NO synthase. Non-nuclear ER signaling entails unique post-translational modifications and protein-protein interactions of the receptor with adaptor molecules, kinases, and G proteins. Recent in vitro and in vivo studies in mice using an estrogen-dendrimer conjugate that is excluded from the nucleus indicate that non-nuclear ER activation underlies the migration and growth responses of endothelial cells to estrogen but not the growth responses of endometrial or breast cancer cells to the hormone. In this minireview, the features of ERα and protein-protein interactions that enable it to invoke extranuclear signaling in the endothelium and the consequences of that signaling are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.R110.191791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083154PMC
April 2011

Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice.

J Clin Invest 2010 Jul 23;120(7):2319-30. Epub 2010 Jun 23.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA.

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI38291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898582PMC
July 2010

The endogenous selective estrogen receptor modulator 27-hydroxycholesterol is a negative regulator of bone homeostasis.

Endocrinology 2010 Aug 25;151(8):3675-85. Epub 2010 May 25.

Department of Pharmacology, Duke University Medical Center, Pharmacology and Cancer Biology, Durham, North Carolina 27710, USA.

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2010-0080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940523PMC
August 2010

27-Hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen.

Nat Med 2007 Oct 16;13(10):1185-92. Epub 2007 Sep 16.

Department of Pharmacology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050, USA.

The cardioprotective effects of estrogen are mediated by receptors expressed in vascular cells. Here we show that 27-hydroxycholesterol (27HC), an abundant cholesterol metabolite that is elevated with hypercholesterolemia and found in atherosclerotic lesions, is a competitive antagonist of estrogen receptor action in the vasculature. 27HC inhibited both the transcription-mediated and the non-transcription-mediated estrogen-dependent production of nitric oxide by vascular cells, resulting in reduced estrogen-induced vasorelaxation of rat aorta. Furthermore, increasing 27HC levels in mice by diet-induced hypercholesterolemia, pharmacologic administration or genetic manipulation (by knocking out the gene encoding the catabolic enzyme CYP7B1) decreased estrogen-dependent expression of vascular nitric oxide synthase and repressed carotid artery reendothelialization. As well as antiestrogenic effects, there were proestrogenic actions of 27HC that were cell-type specific, indicating that 27HC functions as an endogenous selective estrogen receptor modulator (SERM). Taken together, these studies point to 27HC as a contributing factor in the loss of estrogen protection from vascular disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm1641DOI Listing
October 2007

27-hydroxycholesterol is an endogenous selective estrogen receptor modulator.

Mol Endocrinol 2008 Jan 13;22(1):65-77. Epub 2007 Sep 13.

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

Selective estrogen receptor (ER) modulators (SERMs) are ER ligands whose relative agonist/antagonist activities vary in a cell- and promoter-dependent manner. The molecular basis underlying this selectivity can be attributed to the ability of these ligands to induce distinct alterations in ER structure leading to differential recruitment of coactivators and corepressors. Whether SERM activity is restricted to synthetic ligands or whether molecules exist in vivo that function in an analogous manner remains unresolved. However, the recent observation that oxysterols bind ER and antagonize the actions of 17beta-estradiol (E2) on the vascular wall suggests that this class of ligands may possess SERM activity. We demonstrate here that 27-hydroxycholesterol (27HC), the most prevalent oxysterol in circulation, functions as a SERM, the efficacy of which varies when assessed on different endpoints. Importantly, 27HC positively regulates both gene transcription and cell proliferation in cellular models of breast cancer. Using combinatorial peptide phage display, we have determined that 27HC induces a unique conformational change in both ERalpha and ERbeta, distinguishing it from E2 and other SERMs. Thus, as with other ER ligands, it appears that the unique pharmacological activity of 27HC relates to its ability to impact ER structure and modulate cofactor recruitment. Cumulatively, these data indicate that 27HC is an endogenous SERM with partial agonist activity in breast cancer cells and suggest that it may influence the pathology of breast cancer. Moreover, given the product-precursor relationship between 27HC and cholesterol, our findings have implications with respect to breast cancer risk in obese/hypercholesteremic individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2007-0383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194632PMC
January 2008

Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA.

Proc Natl Acad Sci U S A 2006 Nov 6;103(46):17278-83. Epub 2006 Nov 6.

Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Meguro, Tokyo 153-8904, Japan.

Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3' UTR. WTAP knockdown induced G2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0608357103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1634838PMC
November 2006

Identification of a hormonal basis for gallbladder filling.

Nat Med 2006 Nov 29;12(11):1253-5. Epub 2006 Oct 29.

Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

The cycle of gallbladder filling and emptying controls the flow of bile into the intestine for digestion. Here we show that fibroblast growth factor-15, a hormone made by the distal small intestine in response to bile acids, is required for gallbladder filling. These studies demonstrate that gallbladder filling is actively regulated by an endocrine pathway and suggest a postprandial timing mechanism that controls gallbladder motility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm1501DOI Listing
November 2006