Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1–Mediated Cholesterol Removal
2018; Elsevier BV; Volume: 189; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2018.10.013
ISSN1525-2191
AutoresAicha Saadane, Natalia Mast, George Trichonas, Dibyendu Chakraborty, Sandra S. Hammer, Julia V. Busik, Maria B. Grant, Irina A. Pikuleva,
Tópico(s)Hormonal Regulation and Hypertension
ResumoCYP46A1 is the cytochrome P450 enzyme that converts cholesterol to 24-hydroxycholesterol, a cholesterol elimination product and a potent liver X receptor (LXR) ligand. We conducted retinal characterizations of Cyp46a1−/− mice that had normal fasting blood glucose levels but up to a 1.8-fold increase in retinal cholesterol. The retina of Cyp46a1−/− mice exhibited venous beading and tortuosity, microglia/macrophage activation, and increased vascular permeability, features commonly associated with diabetic retinopathy. The expression of Lxrα and Lxrβ was increased in both the whole Cyp46a1−/− retina and retinal macroglia/macrophages. The LXR-target genes were affected as well, primarily in activated microglial cells and macrophages. In the latter, the LXR-transactivated genes (Abca1, Abcg1, Apod, Apoe, Mylip, and Arg2) were up-regulated; similarly, there was an up-regulation of the LXR-transrepressed genes (Ccl2, Ptgs2, Cxcl1, Il1b, Il6, Nos2, and Tnfa). For comparison, gene expression was investigated in bone marrow–derived macrophages from Cyp46a1−/− mice as well as retinal and bone marrow–derived macrophages from Cyp27a1−/− and Cyp27a1−/−Cyp46a1−/− mice. CYP46A1 expression was detected in retinal endothelial cells, and this expression was increased in the proinflammatory environment. Retinal Cyp46a1−/− phosphoproteome revealed altered phosphorylation of 30 different proteins, including tight junction protein zonula occludens 1 and aquaporin 4. Collectively, the data obtained establish metabolic and regulatory significance of CYP46A1 for the retina and suggest pharmacologic activation of CYP46A1 as a potential therapeutic approach to dyslipidemia-induced retinal damage. CYP46A1 is the cytochrome P450 enzyme that converts cholesterol to 24-hydroxycholesterol, a cholesterol elimination product and a potent liver X receptor (LXR) ligand. We conducted retinal characterizations of Cyp46a1−/− mice that had normal fasting blood glucose levels but up to a 1.8-fold increase in retinal cholesterol. The retina of Cyp46a1−/− mice exhibited venous beading and tortuosity, microglia/macrophage activation, and increased vascular permeability, features commonly associated with diabetic retinopathy. The expression of Lxrα and Lxrβ was increased in both the whole Cyp46a1−/− retina and retinal macroglia/macrophages. The LXR-target genes were affected as well, primarily in activated microglial cells and macrophages. In the latter, the LXR-transactivated genes (Abca1, Abcg1, Apod, Apoe, Mylip, and Arg2) were up-regulated; similarly, there was an up-regulation of the LXR-transrepressed genes (Ccl2, Ptgs2, Cxcl1, Il1b, Il6, Nos2, and Tnfa). For comparison, gene expression was investigated in bone marrow–derived macrophages from Cyp46a1−/− mice as well as retinal and bone marrow–derived macrophages from Cyp27a1−/− and Cyp27a1−/−Cyp46a1−/− mice. CYP46A1 expression was detected in retinal endothelial cells, and this expression was increased in the proinflammatory environment. Retinal Cyp46a1−/− phosphoproteome revealed altered phosphorylation of 30 different proteins, including tight junction protein zonula occludens 1 and aquaporin 4. Collectively, the data obtained establish metabolic and regulatory significance of CYP46A1 for the retina and suggest pharmacologic activation of CYP46A1 as a potential therapeutic approach to dyslipidemia-induced retinal damage. Changes in retinal microcirculation are the early manifestations of diabetic retinopathy, the most common microvascular complications in type 1 diabetes and type 2 diabetes.1Tang J. Kern T.S. Inflammation in diabetic retinopathy.Prog Retin Eye Res. 2011; 30: 343-358Crossref PubMed Scopus (590) Google Scholar, 2Durham J.T. Herman I.M. Microvascular modifications in diabetic retinopathy.Curr Diab Rep. 2011; 11: 253-264Crossref PubMed Scopus (116) Google Scholar, 3Stitt A.W. Lois N. Medina R.J. Adamson P. Curtis T.M. Advances in our understanding of diabetic retinopathy.Clin Sci (Lond). 2013; 125: 1-17Crossref PubMed Scopus (105) Google Scholar These changes include retinal microaneurysms, capillary nonperfusion and degeneration, venous beading and looping, intraretinal microvascular abnormalities (large-caliber shunt vessels within nonperfused regions of the capillary bed), excessive vasopermeability, retinal edema, and impairment of neural function.1Tang J. Kern T.S. Inflammation in diabetic retinopathy.Prog Retin Eye Res. 2011; 30: 343-358Crossref PubMed Scopus (590) Google Scholar, 3Stitt A.W. Lois N. Medina R.J. Adamson P. Curtis T.M. Advances in our understanding of diabetic retinopathy.Clin Sci (Lond). 2013; 125: 1-17Crossref PubMed Scopus (105) Google Scholar Remarkably, increased vascular permeability, dilation, nonperfusion, capillary degeneration, and arteriovenous shunts were observed in the retina of Cyp27a1−/−Cyp46a1−/− mice4Saadane A. Mast N. Charvet C. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1-/- Cyp64a1-/- mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar but not Cyp27a1−/− mice, the two genotypes that had normal blood glucose levels but increased total retinal cholesterol.4Saadane A. Mast N. Charvet C. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1-/- Cyp64a1-/- mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Cytochrome P450 27A1 (CYP27A1) is a sterol 27-hydroxylase,5Wikvall K. Hydroxylations in biosynthesis of bile acids: isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids.J Biol Chem. 1984; 259: 3800-3804Abstract Full Text PDF PubMed Google Scholar whereas cytochrome P450 46A1 (CYP46A1) catalyzes cholesterol 24-hydroxylation.6Lund E.G. Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc Natl Acad Sci U S A. 1999; 96: 7238-7243Crossref PubMed Scopus (465) Google Scholar Both P450s are expressed in the retina7Liao W.L. Heo G.Y. Dodder N.G. Reem R.E. Mast N. Huang S. Dipatre P.L. Turko I.V. Pikuleva I.A. Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.J Proteome Res. 2011; 10: 241-248Crossref PubMed Scopus (34) Google Scholar, 8Zheng W. Reem R.E. Omarova S. Huang S. DiPatre P.L. Charvet C.D. Curcio C.A. Pikuleva I.A. Spatial distribution of the pathways of cholesterol homeostasis in human retina.PLoS One. 2012; 7: e37926Crossref PubMed Scopus (66) Google Scholar, 9Mast N. Reem R. Bederman I. Huang S. DiPatre P.L. Bjorkhem I. Pikuleva I.A. Cholestenoic acid is an important elimination product of cholesterol in the retina: comparison of retinal cholesterol metabolism with that in the brain.Invest Ophthalmol Vis Sci. 2011; 52: 594-603Crossref PubMed Scopus (57) Google Scholar and are important for retinal cholesterol elimination.4Saadane A. Mast N. Charvet C. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1-/- Cyp64a1-/- mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 10Omarova S. Charvet C.D. Reem R.E. Mast N. Zheng W. Huang S. Peachey N.S. Pikuleva I.A. Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis.J Clin Invest. 2012; 122: 3012-3023Crossref PubMed Scopus (34) Google Scholar CYP27A1 is ubiquitous and is highly abundant in the photoreceptor inner segments, Muller cells, and retinal pigment epithelium (RPE).11Lee J.W. Fuda H. Javitt N.B. Strott C.A. Rodriguez I.R. Expression and localization of sterol 27-hydroxylase (CYP27A1) in monkey retina.Exp Eye Res. 2006; 83: 465-469Crossref PubMed Scopus (41) Google Scholar CYP46A1 is less abundant in the retina7Liao W.L. Heo G.Y. Dodder N.G. Reem R.E. Mast N. Huang S. Dipatre P.L. Turko I.V. Pikuleva I.A. Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.J Proteome Res. 2011; 10: 241-248Crossref PubMed Scopus (34) Google Scholar and is mainly found in the neurons of the ganglion cell layer with a lower expression in the RPE.12Ramirez D.M. Andersson S. Russell D.W. Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain.J Comp Neurol. 2008; 507: 1676-1693Crossref PubMed Scopus (111) Google Scholar CYP46A1 and CYP27A1 produce 24-hydroxycholesterol (24HC) and 27-hydroxycholesterol (27HC), respectively, oxysterols, which are the transport forms of cholesterol9Mast N. Reem R. Bederman I. Huang S. DiPatre P.L. Bjorkhem I. Pikuleva I.A. Cholestenoic acid is an important elimination product of cholesterol in the retina: comparison of retinal cholesterol metabolism with that in the brain.Invest Ophthalmol Vis Sci. 2011; 52: 594-603Crossref PubMed Scopus (57) Google Scholar, 13Meaney S. Bodin K. Diczfalusy U. Bjorkhem I. On the rate of translocation in vitro and kinetics in vivo of the major oxysterols in human circulation: critical importance of the position of the oxygen function.J Lipid Res. 2002; 43: 2130-2135Crossref PubMed Scopus (113) Google Scholar from the retina to the systemic circulation. In addition, 24HC and 27HC are biologically active molecules that can interact with different regulatory proteins, including the liver X receptors (LXRs), a family of transcription factors.14Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.Nature. 1996; 383: 728-731Crossref PubMed Scopus (1343) Google Scholar 24HC is a more potent LXR agonist than 27HC14Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.Nature. 1996; 383: 728-731Crossref PubMed Scopus (1343) Google Scholar; hence, we hypothesized that some of the abnormalities in Cyp27a1−/−Cyp46a1−/− mice were due to deficiency in CYP46A1 and a lack of 24HC activation of LXR. Indeed, Lxrα/β−/− mice showed vascular changes similar to those observed in the diabetic retina with increases in acellular capillaries despite the lack of metabolic dysfunction.15Hazra S. Rasheed A. Bhatwadekar A. Wang X. Shaw L.C. Patel M. Caballero S. Magomedova L. Solis N. Yan Y. Wang W. Thinschmidt J.S. Verma A. Li Q. Levi M. Cummins C.L. Grant M.B. Liver X receptor modulates diabetic retinopathy outcome in a mouse model of streptozotocin-induced diabetes.Diabetes. 2012; 61: 3270-3279Crossref PubMed Scopus (41) Google Scholar Also, the expression of Lxr was shown to be significantly decreased in diabetic human retinas and in a type 2 diabetes animal model.16Hammer S.S. Beli E. Kady N. Wang Q. Wood K. Lydic T.A. Malek G. Saban D.R. Wang X.X. Hazra S. Levi M. Busik J.V. Grant M.B. The mechanism of diabetic retinopathy pathogenesis unifying key lipid regulators, sirtuin 1 and liver X receptor.EBioMedicine. 2017; 22: 181-190Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Conversely, LXR activation by synthetic ligands was found to prevent retinal inflammation and diabetic retinopathy in diabetic animal models15Hazra S. Rasheed A. Bhatwadekar A. Wang X. Shaw L.C. Patel M. Caballero S. Magomedova L. Solis N. Yan Y. Wang W. Thinschmidt J.S. Verma A. Li Q. Levi M. Cummins C.L. Grant M.B. Liver X receptor modulates diabetic retinopathy outcome in a mouse model of streptozotocin-induced diabetes.Diabetes. 2012; 61: 3270-3279Crossref PubMed Scopus (41) Google Scholar, 16Hammer S.S. Beli E. Kady N. Wang Q. Wood K. Lydic T.A. Malek G. Saban D.R. Wang X.X. Hazra S. Levi M. Busik J.V. Grant M.B. The mechanism of diabetic retinopathy pathogenesis unifying key lipid regulators, sirtuin 1 and liver X receptor.EBioMedicine. 2017; 22: 181-190Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar and to reduce proinflammatory macrophage activity.16Hammer S.S. Beli E. Kady N. Wang Q. Wood K. Lydic T.A. Malek G. Saban D.R. Wang X.X. Hazra S. Levi M. Busik J.V. Grant M.B. The mechanism of diabetic retinopathy pathogenesis unifying key lipid regulators, sirtuin 1 and liver X receptor.EBioMedicine. 2017; 22: 181-190Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar There are two LXR isoforms, LXRα and LXRβ, that share high sequence identity (approximately 80%) and are activated by the same ligands, typically oxygenated metabolites of cholesterol (eg, 24HC and 27HC) as well as cholesterol precursor desmosterol.14Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.Nature. 1996; 383: 728-731Crossref PubMed Scopus (1343) Google Scholar, 17Chen W. Chen G. Head D.L. Mangelsdorf D.J. Russell D.W. Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice.Cell Metab. 2007; 5: 73-79Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 18Spann N.J. Garmire L.X. McDonald J.G. Myers D.S. Milne S.B. Shibata N. Reichart D. Fox J.N. Shaked I. Heudobler D. Raetz C.R. Wang E.W. Kelly S.L. Sullards M.C. Murphy R.C. Merrill Jr., A.H. Brown H.A. Dennis E.A. Li A.C. Ley K. Tsimikas S. Fahy E. Subramaniam S. Quehenberger O. Russell D.W. Glass C.K. Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.Cell. 2012; 151: 138-152Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar LXRβ is ubiquitous, whereas LXRα is tissue specific and is highly expressed in the liver, intestine, kidney, adipose tissue, and macrophages.19Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. LXR, a nuclear receptor that defines a distinct retinoid response pathway.Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (832) Google Scholar, 20Auboeuf D. Rieusset J. Fajas L. Vallier P. Frering V. Riou J.P. Staels B. Auwerx J. Laville M. Vidal H. Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of obese and NIDDM patients.Diabetes. 1997; 46: 1319-1327Crossref PubMed Scopus (604) Google Scholar, 21Apfel R. Benbrook D. Lernhardt E. Ortiz M.A. Salbert G. Pfahl M. A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily.Mol Cell Biol. 1994; 14: 7025-7035Crossref PubMed Scopus (273) Google Scholar Activation of LXRs leads either to gene transactivation or to gene transrepression. In transactivation, the basal condition is gene silencing by a complex of LXR with retinoid X receptor and corepressors bound to the promoter of the target gene.22Wagner B.L. Valledor A.F. Shao G. Daige C.L. Bischoff E.D. Petrowski M. Jepsen K. Baek S.H. Heyman R.A. Rosenfeld M.G. Schulman I.G. Glass C.K. Promoter-specific roles for liver X receptor/corepressor complexes in the regulation of ABCA1 and SREBP1 gene expression.Mol Cell Biol. 2003; 23: 5780-5789Crossref PubMed Scopus (179) Google Scholar Ligand binding to LXR leads to release of corepressors in exchange for coactivators, thus initiating gene transcription.23Glass C.K. Rosenfeld M.G. The coregulator exchange in transcriptional functions of nuclear receptors.Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar Many of the cholesterol-related genes [eg, ATP-binding cassette subfamily members A1 (Abca1) and G1 (Abcg1), apolipoprotein E (Apoe), inducible degrader of low-density lipoprotein receptor (Idol; official name, Mylip), and sterol regulatory element-binding protein 1c (Srebp1c)] along with the genes involved in fatty acid synthesis [eg, elongation of long chain fatty acids family member 5 (Elovl5), fatty acid synthase (Fasn), fatty acid desaturase 2 (Fads2), and stearoyl–coenzyme A desaturases 1 and 2 (Scd1 and Scd2, respectively)] as well as other processes [eg, apoptosis inhibitor of macrophages (Aim), apolipoprotein D (Apod), arginase 2 (Arg2), glucose transporter type 4 (Glut4), and Mer receptor tyrosine kinase (Mertk)] are regulated by this mechanism.24Spann N.J. Glass C.K. Sterols and oxysterols in immune cell function.Nat Immunol. 2013; 14: 893-900Crossref PubMed Scopus (138) Google Scholar, 25Calkin A.C. Tontonoz P. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR.Nat Rev Mol Cell Biol. 2012; 13: 213-224Crossref PubMed Scopus (424) Google Scholar In transrepression, ligand binding leads to LXR sumoylation, which prevents the corepressor release and gene expression.26Ghisletti S. Huang W. Ogawa S. Pascual G. Lin M.E. Willson T.M. Rosenfeld M.G. Glass C.K. Parallel SUMOylation-dependent pathways mediate gene- and signal-specific transrepression by LXRs and PPARgamma.Mol Cell. 2007; 25: 57-70Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar This mechanism, shown to be operative in the liver and macrophages, blocks the transcription of NF-κB and other transcription factors and thereby the expression of the proinflammatory genes [eg, C-C motif chemokine 2 (Ccl2), prostaglandin G/H synthase 2 (Cox-2; official name, Ptgs2), C-X-C motif chemokine ligand 1 (Cxcl1), IL-1β (Il1b), IL-6 (Il6), inducible nitric oxide synthase (iNos), and tumor necrosis factor-α (Tnfa)], which are controlled by these factors.25Calkin A.C. Tontonoz P. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR.Nat Rev Mol Cell Biol. 2012; 13: 213-224Crossref PubMed Scopus (424) Google Scholar, 26Ghisletti S. Huang W. Ogawa S. Pascual G. Lin M.E. Willson T.M. Rosenfeld M.G. Glass C.K. Parallel SUMOylation-dependent pathways mediate gene- and signal-specific transrepression by LXRs and PPARgamma.Mol Cell. 2007; 25: 57-70Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 27Jakobsson T. Treuter E. Gustafsson J.A. Steffensen K.R. Liver X receptor biology and pharmacology: new pathways, challenges and opportunities.Trends Pharmacol Sci. 2012; 33: 394-404Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 28Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors.Nat Med. 2003; 9: 213-219Crossref PubMed Scopus (908) Google Scholar LXRα and LXRβ appear to be largely interchangeable in transactivation, with their contribution determined by relative expression level.29Bischoff E.D. Daige C.L. Petrowski M. Dedman H. Pattison J. Juliano J. Li A.C. Schulman I.G. Non-redundant roles for LXRalpha and LXRbeta in atherosclerosis susceptibility in low density lipoprotein receptor knockout mice.J Lipid Res. 2010; 51: 900-906Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar However, in macrophages, LXRα may be more robust at transactivation and LXRβ more potent at basal target repression.29Bischoff E.D. Daige C.L. Petrowski M. Dedman H. Pattison J. Juliano J. Li A.C. Schulman I.G. Non-redundant roles for LXRalpha and LXRbeta in atherosclerosis susceptibility in low density lipoprotein receptor knockout mice.J Lipid Res. 2010; 51: 900-906Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar LXRα and LXRβ are both present in the neural retina and RPE and were immunolocalized to retinal layers: LXRβ appears to be ubiquitous, and LXRα seems to be expressed in the cells of the ganglion cell layer and RPE.8Zheng W. Reem R.E. Omarova S. Huang S. DiPatre P.L. Charvet C.D. Curcio C.A. Pikuleva I.A. Spatial distribution of the pathways of cholesterol homeostasis in human retina.PLoS One. 2012; 7: e37926Crossref PubMed Scopus (66) Google Scholar, 30Dwyer M.A. Kazmin D. Hu P. McDonnell D.P. Malek G. Research resource: nuclear receptor atlas of human retinal pigment epithelial cells: potential relevance to age-related macular degeneration.Mol Endocrinol. 2011; 25: 360-372Crossref PubMed Scopus (41) Google Scholar However, knowledge of cell-specific retinal LXR localizations is still lacking, including retinal endothelial cells and microglia, which may be missed on retinal cross sections because of the small numbers of these cells. Herein, we characterized the ocular phenotype of Cyp46a1−/− mice and obtained evidence that both metabolic and regulatory CYP46A1 activities are of significance for the retina and retinal blood vessels. We also performed multicolor immunohistochemistry labeling of vascular endothelial cells and CYP46A1. Our findings suggest that CYP46A1 may represent a new pharmacologic target for early-stage diabetic retinopathy treatment. Animals were 6- to 9-month–old female or male mice. In both sexes, retinal vascular abnormalities on fluorescein angiography (FA) were detected starting from the age of 6 months. Furthermore, electroretinography (ERG) responses, conducted for both sexes at 6 months of age, were similar, as were the levels of mouse retinal and serum sterols. Hence, all subsequent experiments used male mice. Cyp46a1+/− mice (on the mixed C57BL/6J; 129S6/SvEv background) were provided by Dr. David Russell (UT Southwestern, Dallas, TX).31Lund E.G. Xie C. Kotti T. Turley S.D. Dietschy J.M. Russell D.W. Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover.J Biol Chem. 2003; 278: 22980-22988Crossref PubMed Scopus (257) Google Scholar Cyp27a1+/− mice (on the C57BL/6J background) were provided by Dr. Sandra Erickson (University of California, San Francisco, San Francisco, CA).32Dubrac S. Lear S.R. Ananthanarayanan M. Balasubramaniyan N. Bollineni J. Shefer S. Hyogo H. Cohen D.E. Blanche P.J. Krauss R.M. Batta A.K. Salen G. Suchy F.J. Maeda N. Erickson S.K. Role of CYP27A in cholesterol and bile acid metabolism.J Lipid Res. 2005; 46: 76-85Crossref PubMed Scopus (40) Google Scholar The heterozygous animals obtained were crossed to generate the Cyp46a1−/−, Cyp46a1+/+, Cyp27a1−/−, and Cyp27a1+/+ breeding pairs, which established the Cyp46a1−/−, Cyp46a1+/+, Cyp27a1−/−, and Cyp27a1+/+ colonies. The Cyp27a1−/−Cyp46a1−/− strain and Cyp27a+/+Cyp46a1+/+ controls were generated by crossing Cyp27a1+/− and Cyp46a1+/− mice.4Saadane A. Mast N. Charvet C. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1-/- Cyp64a1-/- mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar All mice were free of the Crblrd8 mutation, which was bred out of our colonies. Cyp46a1−/− mice had normal fertility, body weight, food consumption, and appearance/weight of difference organs (data are not shown), consistent with previous genotype characterizations.31Lund E.G. Xie C. Kotti T. Turley S.D. Dietschy J.M. Russell D.W. Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover.J Biol Chem. 2003; 278: 22980-22988Crossref PubMed Scopus (257) Google Scholar, 33Russell D.W. Halford R.W. Ramirez D.M. Shah R. Kotti T. Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain.Annu Rev Biochem. 2009; 78: 1017-1040Crossref PubMed Scopus (159) Google Scholar All animals were maintained on a standard 12-hour light (approximately 10 lux)–dark cycle and were fed standard rodent chow and water ad libitum. All animal procedures were approved by the Case Western Reserve University (Cleveland, OH) Institutional Animal Care and Use Committee and conformed to recommendations of the American Veterinary Association Panel on Euthanasia and the Association for Research in Vision and Ophthalmology. ERG was performed as previously described.4Saadane A. Mast N. Charvet C. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1-/- Cyp64a1-/- mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar The scotopic a- and b-waves or the photopic b-wave was recorded in response to strobe flash stimuli presented after overnight dark adaptation (scotopic) or adaptation to a steady adapting field (photopic). FA was as described10Omarova S. Charvet C.D. Reem R.E. Mast N. Zheng W. Huang S. Peachey N.S. Pikuleva I.A. Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis.J Clin Invest. 2012; 122: 3012-3023Crossref PubMed Scopus (34) Google Scholar after a bolus (0.1-mL) i.p. injection of 1.6% sodium fluorescein in phosphate-buffered saline (PBS). Mice were fasted overnight and anesthetized via i.p. injection of 80 mg/kg ketamine (Fort Dodge Animal Health, Overland Park, KS) and 15 mg/kg xylazine (Akorn Inc., Lake Forest, IL) in PBS, pH 7.4. Blood was withdrawn via cardiac puncture and used for serum isolation, as described.34Mast N. Shafaati M. Zaman W. Zheng W. Prusak D. Wood T. Ansari G.A. Lovgren-Sandblom A. Olin M. Bjorkhem I. Pikuleva I. Marked variability in hepatic expression of cytochromes CYP7A1 and CYP27A1 as compared to cerebral CYP46A1: lessons from a dietary study with omega 3 fatty acids in hamsters.Biochim Biophys Acta. 2010; 1801: 674-681Crossref PubMed Scopus (16) Google Scholar Serum was analyzed for total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and free fatty acids by Marshfield Labs (Marshfield, WI). For the glucose tolerance test, mice were fasted overnight and injected with a solution of 50% d-glucose (2 g/kg body weight; Hospira, Lake Forest, IL) into the peritoneum. The blood was withdrawn from the tail vein and assayed for glucose by an Elite XL Glucometer (Bayer Contour, Parsippany, NJ) before and after the injection (30, 60, 120, and 150 minutes). Retinal isolation and processing were as described,4Saadane A. Mast N. Charvet C. Omarova S. Zheng W. Huang S.S. Kern T.S. Peachey N.S. Pikuleva I.A. Retinal and non-ocular abnormalities in Cyp27a1-/- Cyp64a1-/- mice with dysfunctional metabolism of cholesterol.Am J Pathol. 2014; 184: 2403-2419Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar as are subsequent sterol quantifications by isotope dilution gas chromatography–mass spectroscopy.9Mast N. Reem R. Bederman I. Huang S. DiPatre P.L. Bjorkhem I. Pikuleva I.A. Cholestenoic acid is an important elimination product of cholesterol in the retina: comparison of retinal cholesterol metabolism with that in the brain.Invest Ophthalmol Vis Sci. 2011; 52: 594-603Crossref PubMed Scopus (57) Google Scholar Primary retinal microglia/macrophages (RMMs) and bone marrow–derived macrophages (BMDMs) were prepared from 1.5- to 2.5-month–old 8 to 12 male and female mice of the same genotype, as described35Kohno H. Chen Y. Kevany B.M. Pearlman E. Miyagi M. Maeda T. Palczewski K. Maeda A. Photoreceptor proteins initiate microglial activation via Toll-like receptor 4 in retinal degeneration mediated by all-trans-retinal.J Biol Chem. 2013; 288: 15326-15341Crossref PubMed Scopus (99) Google Scholar with the following modifications. After isolation, mouse eyes were first placed in PBS containing 1% of penicillin/streptomycin (ThermoFisher, Waltham, MA) and then transferred to the ice-cold Dulbecco's modified Eagle's medium (DMEM; ThermoFisher) supplemented with 1% of penicillin/streptomycin. Next, eyes were cleaned from the connectives tissues and incubated for 30 minutes at 37°C and 60 rpm shaking in DMEM containing 2% dispase (Invitrogen, Waltham, MA) and 100 mg/mL collagenase IV (Invitrogen). The cornea, lens, and vitreous were removed, and the retinas were isolated from the eye cup under a surgical microscope (High Illuminator NI-5; Nikon Instruments Inc., Melville, NY) using tweezers. The retinas were then placed back in DMEM containing 2% dispase and 100 mg/mL collagenase IV and incubated for additional 15 minutes at 37°C and 60 rpm shaking. The tissue was transferred to a culture dish to peel off the RPE. The remaining neural retinas were homogenized by pipetting and cultured in T25 or T75 flasks (Falcon, Waltham, MA) for 7 days at 37°C in DMEM containing 1% penicillin/streptomycin and 20% fetal bovine serum (FBS; ThermoFisher). After 1 week, the culture medium was changed and the remaining adherent cells were left to replicate for another 7 to 10 days. The medium was changed every other day. After the last medium change, cells adherent to the plastic surface were treated with 0.05% trypsin (Invitrogen) for 2 to 3 minutes, and the less adhesive cells were collected as RMM. A small portion of RMM (approximately 25,000 cells) was plated on a μ-slide (Ibidi, Fitchburg, WI) and kept overnight in DMEM containing 1% penicillin/streptomycin and 20% FBS. These cells were used for the characterization of preparation homogeneity. The remaining RMMs were plated (no less than 250,000 cells per well) in 6-well tissue culture plates (Falcon) and allowed to adhere for 5 to 6 hours. Cells were then starved overnight in DMEM containing 1% penicillin/streptomycin without FBS; the next morning, they were stimulated for 6 hours with or without 100 ng/mL lipopolysaccharide (LPS; Escherichia coli O111:B4; InvivoGen, San Diego, CA). At the end of stimulation, cells were washed with ice-cold PBS and lysed with TRIzol reagent (ThermoFisher) for RNA isolation. The isolation of BMDM was as described.35Kohno H. Chen Y. Kevany B.M. Pearlman E. Miyagi M. Maeda T. Palczewski K. Maeda A. Photoreceptor proteins initiate microglial activation via Tol
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