FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034672127> ?p ?o ?g. }

• W2034672127 endingPage "2253" @default.
• W2034672127 startingPage "2242" @default.
• W2034672127 abstract "Hyperlipidemia and arterial cholesterol accumulation are primary causes of cardiovascular events. Monogenic forms of hyperlipidemia and recent genome-wide association studies indicate that genetics plays an important role. Zebrafish are a useful model for studying the genetic susceptibility to hyperlipidemia owing to conservation of many components of lipoprotein metabolism, including those related to LDL, ease of genetic manipulation, and in vivo observation of lipid transport and vascular calcification. We sought to develop a genetic model for lipid metabolism in zebrafish, capitalizing on one well-understood player in LDL cholesterol (LDL-c) transport, the LDL receptor (ldlr), and an established in vivo model of hypercholesterolemia. We report that morpholinos targeted against the gene encoding ldlr effectively suppressed its expression in embryos during the first 8 days of development. The ldlr morphants exhibited increased LDL-c levels that were exacerbated by feeding a high cholesterol diet. Increased LDL-c was ameliorated in morphants upon treatment with atorvastatin. Furthermore, we observed significant vascular and liver lipid accumulation, vascular leakage, and plaque oxidation in ldlr-deficient embryos. Finally, upon transcript analysis of several cholesterol-regulating genes, we observed changes similar to those seen in mammalian systems, suggesting that cholesterol regulation may be conserved in zebrafish. Taken together, these observations indicate conservation of ldlr function in zebrafish and demonstrate the utility of transient gene knockdown in embryos as a genetic model for hyperlipidemia. Hyperlipidemia and arterial cholesterol accumulation are primary causes of cardiovascular events. Monogenic forms of hyperlipidemia and recent genome-wide association studies indicate that genetics plays an important role. Zebrafish are a useful model for studying the genetic susceptibility to hyperlipidemia owing to conservation of many components of lipoprotein metabolism, including those related to LDL, ease of genetic manipulation, and in vivo observation of lipid transport and vascular calcification. We sought to develop a genetic model for lipid metabolism in zebrafish, capitalizing on one well-understood player in LDL cholesterol (LDL-c) transport, the LDL receptor (ldlr), and an established in vivo model of hypercholesterolemia. We report that morpholinos targeted against the gene encoding ldlr effectively suppressed its expression in embryos during the first 8 days of development. The ldlr morphants exhibited increased LDL-c levels that were exacerbated by feeding a high cholesterol diet. Increased LDL-c was ameliorated in morphants upon treatment with atorvastatin. Furthermore, we observed significant vascular and liver lipid accumulation, vascular leakage, and plaque oxidation in ldlr-deficient embryos. Finally, upon transcript analysis of several cholesterol-regulating genes, we observed changes similar to those seen in mammalian systems, suggesting that cholesterol regulation may be conserved in zebrafish. Taken together, these observations indicate conservation of ldlr function in zebrafish and demonstrate the utility of transient gene knockdown in embryos as a genetic model for hyperlipidemia. Hypercholesterolemia is a primary cause of atherosclerosis and subsequent cardiovascular events. Genetic factors play a major role in regulation of plasma cholesterol levels, with heritability estimated to range from 40 to 70% [reviewed in (1Dong C. Beecham A. Wang L. Slifer S. Wright C.B. Blanton S.H. Rundek T. Sacco R.L. Genetic loci for blood lipid levels identified by linkage and association analyses in Caribbean Hispanics.J. Lipid Res. 2011; 52: 1411-1419Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar)]. Over 100 genomic regions have been associated with changes in plasma lipid levels (2Willer C.J. Schmidt E.M. Sengupta S. Peloso G.M. Gustafsson S. Kanoni S. Ganna A. Chen J. Buchkovich M.L. Mora S. Global Lipids Genetics Consortium et al.Discovery and refinement of loci associated with lipid levels.Nat. Genet. 2013; 45: 1274-1283Crossref PubMed Scopus (1874) Google Scholar, 3Teslovich T.M. Musunuru K. Smith A.V. Edmondson A.C. Stylianou I.M. Koseki M. Pirruccello J.P. Ripatti S. Chasman D.I. Willer C.J. et al.Biological, clinical and population relevance of 95 loci for blood lipids.Nature. 2010; 466: 707-713Crossref PubMed Scopus (2780) Google Scholar, 4Willer C.J. Mohlke K.L. Finding genes and variants for lipid levels after genome-wide association analysis.Curr. Opin. Lipidol. 2012; 23: 98-103Crossref PubMed Scopus (41) Google Scholar, 5Hegele R.A. Genome-wide association studies of plasma lipids: have we reached the limit?.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2084-2086Crossref PubMed Scopus (6) Google Scholar). Despite the large number of putative candidate genes identified, the functions of many remain largely unknown, owing in part to the lack of physiologically relevant in vivo models with which to validate and characterize the functionality of associated variants (3Teslovich T.M. Musunuru K. Smith A.V. Edmondson A.C. Stylianou I.M. Koseki M. Pirruccello J.P. Ripatti S. Chasman D.I. Willer C.J. et al.Biological, clinical and population relevance of 95 loci for blood lipids.Nature. 2010; 466: 707-713Crossref PubMed Scopus (2780) Google Scholar, 4Willer C.J. Mohlke K.L. Finding genes and variants for lipid levels after genome-wide association analysis.Curr. Opin. Lipidol. 2012; 23: 98-103Crossref PubMed Scopus (41) Google Scholar, 6Rajan A. Perrimon N. Of flies and men: insights on organismal metabolism from fruit flies.BMC Biol. 2013; 11: 38Crossref PubMed Scopus (63) Google Scholar, 7Zhang Y. Zou X. Ding Y. Wang H. Wu X. Liang B. Comparative genomics and functional study of lipid metabolic genes in Caenorhabditis elegans.BMC Genomics. 2013; 14: 164Crossref PubMed Scopus (69) Google Scholar, 8Anderson J.L. Carten J.D. Farber S.A. Zebrafish lipid metabolism: from mediating early patterning to the metabolism of dietary fat and cholesterol.in: Detrich III H.W. Westerfield M. Zon L.I. In The Zebrafish: Cellular and Developmental Biology, Part B. 3rd edition. Routledge, 2011: 111-141Crossref Scopus (123) Google Scholar, 9Babin P.J. Thisse C. Durliat M. Andre M. Akimenko M-A. Thisse B. Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development.Proc. Natl. Acad. Sci. USA. 1997; 94: 8622-8627Crossref PubMed Scopus (144) Google Scholar, 10Barter P.J. Brewer H.B. Chapman M.J. Hennekens C.H. Rader D.J. Tall A.R. Cholesteryl ester transfer protein: a novel target for raising HDL and inhibiting atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 160-167Crossref PubMed Scopus (708) Google Scholar, 11Kotokorpi P. Ellis E. Parini P. Nilsson L-M. Strom S. Steffensen K.R. Gustafsson J-Å. Mode A. Physiological differences between human and rat primary hepatocytes in response to liver X receptor activation by 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride (GW3965).Mol. Pharmacol. 2007; 72: 947-955Crossref PubMed Scopus (37) Google Scholar, 12Wang X. Paigen B. Genetics of variation in HDL cholesterol in humans and mice.Circ. Res. 2005; 96: 27-42Crossref PubMed Scopus (120) Google Scholar, 13Bergen W.G. Mersmann H.J. Comparative aspects of lipid metabolism: impact on contemporary research and use of animal models.J. Nutr. 2005; 135: 2499-2502Crossref PubMed Scopus (204) Google Scholar). Development of novel models in which genes can be individually targeted will likely reveal functional roles for genetic determinants of blood lipid levels and may facilitate discovery of novel targets for drug development. The zebrafish is an excellent candidate for establishing genetic models of hyperlipidemia due to conservation of cell types and molecular pathways involved in lipid metabolism and cholesterol synthesis (8Anderson J.L. Carten J.D. Farber S.A. Zebrafish lipid metabolism: from mediating early patterning to the metabolism of dietary fat and cholesterol.in: Detrich III H.W. Westerfield M. Zon L.I. In The Zebrafish: Cellular and Developmental Biology, Part B. 3rd edition. Routledge, 2011: 111-141Crossref Scopus (123) Google Scholar, 14Lieschke G.J. Currie P.D. Animal models of human disease: zebrafish swim into view.Nat. Rev. Genet. 2007; 8: 353-367Crossref PubMed Scopus (1574) Google Scholar, 15Pack M. Solnica-Krezel L. Malicki J. Neuhauss S.C. Schier A.F. Stemple D.L. Driever W. Fishman M.C. Mutations affecting development of zebrafish digestive organs.Development. 1996; 123: 321-328Crossref PubMed Google Scholar, 16Schlegel A. Stainier D.Y.R. Microsomal triglyceride transfer protein is required for yolk lipid utilization and absorption of dietary lipids in zebrafish larvae.Biochemistry. 2006; 45: 15179-15187Crossref PubMed Scopus (122) Google Scholar, 17Wallace K.N. Akhter S. Smith E.M. Lorent K. Pack M. Intestinal growth and differentiation in zebrafish.Mech. Dev. 2005; 122: 157-173Crossref PubMed Scopus (354) Google Scholar, 18Wallace K.N. Pack M. Unique and conserved aspects of gut development in zebrafish.Dev. Biol. 2003; 255: 12-29Crossref PubMed Scopus (265) Google Scholar, 19Walters J.W. Anderson J.L. Bittman R. Pack M. Farber S.A. Visualization of lipid metabolism in the zebrafish intestine reveals a relationship between NPC1L1-mediated cholesterol uptake and dietary fatty acid.Chem. Biol. 2012; 19 (. [Erratum. 2012. Chem. Biol.19: 1073.]): 913-925Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 20Hölttä‑Vuori M. Salo V.T.V. Nyberg L. Brackmann C. Enejder A. Panula P. Ikonen E. Zebrafish: gaining popularity in lipid research.Biochem. J. 2010; 429: 235-242Crossref PubMed Scopus (130) Google Scholar). These include hepatocytes, adipocytes, and pancreatic acinar cells (17Wallace K.N. Akhter S. Smith E.M. Lorent K. Pack M. Intestinal growth and differentiation in zebrafish.Mech. Dev. 2005; 122: 157-173Crossref PubMed Scopus (354) Google Scholar) as well as expression of genes involved in lipid transport and metabolism, such as LDL receptor (ldlr), sterol regulatory element binding protein (srebp)1/2, and HMG-CoA reductase (Hmgcr) (18Wallace K.N. Pack M. Unique and conserved aspects of gut development in zebrafish.Dev. Biol. 2003; 255: 12-29Crossref PubMed Scopus (265) Google Scholar, 19Walters J.W. Anderson J.L. Bittman R. Pack M. Farber S.A. Visualization of lipid metabolism in the zebrafish intestine reveals a relationship between NPC1L1-mediated cholesterol uptake and dietary fatty acid.Chem. Biol. 2012; 19 (. [Erratum. 2012. Chem. Biol.19: 1073.]): 913-925Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 20Hölttä‑Vuori M. Salo V.T.V. Nyberg L. Brackmann C. Enejder A. Panula P. Ikonen E. Zebrafish: gaining popularity in lipid research.Biochem. J. 2010; 429: 235-242Crossref PubMed Scopus (130) Google Scholar, 21Wallace K.N. Yusuff S. Sonntag J.M. Chin A.J. Pack M. Zebrafish hhex regulates liver development and digestive organ chirality.genesis. 2001; 30: 141-143Crossref PubMed Scopus (75) Google Scholar, 22Yee N.S. Lorent K. Pack M. Exocrine pancreas development in zebrafish.Dev. Biol. 2005; 284: 84-101Crossref PubMed Scopus (99) Google Scholar, 23Apelqvist A. Li H. Sommer L. Beatus P. Anderson D.J. Honjo T. de Angelis M.H. Lendahl U. Edlund H. Notch signalling controls pancreatic cell differentiation.Nature. 1999; 400: 877-881Crossref PubMed Scopus (975) Google Scholar, 24Esni F. Ghosh B. Biankin A.V. Lin J.W. Albert M.A. Yu X. MacDonald R.J. Civin C.I. Real F.X. Pack M.A. et al.Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.Development. 2004; 131: 4213-4224Crossref PubMed Scopus (178) Google Scholar, 25Zecchin E. Mavropoulos A. Devos N. Filippi A. Tiso N. Meyer D. Peers B. Bortolussi M. Argenton F. Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates.Dev. Biol. 2004; 268: 174-184Crossref PubMed Scopus (94) Google Scholar, 26Garcia-Calvo M. Lisnock J. Bull H.G. Hawes B.E. Burnett D.A. Braun M.P. Crona J.H. Davis H.R. Dean D.C. Detmers P.A. et al.The target of ezetimibe is Niemann-Pick C1-like 1 (NPC1L1).Proc. Natl. Acad. Sci. USA. 2005; 102: 8132-8137Crossref PubMed Scopus (650) Google Scholar, 27Gregg R.G. Willer G.B. Fadool J.M. Dowling J.E. Link B.A. Positional cloning of the young mutation identifies an essential role for the Brahma chromatin remodeling complex in mediating retinal cell differentiation.Proc. Natl. Acad. Sci. USA. 2003; 100: 6535-6540Crossref PubMed Scopus (82) Google Scholar, 28Bauer M.P. Bridgham J.T. Langenau D.M. Johnson A.L. Goetz F.W. Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates.Mol. Cell. Endocrinol. 2000; 168: 119-125Crossref PubMed Scopus (101) Google Scholar, 29Archer A. Srinivas Kitambi S. Hallgren S.L. Pedrelli M. Håkan Olsén K. Mode A. Gustafsson J. The liver X-receptor (Lxr) governs lipid homeostasis in zebrafish during development.Open J. Endocr. Metab. Dis. 2012; 2: 74-81Crossref Google Scholar). Consequently, mechanisms of lipid metabolism, storage, absorption, and transport are highly conserved in zebrafish, allowing for physiologically relevant investigation of these processes (20Hölttä‑Vuori M. Salo V.T.V. Nyberg L. Brackmann C. Enejder A. Panula P. Ikonen E. Zebrafish: gaining popularity in lipid research.Biochem. J. 2010; 429: 235-242Crossref PubMed Scopus (130) Google Scholar, 28Bauer M.P. Bridgham J.T. Langenau D.M. Johnson A.L. Goetz F.W. Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates.Mol. Cell. Endocrinol. 2000; 168: 119-125Crossref PubMed Scopus (101) Google Scholar, 30Clifton J.D. Lucumi E. Myers M.C. Napper A. Hama K. Farber S.A. Smith III, A.B. Huryn D.M. Diamond S.L. Pack M. Identification of novel inhibitors of dietary lipid absorption using zebrafish.PLoS ONE. 2010; 5: e12386Crossref PubMed Scopus (68) Google Scholar, 31Marza E. Barthe C. André M. Villeneuve L. Hélou C. Babin P.J. Developmental expression and nutritional regulation of a zebrafish gene homologous to mammalian microsomal triglyceride transfer protein large subunit.Dev. Dyn. 2005; 232: 506-518Crossref PubMed Scopus (70) Google Scholar, 32Ho S-Y. Lorent K. Pack M. Farber S.A. Zebrafish fat-free is required for intestinal lipid absorption and Golgi apparatus structure.Cell Metab. 2006; 3: 289-300Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 33Nixon S.J. Wegner J. Ferguson C. Méry P-F. Hancock J.F. Currie P.D. Key B. Westerfield M. Parton R.G. Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning.Hum. Mol. Genet. 2005; 14: 1727-1743Crossref PubMed Scopus (75) Google Scholar, 34Annilo T. Chen Z-Q. Shulenin S. Costantino J. Thomas L. Lou H. Stefanov S. Dean M. Evolution of the vertebrate ABC gene family: analysis of gene birth and death.Genomics. 2006; 88: 1-11Crossref PubMed Scopus (132) Google Scholar). Moreover, targeted suppression of gene expression can be easily achieved in zebrafish embryos, making assessment of the resulting larval phenotypes feasible for a large number of candidate genes. This is particularly useful in light of the recent development of a zebrafish model for diet-induced hypercholesterolemia characterized by elevated LDL cholesterol (LDL-c) levels and accumulation of vascular lipid deposits in larvae (35Stoletov K. Fang L. Choi S-H. Hartvigsen K. Hansen L.F. Hall C. Pattison J. Juliano J. Miller E.R. Almazan F. et al.Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.Circ. Res. 2009; 104: 952-960Crossref PubMed Scopus (179) Google Scholar). A genetic model of hypercholesterolemia in zebrafish has not previously been reported, however. Here, we report the expansion of the zebrafish hypercholesterolemia model to include hypercholesterolemia as a result of genetic manipulation. The LDLR plays a well-understood role in LDL endocytosis, and mutations in the LDLR-encoding gene (LDLR) are associated with elevated plasma LDL-c levels and familial hypercholesterolemia (36Goldstein J.L. Brown M.S. Lipoprotein receptors and the control of plasma LDL cholesterol levels.Eur. Heart J. 1992; 13: 34-36Crossref PubMed Google Scholar, 37Goldstein J.L. Brown M.S. The LDL receptor.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 431-438Crossref PubMed Scopus (871) Google Scholar, 38Hobbs H.H. Brown M.S. Goldstein J.L. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia.Hum. Mutat. 1992; 1: 445-466Crossref PubMed Scopus (934) Google Scholar, 39Varret M. Rabes J.P. Boileau C. Familial hypercholesterolemia 25 years after. 1. LDL receptor defects.Med. Sci. (Paris). 1997; 13: 1399-1408Crossref Scopus (5) Google Scholar, 40Varret M. Rabés J-P. Missense mutation in the LDLR gene: a wide spectrum in the severity of familial hypercholesterolemia.in: Cooper D.N.C. In Mutations in Human Genetic Disease. InTech, Rijeka, Croatia2012: 55-74Crossref Google Scholar). Our results demonstrate the feasibility of the zebrafish model through transient knockdown of the zebrafish homolog of LDLR, ldlr, by morpholino (MO), which resulted in hypercholesterolemia. These effects were significantly exacerbated when compounded with a high cholesterol diet (HCD) and were ameliorated by exogenous treatment with atorvastatin. Finally, we provide evidence that elevated LDL-c in ldlr-deficient larvae results in vascular and liver lipid accumulation, vascular leakage, increased plaque oxidation, macrophage recruitment, and hepatomegaly in larvae. Taken together, these data indicate conservation of ldlr function in zebrafish and support the use of this model to investigate genetic mechanisms of hypercholesterolemia. Embryos were collected from natural matings of adult wild-type Tubingen zebrafish stocks maintained at 28.5°C. Embryos were cultured in embryo medium (41Westerfield M. The Zebrafish BookA Guide for the Laboratory Use of Zebrafish (Danio rerio). 4th edition. University of Oregon Press, Eugene, OR2000Google Scholar) at 28.5°C until harvesting at time points between 1 and 12 days postfertilization (dpf). All zebrafish procedures were approved by the University of Maryland Animal Care and Use Committee (protocol 0313013). A splice-blocking (SB) MO (Gene Tools, LLC) was designed (5′-AGATCACATTTCATTTCTTACAGCA-3′) to target the intron 4-exon 4 splice junction of zebrafish ldlr. A translation-blocking (TB) MO was also used (5′-GATCTCCATAGTCTCTAAGCGAGCT-3′) to inhibit translation of zebrafish ldlr. Five nanograms of ldlr SB or TB MO, diluted in nuclease-free (DEPC-free) water containing 0.1% phenol red, was injected into one- or two-cell stage embryos. Five nanograms of nonspecific standard control MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used for control embryos. Embryos were injected with each MO and cultured at 28.5°C. Approximately 4,000 viable embryos were collected per MO per experiment for use in subsequent assays. To validate disruption of splicing and removal of ldlr exon 4, cDNA was generated from ldlr (SB) and control morphants by isolation of whole embryo RNA and subsequent reverse transcription (RevertAid First Strand cDNA synthesis kit, Thermo Scientific) at each day of development starting at 24 h postfertilization (hpf) through 10 dpf. A total of 20 embryos were pooled for extraction of RNA at each time point. Experiments were repeated three times. Primers flanking ldlr exon 4 were designed with the following sequences: ldlr_SB_KD_forward primer, 5'-TGCAGACCCAGTCAGTTCAG-3' ldlr_SB_KD_reverse primer, 5'-TCCATCTGGTAGCCATCCTC-3'. To determine the nucleotides excised by the ldlr SB MO, PCR product generated using the same primers was sequenced using an Applied Biosystems model 3730XL 96-capillary high-throughput sequencer. Both the wild-type and aberrantly spliced ldlr transcripts were isolated from cDNA generated from zebrafish control and ldlr SB embryo homogenates, respectively. The open reading frame of each was amplified by PCR using the following primers: ldlr_gene_F, 5'-ATGGAGATCTCATTCATGCTTTTA-3' ldlr_gene_R, 5'-TCATACCTGAGGGTAAAAATAGC-3'. Products were gel-purified (QIAquick gel extraction kit) and cloned into the pCR2.1-TOPO TA vector (Life Technologies). Proper transcript length and sequence were confirmed via DNA sequencing. mRNA was transcribed from each construct using the mMESSAGE mMACHINE T7 transcription kit (Ambion) after linearization with SacI. The ldlr mRNA was injected into embryos either alone or along with 5 ng ldlr SB MO. Larvae (5 dpf) were fed either control diet (CD) [Zeigler AP100 (Aquatic Habitats, Inc.) + 10 μg/g BODIPY 576/589 C11 (Molecular Probes, Inc.)] or the same diet supplemented with 4% w/w cholesterol (HCD), as previously described (35Stoletov K. Fang L. Choi S-H. Hartvigsen K. Hansen L.F. Hall C. Pattison J. Juliano J. Miller E.R. Almazan F. et al.Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.Circ. Res. 2009; 104: 952-960Crossref PubMed Scopus (179) Google Scholar). Diets were replaced twice daily until 7 dpf (48 h of feeding) or until 12 dpf (7 days of feeding). LDL-c levels were quantified from whole embryo homogenates consisting of 100 embryos per diet group homogenized in 400 μl of ice-cold 10 μM butylated hydroxytoluene. Homogenate was filtered through a 0.45 μm Dura PVDF membrane filter (Millipore) in preparation for lipid extraction and processed using the HDL and LDL/VLDL cholesterol assay kit (Cell Biolabs, Inc.) as per the manufacturer's protocol. After precipitation and dilution, samples were analyzed by fluorimetric analysis using a SpectraMax Gemini EM plate reader and SoftMax Pro microplate data acquisition and analysis software (Molecular Devices). Experiments were repeated at least three times. Values obtained by fluorimetric analysis were calculated relative to total protein concentration. Total protein was isolated from homogenates of 100 embryos in microcentrifuge tubes placed on ice after aspiration of embryo media and replacement with 500 μl cold RIPA buffer [150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.5), 1% NP-40] containing 1:100 protease inhibitor cocktail (Sigma P8340). After homogenization, tubes were rotated for 10 min at 4°C, then centrifuged at 12,000 g at 4°C for 10 min. Supernatant (20 μl) was transferred to a fresh tube and the protein concentration was quantified by BCA protein assay (Sigma C2284 and B9643). CD- or HCD-fed larvae were immobilized at 7 or 12 dpf by placement in one drop of 0.02% tricaine (Sigma-Aldrich) diluted in water on a glass depression microscope slide. Larvae were imaged at 20× magnification using an Olympus IX50 epifluorescent inverted microscope and cellSens Dimension software. The total number of BODIPY 576/589 C11-positive plaques was quantified in a 438.6 × 330.2 μm window along the caudal vein for a minimum of 25 larvae per MO per diet, as per published protocols (35Stoletov K. Fang L. Choi S-H. Hartvigsen K. Hansen L.F. Hall C. Pattison J. Juliano J. Miller E.R. Almazan F. et al.Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.Circ. Res. 2009; 104: 952-960Crossref PubMed Scopus (179) Google Scholar, 42Fang L. Green S.R. Baek J.S. Lee S-H. Ellett F. Deer E. Lieschke G.J. Witztum J.L. Tsimikas S. Miller Y.I. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.J. Clin. Invest. 2011; 121: 4861-4869Crossref PubMed Scopus (68) Google Scholar). cDNA derived from mRNA isolated from pooled embryos (n = 20) of either ldlr SB or control morphants at 1–10 dpf were used for quantification of gene expression using SYBR Green I (Roche). Samples and controls/standards were run in triplicates on a Roche LightCycler 480 instrument and mRNA expression levels were quantified relative to β-actin. Experiments were repeated at least three times. Primer sequences are available upon request. Embryo medium from CD- or HCD-fed larvae was removed after 24 h of feeding and replaced with atorvastatin-containing medium at a concentration of 50 μM (LKT Laboratories). Larvae were cultured at 28.5°C for an additional 24 h and then utilized in subsequent assays. Larvae (5 and 7 dpf) were staged and fixed in 4% paraformaldehyde in PBS and incubated overnight at 4°C. Embryos were subsequently removed from the fixative and washed in 60% 2-propanol at room temperature; this was followed by a 3 h incubation in freshly filtered 0.3% Oil Red O (ORO) (Sigma, O0625) in 60% 2-propanol. To remove background staining prior to imaging, embryos were washed twice with 60% 2-propanol for 10 min and transferred to DEPC-treated water. Embryos were imaged using a Zeiss Lumar.V12 microscope and assessed for lipid density content. The size of the liver for ORO-stained control MOs (n = 20) and ldlr SB MO embryos (n = 20) at 4, 5, and 7 dpf was determined using ImageJ software. Protein was extracted from pooled (n = 10) deyolked control MO-, ldlr SB MO-, and ldlr TB MO-injected embryos at 3, 5, and 7 dpf. Embryos were homogenized in 100 μl 2× SDS buffer [0.125 M Tris-Cl, 4% SDS, 20% v/v glycerol, 0.2 M DTT, 0.02% bromophenol blue, (pH 6.8)] and 2 μl protease inhibitor cocktail (Sigma) using a #23 syringe. PMSF (2 μl) was added and samples were then boiled for 5 min, cooled on ice for 5 min, and centrifuged for 3 min at 12,000 g. Ten microliters of each sample and protein ladder (Precision Plus Protein dual color standards #161-0374, Bio-Rad) were separated using 7.5% Criterion Tris-HCD gels (345-0007, Bio-Rad) at 100 V for 1 h. Proteins were transferred using a Criterion blotter (Bio-Rad) at 115 V for 1 h to Immobilon-P transfer membranes (IPVH00010, Millipore). The membranes were placed in blocking buffer for 1 h. The membranes were cut at 50 kDa in order to probe for both Ldlr (>50 kDa) and Actin (<50 kDa). The immunoblotting protocol was performed as follows: a 2 h room temperature incubation of anti-LDLR antibody (Sigma-Aldrich SAB3500286; 1:3,000) was followed by three 5 min washes using PBS/Tween; the secondary antibody, anti-chicken IgY (Sigma-Aldrich A9046; 1:160,000 dilution) was incubated for 2 h at room temperature followed by three PBS/Tween 15 min washes. Actin was detected by incubation with primary antibody (Sigma A2066; 1:3,000) for 2 h followed by secondary polyclonal goat anti-IgG antibody (Jackson ImmunoResearch Laboratories 111-035-003; 1:5,000). Chemiluminescence was enhanced using SuperSignal West Dura extended duration substrate (Thermo Scientific) following the manufacturer's protocol, and imaged using Alpha View-Fluor ChemQ software (Alpha Innotech Gel Imaging System for Life Science). Transgenic heat shock cognate 70 kDa protein (hsp70):IK17-eGFP zebrafish embryos (kindly received from Yury I. Miller, University of California, San Diego) were heat shocked for 1 h in a 37°C water bath prior to treatment with CD or HCD supplemented with cholesteryl BODIPY 576/589 at 5 dpf (42Fang L. Green S.R. Baek J.S. Lee S-H. Ellett F. Deer E. Lieschke G.J. Witztum J.L. Tsimikas S. Miller Y.I. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.J. Clin. Invest. 2011; 121: 4861-4869Crossref PubMed Scopus (68) Google Scholar). At 7 dpf, embryos were immobilized by placement in one drop of 0.02% tricaine (Sigma-Aldrich) diluted in water on a glass depression microscope slide and imaged using an Olympus IX50 epifluorescent inverted microscope and cellSens Dimension software. Oxidized lipoprotein (OxLDL) was determined by colocalization of red BODIPY 576/589 and green IK17-eGFP. Experiments were repeated twice to confirm results and to obtain a total of 20 embryos per treatment. HCD- or CD-fed embryos (7 dpf, control and morphant) were fixed in 10% neutral buffer formalin overnight and washed in PBS three times for 5 min prior to paraffin-embedding and collection of 10 μm thick transverse sections from 10 embryos per treatment group. Slides were washed three times in CitriSolv Hybrid (Decon Labs, Inc.) for 5 min each, followed by rehydration in 100, 95, 70, 50, and 25% ethanol (2 min each). Slides were then washed with PBS (5 min), treated in 0.1% Triton X-100 (5 min), and rewashed in PBS (5 min) prior to blocking (PBST, 1% BSA, 10% FBS) for 1 h at room temperature. Slides were immunostained using anti-human L-plastin (D-16) primary antibody (Santa Cruz Biotechnology sc-16657; 1:50) overnight in a humidity chamber at 4°C followed by two washes in PBS (5 min each) and 1 h incubation with Alexa Fluor® 594 donkey anti-goat IgG secondary antibody (Life Technologies A11058; 1:800), and 10 min counterstaining with DAPI (4′,6-diamidino-2-phenylindole) after two additional PBS washes (5 min each). Sections were imaged using an Olympus IX50 epifluorescent inverted microscope and cellSens Dimension software. To assess the colocalization of macrophages with cholesterol plaques, coverslips were removed by incubation at −20°C (25 min) followed by a PBS wash (5 min) and 10 min incubation with BODIPY 493/503 (Life Technologies; 1 mg/ml). Sections were washed again in PBS (5 min) and reimaged. Larvae (7 dpf) were immobilized by anesthetization with 0.02% tricaine and placed in 0.3% methyl cellulose. Individual larvae were injected in the cardinal vein with 0.5 mg/ml dextran labeled with Alexa Fluor 594 (Life Technologies) as described in Stoletov et al. (35Stoletov K. Fang L. Choi S-H. Hartvigsen K. Hansen L.F. Hall C. Pattison J. Juliano J. Miller E.R. Almazan F. et al.Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in h" @default.
• W2034672127 created "2016-06-24" @default.
• W2034672127 creator A5004416446 @default.
• W2034672127 creator A5008844169 @default.
• W2034672127 creator A5021956611 @default.
• W2034672127 creator A5027947946 @default.
• W2034672127 creator A5038187262 @default.
• W2034672127 creator A5077733401 @default.
• W2034672127 date "2014-11-01" @default.
• W2034672127 modified "2023-10-16" @default.
• W2034672127 title "Disruption of ldlr causes increased LDL-c and vascular lipid accumulation in a zebrafish model of hypercholesterolemia" @default.
• W2034672127 cites W1510378561 @default.
• W2034672127 cites W1535964227 @default.
• W2034672127 cites W1575893908 @default.
• W2034672127 cites W1650965741 @default.
• W2034672127 cites W1721336579 @default.
• W2034672127 cites W1860242155 @default.
• W2034672127 cites W1904804142 @default.
• W2034672127 cites W1939684610 @default.
• W2034672127 cites W1964639766 @default.
• W2034672127 cites W1966650064 @default.
• W2034672127 cites W1968483542 @default.
• W2034672127 cites W1969219820 @default.
• W2034672127 cites W1974089312 @default.
• W2034672127 cites W1975411923 @default.
• W2034672127 cites W1981335859 @default.
• W2034672127 cites W1981616219 @default.
• W2034672127 cites W1989011678 @default.
• W2034672127 cites W1989312217 @default.
• W2034672127 cites W1990484548 @default.
• W2034672127 cites W1992842248 @default.
• W2034672127 cites W1994412419 @default.
• W2034672127 cites W1996904509 @default.
• W2034672127 cites W1997016486 @default.
• W2034672127 cites W2008880591 @default.
• W2034672127 cites W2015376110 @default.
• W2034672127 cites W2018970660 @default.
• W2034672127 cites W2021043456 @default.
• W2034672127 cites W2023899667 @default.
• W2034672127 cites W2027713980 @default.
• W2034672127 cites W2029070177 @default.
• W2034672127 cites W2029187912 @default.
• W2034672127 cites W2036470826 @default.
• W2034672127 cites W2039431622 @default.
• W2034672127 cites W2039999119 @default.
• W2034672127 cites W2044679605 @default.
• W2034672127 cites W2048978769 @default.
• W2034672127 cites W2053383361 @default.
• W2034672127 cites W2057394745 @default.
• W2034672127 cites W2058108347 @default.
• W2034672127 cites W2062716507 @default.
• W2034672127 cites W2065000758 @default.
• W2034672127 cites W2065527687 @default.
• W2034672127 cites W2067504916 @default.
• W2034672127 cites W2070978553 @default.
• W2034672127 cites W2073119213 @default.
• W2034672127 cites W2075224548 @default.
• W2034672127 cites W2078095032 @default.
• W2034672127 cites W2080118521 @default.
• W2034672127 cites W2084744450 @default.
• W2034672127 cites W2090050647 @default.
• W2034672127 cites W2093683138 @default.
• W2034672127 cites W2093913603 @default.
• W2034672127 cites W2094788334 @default.
• W2034672127 cites W2102468628 @default.
• W2034672127 cites W2102491464 @default.
• W2034672127 cites W2102805985 @default.
• W2034672127 cites W2105958180 @default.
• W2034672127 cites W2106069121 @default.
• W2034672127 cites W2107841008 @default.
• W2034672127 cites W2108333200 @default.
• W2034672127 cites W2113284407 @default.
• W2034672127 cites W2114835891 @default.
• W2034672127 cites W2115801316 @default.
• W2034672127 cites W2124130781 @default.
• W2034672127 cites W2127690615 @default.
• W2034672127 cites W2127791131 @default.
• W2034672127 cites W2128007109 @default.
• W2034672127 cites W2131834725 @default.
• W2034672127 cites W2135152784 @default.
• W2034672127 cites W2137694414 @default.
• W2034672127 cites W2138704564 @default.
• W2034672127 cites W2148463229 @default.
• W2034672127 cites W2148536887 @default.
• W2034672127 cites W2148609678 @default.
• W2034672127 cites W2152415811 @default.
• W2034672127 cites W2153118028 @default.
• W2034672127 cites W2156818917 @default.
• W2034672127 cites W2159516023 @default.
• W2034672127 cites W2166594345 @default.
• W2034672127 cites W2167994535 @default.
• W2034672127 cites W2329905543 @default.
• W2034672127 cites W2404964590 @default.
• W2034672127 cites W4243805969 @default.
• W2034672127 cites W4252021281 @default.
• W2034672127 cites W2098022640 @default.
• W2034672127 doi "https://doi.org/10.1194/jlr.m046540" @default.
• W2034672127 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4617127" @default.

• next