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• W2000939135 abstract "The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2–4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2 × 106 Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains. The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2–4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2 × 106 Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains. Hyaluronan (HA) 2The abbreviations used are: HAhyaluronanGAGglycosaminoglycanECMextracellular matrixHASthe gene that encodes HA synthase (the enzyme)ASMCaortic smooth muscle cellsLDLlow density lipoproteinWHHLWatanabe heritable hyperlipidemicFHfamilial hypercholesterolemiaNZWNew Zealand WhiteDMEMDulbecco's modified Eagle's mediumSFMserum-free mediumFBSfetal bovine serumPBSphosphate-buffered salineMeβCDmethyl-β-cyclodextrinbPGbiotinylated HA-binding protein isolated from bovine cartilageCPCcetylpyridinium chlorideUCunesterified cholesterolHMG-CoAhydroxymethylglutaryl coenzyme ASREBPssterol regulatory element-binding proteinsELISAenzyme-linked immunosorbent assayCHAPS3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonic acid 2The abbreviations used are: HAhyaluronanGAGglycosaminoglycanECMextracellular matrixHASthe gene that encodes HA synthase (the enzyme)ASMCaortic smooth muscle cellsLDLlow density lipoproteinWHHLWatanabe heritable hyperlipidemicFHfamilial hypercholesterolemiaNZWNew Zealand WhiteDMEMDulbecco's modified Eagle's mediumSFMserum-free mediumFBSfetal bovine serumPBSphosphate-buffered salineMeβCDmethyl-β-cyclodextrinbPGbiotinylated HA-binding protein isolated from bovine cartilageCPCcetylpyridinium chlorideUCunesterified cholesterolHMG-CoAhydroxymethylglutaryl coenzyme ASREBPssterol regulatory element-binding proteinsELISAenzyme-linked immunosorbent assayCHAPS3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonic acid is a high molecular mass (1 × 105–1 × 107 Da) unbranched glycosaminoglycan (GAG), composed of repeating disaccharides of N-acetyl-d-glucosamine and d-glucuronic acid (1Laurent T.C. Fraser J.R. F. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2057) Google Scholar, 2Lapcik Jr., L. Lapcik L. De Smedt S. Demeester J. Chabrecek P. Chem. Rev. 1998; 98: 2663-2684Crossref PubMed Scopus (400) Google Scholar). It is synthesized at the cytoplasmic surface of the plasma membrane by a family of HA synthases (HASs) (3Prehm P. Biochem. J. 1984; 220: 597-600Crossref PubMed Scopus (292) Google Scholar, 4Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Abstract Full Text PDF PubMed Google Scholar), and it is subsequently extruded through the plasma membrane into the pericellular matrix (3Prehm P. Biochem. J. 1984; 220: 597-600Crossref PubMed Scopus (292) Google Scholar). HAS enzymes are encoded by three highly conserved mammalian genes, HAS1, HAS2, and HAS3, and are predicted integral plasma membrane proteins with multiple transmembrane domains (5Itano N. Sawai T. Yoshida M. Lenas P. Yamada Y. Imagawa M. Shinomura T. Hamaguchi M. Yoshida Y. Ohnuki Y. Miyauchi S. Spicer A.P. McDonald J.A. Kimata K. J. Biol. Chem. 1999; 274: 25085-25092Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar, 6Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar) that partially co-localize with membrane lipid microdomains (7Kultti A. Rilla K. Tiihonen R. Spicer A.P. Tammi R.H. Tammi M.I. J. Biol. Chem. 2006; 281: 15821-15828Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). HA degradation occurs locally through at least two catabolic pathways as follows: internalization by an endocytic pathway that requires functional CD44 (8Knudson W. Chow G. Knudson C.B. Matrix Biol. 2002; 21: 15-23Crossref PubMed Scopus (203) Google Scholar), a membrane microdomain-associated protein (9Ilangumaran S. Briol A. Hoessli D.C. Blood. 1998; 91: 3901-3908Crossref PubMed Google Scholar) and the principal cell-surface receptor for HA (10Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2141) Google Scholar); and an extracellular catalysis mediated by hyaluronidase-2, a glycerophosphoinositol-anchored plasma membrane protein (11Stern R. Glycobiology. 2003; 13: R105-R115Crossref PubMed Scopus (286) Google Scholar). The half-life of HA in tissues ranges from a few hours to several days. Interstitial HA is catabolized either locally or, very rapidly, in the lymphatics and by endothelial cells of the liver sinusoids (1Laurent T.C. Fraser J.R. F. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2057) Google Scholar, 12Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1453) Google Scholar). Thus, in tissues where interstitial fluid normally drains into the lymphatic system, a rapid rate of turnover by local cells is not necessary to avoid excessive accumulation of HA. hyaluronan glycosaminoglycan extracellular matrix the gene that encodes HA synthase (the enzyme) aortic smooth muscle cells low density lipoprotein Watanabe heritable hyperlipidemic familial hypercholesterolemia New Zealand White Dulbecco's modified Eagle's medium serum-free medium fetal bovine serum phosphate-buffered saline methyl-β-cyclodextrin biotinylated HA-binding protein isolated from bovine cartilage cetylpyridinium chloride unesterified cholesterol hydroxymethylglutaryl coenzyme A sterol regulatory element-binding proteins enzyme-linked immunosorbent assay 3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonic acid hyaluronan glycosaminoglycan extracellular matrix the gene that encodes HA synthase (the enzyme) aortic smooth muscle cells low density lipoprotein Watanabe heritable hyperlipidemic familial hypercholesterolemia New Zealand White Dulbecco's modified Eagle's medium serum-free medium fetal bovine serum phosphate-buffered saline methyl-β-cyclodextrin biotinylated HA-binding protein isolated from bovine cartilage cetylpyridinium chloride unesterified cholesterol hydroxymethylglutaryl coenzyme A sterol regulatory element-binding proteins enzyme-linked immunosorbent assay 3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonic acid Considerable work indicates that HA, a widely distributed component of the extracellular matrix (ECM) of vertebrate tissues (1Laurent T.C. Fraser J.R. F. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2057) Google Scholar, 2Lapcik Jr., L. Lapcik L. De Smedt S. Demeester J. Chabrecek P. Chem. Rev. 1998; 98: 2663-2684Crossref PubMed Scopus (400) Google Scholar), accumulates in human atherosclerotic lesions (13Levesque H. Girard N. Maingonnat C. Delpech A. Chauzy C. Tayot J. Courtois H. Delpech B. Atherosclerosis. 1994; 105: 51-62Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 14Riessen R. Wight T.N. Pastore C. Henley C. Isner J.M. Circulation. 1996; 93: 1141-1147Crossref PubMed Scopus (148) Google Scholar, 15Papakonstantinou E. Roth M. Block L.H. Mirtsou-Fidani V. Argiriadis P. Karakiulakis G. Atherosclerosis. 1998; 138: 79-89Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 17Kolodgie F.D. Burke A.P. Farb A. Weber D.K. Kutys R. Wight T.N. Virmani R. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1642-1648Crossref PubMed Scopus (212) Google Scholar, 18Srinivasan S.R. Yost K. Radhakrishnamurthy B. Dalferes Jr., E.R. Berenson G.S. Atherosclerosis. 1980; 36: 25-37Abstract Full Text PDF PubMed Scopus (37) Google Scholar) and in animal models of vascular injury and atherosclerosis (19Evanko S.P. Raines E.W. Ross R. Gold L.I. Wight T.N. Am. J. Pathol. 1998; 152: 533-546PubMed Google Scholar, 20Kaneko E. Skinner M.P. Raines E.W. Yuan C. Rosenfeld M.E. Wight T.N. Ross R. Coron. Artery Dis. 2000; 11: 599-606Crossref PubMed Scopus (8) Google Scholar, 21Cuff C.A. Kothapalli D. Azonobi I. Chun S. Zhang Y. Belkin R. Yeh C. Secreto A. Assoian R.K. Rader D.J. Pure E. J. Clin. Investig. 2001; 108: 1031-1040Crossref PubMed Scopus (255) Google Scholar). Furthermore, HAS2 transgene overexpression promotes atherosclerosis in apolipoprotein E knock-out mice (22Chai S. Chai Q. Danielsen C.C. Hjorth P. Nyengaard J.R. Ledet T. Yamaguchi Y. Rasmussen L.M. Wogensen L. Circ. Res. 2005; 96: 583-591Crossref PubMed Scopus (110) Google Scholar). HA is frequently found in lipid-enriched areas of atherosclerotic lesions (19Evanko S.P. Raines E.W. Ross R. Gold L.I. Wight T.N. Am. J. Pathol. 1998; 152: 533-546PubMed Google Scholar). It also co-isolates with lipoproteins from human atherosclerotic lesions (18Srinivasan S.R. Yost K. Radhakrishnamurthy B. Dalferes Jr., E.R. Berenson G.S. Atherosclerosis. 1980; 36: 25-37Abstract Full Text PDF PubMed Scopus (37) Google Scholar) and promotes foam cell and cholesterol accumulation in skin xanthoma (23Seike M. Ikeda M. Matsumoto M. Hamada R. Takeya M. Kodama H. J. Dermatol. Sci. 2006; 41: 197-204Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although HA and cholesterol accumulation are both prominent features of atherosclerosis, the link between HA metabolism and cholesterol homeostasis in the cells of vascular lesions has not been adequately addressed. To explore this relationship, we have used aortic smooth muscle cells (ASMC) cultured from low density lipoprotein (LDL) receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbits that exhibit atherosclerosis because of high plasma LDL cholesterol levels (24Watanabe Y. Atherosclerosis. 1980; 36: 261-268Abstract Full Text PDF PubMed Scopus (409) Google Scholar, 25Rosenfeld M.E. Tsukada T. Gown A.M. Ross R. Arteriosclerosis. 1987; 7: 9-23Crossref PubMed Google Scholar, 26Rosenfeld M.E. Tsukada T. Chait A. Bierman E.L. Gown A.M. Ross R. Arteriosclerosis. 1987; 7: 24-34Crossref PubMed Google Scholar, 27Rosenfeld M.E. Chait A. Bierman E.L. King W. Goodwin P. Walden C.E. Ross R. Arteriosclerosis. 1988; 8: 338-347Crossref PubMed Google Scholar). Disrupted cholesterol homeostasis in this model is because of a mutation in the LDL receptor gene (28Yamamoto T. Bishop R.W. Brown M.S. Goldstein J.L. Russell D.W. Science. 1986; 232: 1230-1237Crossref PubMed Scopus (208) Google Scholar) and is similar to the most common class of mutations in the human disease of familial hypercholesterolemia (FH) (29Hobbs H.H. Brown M.S. Goldstein J.L. Hum. Mutat. 1992; 1: 445-466Crossref PubMed Scopus (927) Google Scholar). We report that cultured WHHL ASMC accumulate 2–4-fold more HA than ASMC from normolipidemic, sex- and age-matched New Zealand White (NZW) rabbits. In addition, the HA synthesized by WHHL ASMC is of a larger average hydrodynamic size. These changes appear to be due to a combination of increased synthesis and decreased degradation of HA in WHHL ASMC. Furthermore, we show that removing cholesterol from the WHHL ASMC returns HA accumulation by these cells to control levels, whereas replenishing cholesterol to these cells restores the elevated HA accumulation. These findings directly link cholesterol homeostasis in cells to the metabolism of HA, and may help to explain HA accumulation in atherosclerotic lesions. Materials—Skin fibroblasts from homozygous patients with FH and from matched normolipidemic individuals, and Dulbecco's modified Eagle's medium (DMEM) with Earle's salts twice enriched with vitamins and amino acids were purchased from Coriell Cell Repositories (Camden, NJ). Coriell repository numbers for the normal cell lines were as follows: GM00408D, GM00500B, GM02674B, and GM03651E. Repository numbers for the FH cells were as follows: GM00283, GM00486C, GM00488C, GM00701C, GM02000G, and GM03040C. Rabbit SMC growth medium and rabbit SMC serum- and growth factor-free medium (SFM) were purchased from Cell Applications Inc. (San Diego, CA). Fetal bovine serum (FBS) was from Irvine Scientific (Santa Ana, CA). Phosphate-buffered saline (PBS) and trypsin/EDTA were from Invitrogen. Hepes-buffered saline, 0.025 mg/ml trypsin, 0.25 mm EDTA and trypsin-neutralizing solutions were from Cambrex (Walkersville, MD). d-[3H]Glucosamine HCl (24 Ci/mmol), [3H]acetic acid sodium (4.1 Ci/mmol), and Streptomyces hyaluronidase were purchased from MP Biomedicals (Aurora, OH). Streptomyces griseus Pronase was from Roche Diagnostics. Hyaluronic acid, from human umbilical cord, and all other reagents were products of Sigma. Rabbits—A WHHL rabbit colony was maintained by inbreeding as described previously (25Rosenfeld M.E. Tsukada T. Gown A.M. Ross R. Arteriosclerosis. 1987; 7: 9-23Crossref PubMed Google Scholar). Breeding pairs of the WHHL rabbits were a generous gift of Drs. Daniel Steinberg and Joseph Witztum, University of California, San Diego. In addition, two 4-month-old WHHL rabbits were purchased from Covance Research Products Inc. (Denver, PA). The animals were 4–10-month-old males and females. Age- and sex-matched NZW rabbits were from Western Oregon Rabbit Co. (Philomath, OR). The rabbits were sacrificed by sodium pentobarbital injection (intravenous 0.5 ml/lb body weight), and the aortas were harvested. Animal care and procedures were conducted in accordance with applicable state and federal laws and under protocols approved by the University of Washington Institutional Animal Care and Use Committee. Cell Culture—Primary ASMC were isolated from WHHL and NZW rabbit aortas by the explant outgrowth method (30Ross R. J. Cell Biol. 1971; 50: 172-186Crossref PubMed Scopus (1265) Google Scholar) and grown in rabbit SMC growth medium. All cell cultures were maintained at 37 °C, in a humidified atmosphere containing 5% CO2, and used between the fourth and sixth passage. For cell proliferation experiments, 24 h after seeding in rabbit SMC growth medium, cells were growth-arrested in 0.1–0.5% FBS/SFM for 48 h. Fresh 10% FBS/SFM was then added to growth-arrested ASMC at day 0, and subsequently after 24 h (day 1) and then every 48 h (days 3, 5, 7, and 9) in 10% FBS/SFM. In cell density experiments, growth-arrested ASMC were incubated with fresh 0.1 or 10% FBS medium for 20 h. Methyl-β-cyclodextrin (MeβCD) is commonly used as a cell cholesterol acceptor to deplete cells (31Kojro E. Gimpl G. Lammich S. Marz W. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5815-5820Crossref PubMed Scopus (721) Google Scholar, 32Levitan I. Christian A.E. Tulenko T.N. Rothblat G.H. J. Gen. Physiol. 2000; 115: 405-416Crossref PubMed Scopus (174) Google Scholar) and their plasma membrane (33Oliferenko S. Paiha K. Harder T. Gerke V. Schwarzler C. Schwarz H. Beug H. Gunthert U. Huber L.A. J. Cell Biol. 1999; 146: 843-854Crossref PubMed Scopus (355) Google Scholar) of cholesterol. Over 50% cholesterol depletion can be achieved using MeβCD at concentrations as low as 5 mm during a 15-min incubation (33Oliferenko S. Paiha K. Harder T. Gerke V. Schwarzler C. Schwarz H. Beug H. Gunthert U. Huber L.A. J. Cell Biol. 1999; 146: 843-854Crossref PubMed Scopus (355) Google Scholar). Cholesterol-MeβCD complex is used as a cholesterol donor because it is a water-soluble form of cholesterol that is rapidly and efficiently taken up by cells, thus inducing cholesterol loading of cell membranes (32Levitan I. Christian A.E. Tulenko T.N. Rothblat G.H. J. Gen. Physiol. 2000; 115: 405-416Crossref PubMed Scopus (174) Google Scholar, 34Christian A.E. Haynes M.P. Phillips M.C. Rothblat G.H. J. Lipid Res. 1997; 38: 2264-2272Abstract Full Text PDF PubMed Google Scholar). For cholesterol depletion and replenishing experiments, the indicated amounts of MeβCD were added to growth-arrested ASMC for 1 h in 0.5% FBS/SFM. Medium was removed, and the cells were gently washed with SFM. Fresh medium was then added with or without 0.25 mm cholesterol-MeβCD water-soluble complex in an 8:1 mol:mol ratio for 30 min in 0.5% FBS/SFM (32Levitan I. Christian A.E. Tulenko T.N. Rothblat G.H. J. Gen. Physiol. 2000; 115: 405-416Crossref PubMed Scopus (174) Google Scholar, 34Christian A.E. Haynes M.P. Phillips M.C. Rothblat G.H. J. Lipid Res. 1997; 38: 2264-2272Abstract Full Text PDF PubMed Google Scholar). This complex is a cholesterol donor. The cholesterol complex was also added to control cells. To block cholesterol synthesis, 30 μm lovastatin was added for the last 24 h of the 48-h growth-arrest period. To counteract the lovastatin inhibition of cholesterol synthesis, 30 μm mevalonic acid was added simultaneously with the lovastatin or to controls in parallel dishes. Following the incubations with cyclodextrins or lovastatin (with or without mevalonic acid), the cells were washed gently one time with SFM and then incubated with 1% FBS/SFM for 2.5 h. Skin fibroblasts were maintained in DMEM supplemented with 10% FBS. For experiments, fibroblasts were seeded at the indicated cell density in 10% FBS/DMEM. After 24 h, they were growth-arrested in 0.1% FBS/DMEM for 48 h. Fresh medium containing 10% FBS was then added for 24 h. At the end of each incubation period, the medium was collected, incubated with 100–500 μg/ml Pronase at 37 °C for 2–18 h to release HA from its binding proteins, then heated at 100 °C for 20–30 min to inactivate the Pronase. Cell layers were washed with PBS, incubated in Tris-buffered Pronase (0.5 m Tris, pH 6.5) at 37 °C for 18 h, scraped into the buffer, and heat-inactivated. After centrifugation at 1000 rpm for 5 min, HA in the supernatant was quantified and normalized to cell number. Hyaluronan Assay—We used a modification (35Wilkinson T.S. Potter-Perigo S. Tsoi C. Altman L.C. Wight T.N. Am. J. Respir. Cell Mol. Biol. 2004; 31: 92-99Crossref PubMed Scopus (91) Google Scholar) of a previously described (36Underhill C.B. Nguyen H. Shizari M. Culty M. Dev. Biol. 1993; 155: 324-336Crossref PubMed Scopus (129) Google Scholar) competitive ELISA in which the samples were first mixed with HA-binding protein isolated from bovine cartilage that has been biotinylated (bPG) and then added to human umbilical cord HA-coated microtiter plates, the final signal being inversely proportional to the level of HA added to the bPG. Characterization of Hyaluronan by Hydrodynamic Size—WHHL and NZW rabbit ASMC, seeded and then growth-arrested as described above, were labeled for 6 h with 50 μCi/ml [3H]glucosamine ([3H]GlcN) spiked at 0-, 6-, 12-, and 18-h time points in 0.5% or 10% FBS-containing medium. At the end of each 6-h pulse, the medium was collected, incubated with Pronase, and then heated as for the ELISAs. Heating HA to 100 °C did not change its size as determined by size exclusion chromatography on Sephacryl S-1000 (data not shown). HA and other GAGs were separated from unincorporated [3H]GlcN by chromatography on Sephadex G-50 columns. Macromolecular fractions containing identical 3H counts were incubated with or without 0.5 unit/ml Streptomyces hyaluronidase for 2–18 h at 37 °C, and macromolecular size was analyzed by size exclusion chromatography on a 1.2 × 58-cm Sephacryl S-1000 column, under nondissociative conditions. Fractions (0.5 ml) were eluted in 0.5 m sodium acetate, 0.025% CHAPS, pH 7.0, and the radioactivity was measured by liquid scintillation counting. HA radioactivity for each fraction was determined as hyaluronidase-sensitive GAG, which was calculated by subtracting hyaluronidase-resistant radioactivity from that of the undigested total. Blue dextran with ∼2 × 106 Da was used as a calibration standard. The percent of HA with molecular mass >2 × 106 Da was calculated and used to compare samples. Hyaluronan Degradation—To examine HA degradation, [3H]HA was prepared as described (37Underhill C.B. Toole B.P. J. Cell Biol. 1979; 82: 475-484Crossref PubMed Scopus (163) Google Scholar). Briefly, NZW ASMC were incubated in 135-mm dishes with growth medium for 3 days and then growth-arrested in 0.5% FBS/SFM. After 48 h, 62.5 μCi/ml [3H]acetate (1.25 mCi/dish) was added with fresh 10% FBS/SFM for 3 days. The medium was collected and treated with Pronase and heated as above. After centrifugation at 1000 rpm for 5 min, the medium supernatant was extensively dialyzed against Ca2+- and Mg2+-free PBS and subsequently against distilled water at 4 °C. Solid NaCl was added to a final concentration of 0.03 m, and [3H]GAGs were precipitated with 1% cetylpyridinium chloride (CPC) at room temperature. After centrifugation at 15,000 rpm for 15 min, the pellet was resuspended in 2 ml of 0.1% CPC, 0.4 m NaCl to dissolve HA. Dissolved HA was then precipitated with 4 volumes of 100% ethanol at –20 °C for at least 18 h. The pellet was extensively washed with cold 100% ethanol, air-dried, and then resuspended in 1.5 ml of distilled water. Purity of the [3H]HA preparation was checked by CPC precipitation of hyaluronidase-digested or undigested aliquots using a slot-blot assay as described (38Agren U.M. Tammi R. Tammi M. Anal. Biochem. 1994; 217: 311-315Crossref PubMed Scopus (22) Google Scholar). Ninety one percent of labeled GAGs were hyaluronidase-sensitive. To eliminate residual unincorporated [3H]acetate, the labeled HA preparation was desalted on a Sephadex G-50 size exclusion column. Eluted [3H]HA had a specific activity of 284 dpm/ng and contained at least 34% with a molecular mass >2 × 106 Da. To maximize the accessibility of the HA to its potential degradation pathways, the original [3H]HA preparation was sonicated twice for 30 s at 4 °C (Sonifier 450, output levels were set to the microtip limit, percent duty cycle was constant). This procedure reduced the proportion of HA with mass >2 × 106 Da to 2%. For degradation experiments, sonicated [3H]HA (2.58 × 106 dpm/ml) was added to growth-arrested ASMC in SFM. To determine background degradation, [3H]HA was incubated in SFM in cell-free culture dishes. After 18 h, medium aliquots were ultrafiltered through a 50,000-Da cutoff filter (Centricon, Millipore Inc., Billerica, MA), and the radioactivity in the filtrate was taken as degraded HA. Control background counts, from medium not exposed to cells, were subtracted from each sample medium filtrate. In addition, the labeled HA in the retentate was assayed for hydrodynamic size by chromatography on Sephacryl S-1000, as described above. Statistical Analysis—Data are reported as means of triplicate wells from one representative experiment. Error bars indicate S.D. or S.E. as described in the figure legends. Statistically significant differences between WHHL and NZW or within a cell strain were determined using an unpaired Student's t test, with p < 0.05 considered significant. To quantify the differences in HA accumulation between the WHHL and NZW cells as demonstrated in Figs. 2 and 3, a likelihood ratio test was performed using the R Project for Statistical Computing (Free Software Foundation). This procedure simultaneously evaluates both the slope and intercept estimates. If the results of this test are significant, then it can be concluded that the two groups are statistically different. To achieve a linear relationship between cell density and HA production per cell, the log transform of cell density was performed.FIGURE 3Elevated accumulation of hyaluronan by cultures of skin fibroblasts from patients with FH is independent of cell density. Growth-arrested skin fibroblasts from patients with FH (▴) and normolipidemic subjects (▵) were incubated for 24 h with fresh medium containing 10% FBS. The likelihood ratio test produced p < 0.001, and thus we conclude that the accumulation of HA on a per cell basis by FH and normolipidemic strains is different. This experiment was performed three times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) WHHL ASMC Cultures Accumulate Elevated Levels of Hyaluronan and Grow More Slowly than Controls—In vivo accumulation of HA is a feature of atherosclerosis (16Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), a disease that commonly occurs in LDL receptor-deficient WHHL rabbits (24Watanabe Y. Atherosclerosis. 1980; 36: 261-268Abstract Full Text PDF PubMed Scopus (409) Google Scholar, 26Rosenfeld M.E. Tsukada T. Chait A. Bierman E.L. Gown A.M. Ross R. Arteriosclerosis. 1987; 7: 24-34Crossref PubMed Google Scholar, 39Tanzawa K. Shimada Y. Kuroda M. Tsujita Y. Arai M. Watanabe H. FEBS Lett. 1980; 118: 81-84Crossref PubMed Scopus (124) Google Scholar). To explore the source of HA deposition in the atherosclerotic wall, we isolated ASMC from the aortic arch of WHHL rabbits, quantified HA accumulation in low passage cultures, and compared it with HA production by NZW ASMC. As shown in Fig. 1A, during 2-day incubations, HA accumulates in the culture medium of WHHL ASMC to a significantly greater extent than in that of NZW ASMC, both on a per cell basis and at all time points studied. The maximal increase in HA accumulation in the cultures of WHHL as compared with NZW ASMC was ∼2.5-fold, reached after 3 days of culture, and remained approximately the same over the 11 days tested. In 10% FBS-containing medium, the doubling time for WHHL ASMC was ∼5 days, but only ∼1 day for NZW ASMC (Fig. 1B). Interestingly, WHHL ASMC cultures had higher HA levels than those of NZW ASMC even in the absence of high serum (Fig. 1A, day 0), indicating that this difference is not dependent upon serum stimulation of the cells. These results showed for the first time that lacking a functional LDL receptor can be associated with enhanced HA accumulation in cell cultures over a wide range of incubation periods. Elevated Hyaluronan Accumulation by WHHL ASMC Cultures Is Independent of Cell Density and Serum Level—Because the amount of HA produced in cell culture is inversely proportional to the cell density (40Scott L.J. Merrilees M.J. Atherosclerosis. 1987; 63: 145-152Abstract Full Text PDF PubMed Scopus (10) Google Scholar) and WHHL cells grow more slowly than NZW cells (41Rosenfeld M.E. Ross R. Arteriosclerosis. 1990; 10: 680-687Crossref PubMed Google Scholar, 42Ikeda U. Oguchi A. Okada K. Ishikawa S. Saito T. Ikeda M. Kano S. Takahashi M. Shiomi M. Shimada K. Atherosclerosis. 1994; 110: 87-94Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 43Bjorkerud S. Bjorkerud B. Biochim. Biophys. Acta. 1995; 1268: 237-247Crossref PubMed Scopus (11) Google Scholar) (Fig. 1B), we tested the effect of cell density on HA levels. As shown in Fig. 2, in all cases, increasing the cell density significantly reduced the amount of HA secreted on a per cell basis in the medium, as expected (40Scott L.J. Merrilees M.J. Atherosclerosis. 1987; 63: 145-152Abstract Full Text PDF PubMed Scopus (10) Google Scholar). At each cell density tested, either in the presence (Fig. 2A) or absence (Fig. 2B) of high serum, HA accumulation was significantly elevated in WHHL ASMC medium. Similar results were foun" @default.
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• W2000939135 date "2008-12-01" @default.
• W2000939135 modified "2023-09-27" @default.
• W2000939135 title "Hyaluronan Accumulation Is Elevated in Cultures of Low Density Lipoprotein Receptor-deficient Cells and Is Altered by Manipulation of Cell Cholesterol Content" @default.
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• W2000939135 doi "https://doi.org/10.1074/jbc.m807772200" @default.
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