Caveolin-associated Accumulation of Globotriaosylceramide in the Vascular Endothelium of α-Galactosidase A Null Mice

Cardiovascular complications, including stroke and myocardial infarction, result in premature mortality in patients with Fabry disease, an X-linked deficiency of α-galactosidase A (α-Gal A). The enzymatic defect results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. To better understand the underlying pathogenesis of Fabry disease, the caveolar lipid content of primary cultured mouse aortic endothelial cells isolated from α-Gal A null mice was measured. Lipid mass analysis revealed that the excessive Gb3 in cultured α-Gal A-deficient mouse aortic endothelial cells accumulated in endothelial plasma membrane caveolar fractions. The levels of glucosylceramide and lactosylceramide increased in parallel with Gb3 levels in an age-dependent manner, whereas globotetraosylceramide (Gb4) levels reached maximal levels by 6 months of age and then rapidly decreased at older ages. The levels of cholesterol enriched in caveolar membranes declined in parallel with the progressive deposition of Gb3. Depleting Gb3 with recombinant human α-Gal A protein or d-threo-ethylenedioxyphenyl-P4, an inhibitor of glucosylceramide synthase, restored cholesterol in cultured α-Gal A-deficient mouse aortic endothelial cell caveolae. By contrast, recombinant human α-Gal A was less effective in normalizing the cholesterol content. These results demonstrate the caveolar accumulation of glycosphingolipids in an in vitro model of a lysosomal storage disease and raise the possibility that dynamic changes in the composition of plasma membrane lipid microdomains may mediate the endothelial dysfunction seen in Fabry disease. Cardiovascular complications, including stroke and myocardial infarction, result in premature mortality in patients with Fabry disease, an X-linked deficiency of α-galactosidase A (α-Gal A). The enzymatic defect results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. To better understand the underlying pathogenesis of Fabry disease, the caveolar lipid content of primary cultured mouse aortic endothelial cells isolated from α-Gal A null mice was measured. Lipid mass analysis revealed that the excessive Gb3 in cultured α-Gal A-deficient mouse aortic endothelial cells accumulated in endothelial plasma membrane caveolar fractions. The levels of glucosylceramide and lactosylceramide increased in parallel with Gb3 levels in an age-dependent manner, whereas globotetraosylceramide (Gb4) levels reached maximal levels by 6 months of age and then rapidly decreased at older ages. The levels of cholesterol enriched in caveolar membranes declined in parallel with the progressive deposition of Gb3. Depleting Gb3 with recombinant human α-Gal A protein or d-threo-ethylenedioxyphenyl-P4, an inhibitor of glucosylceramide synthase, restored cholesterol in cultured α-Gal A-deficient mouse aortic endothelial cell caveolae. By contrast, recombinant human α-Gal A was less effective in normalizing the cholesterol content. These results demonstrate the caveolar accumulation of glycosphingolipids in an in vitro model of a lysosomal storage disease and raise the possibility that dynamic changes in the composition of plasma membrane lipid microdomains may mediate the endothelial dysfunction seen in Fabry disease. Fabry disease is one of 42 recognized lysosomal storage disorders. Fabry disease results from the absence or decreased activity of the lysosomal hydrolase α-galactosidase A (α-Gal A). 2The abbreviations used are: α-Gal A, α-galactosidase A; Gb3, globotriaosylceramide; Gb4, globotetraosylceramide; MAEC, mouse aortic endothelial cell; d-t-EtDO-P4, d-threo-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol; FBS, fetal bovine serum; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GlcCer, glucosylceramide; LacCer, lactosylceramide; HPTLC, high performance thin layer chromatography. α-Gal A is transmitted as an X-linked recessive gene (1Anderson W. Br. J. Dermatol. 1898; 10: 113Crossref Scopus (258) Google Scholar, 2Fabry J. Arch. Dermatol. Syphilis. 1898; 43: 187Crossref Scopus (279) Google Scholar). The lack of enzymatic activity of α-Gal A in Fabry patients leads to the progressive accumulation of glycolipids with α-galactosyl linkages, primarily globotriaosylceramide (Gb3). Gb3 accumulates in the lysosomes of numerous tissues throughout the body, most notably the vascular endothelium. Fabry disease is a systemic disorder and functionally affects the skin, eyes, kidney, heart, and autonomic nervous system. The premature mortality of affected patients, however, primarily occurs from cardiovascular catastrophes such as strokes and myocardial infarctions (3Desnick R.J. Wasserstein M.P. Adv. Nephrol. Necker. Hosp. 2001; 31: 317-339PubMed Google Scholar). Although it is well documented that cardiovascular abnormalities are the most common and predominant clinical manifestation of Fabry disease, the precise biological role of Gb3 deposition in the pathogenesis of the large vessel vasculopathy in Fabry disease remains unknown. The generation of knock-out mice lacking α-Gal A activity has provided a useful model for the study of the vasculopathy in Fabry disease (4Ohshima T. Murray G.J. Swaim W.D. Longenecker G. Quirk J.M. Cardarelli C.O. Sugimoto Y. Pastan I. Gottesman M.M. Brady R.O. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2540-2544Crossref PubMed Scopus (293) Google Scholar). Although these mice do not display a spontaneous vasculopathy, they exhibit a robust thrombotic response to oxidant-induced injury (5Eitzman D.T. Bodary P.F. Shen Y. Khairallah C.G. Wild S.R. Abe A. Shaffer-Hartman J. Shayman J.A. J. Am. Soc. Nephrol. 2003; 14: 298-302Crossref PubMed Scopus (64) Google Scholar) and accelerated atherosclerosis when bred on an apo-E1 null background (6Bodary P.F. Shen Y. Vargas F.B. Bi X. Ostenso K.A. Gu S. Shayman J.A. Eitzman D.T. Circulation. 2005; 111: 629-632Crossref PubMed Scopus (75) Google Scholar). In addition, these mice display impaired contraction to phenylephrine and impaired acetylcholine-stimulated vasorelaxation in their aortic rings. 3J. Park and J. A. Shayman, unpublished data. All three findings are age-dependent. Because these phenotypes are observed in either living mice or isolated vessels, establishing a mechanistic basis for the vasculopathy has been difficult. As an initial step in studying potential mechanisms, a method for growing primary cultures of aortic endothelial cells derived from the α-Gal A null mice was established (7Shu L. Murphy H.S. Cooling L.L. Shayman J.A. J. Am. Soc. Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar). These cells retain high levels of Gb3 and thus provide a potentially useful reagent for probing the Fabry phenotype. The vascular models established to date in the α-Gal A null mice are consistent with the presence of an endothelial signaling defect in nitric oxide generation. How an enzyme deficiency resulting in the lysosomal accumulation of Gb3 might result in the impairment in agonist-stimulated nitric oxide formation is unknown. Plasma membrane-associated glycosphingolipids have long been demonstrated to regulate the activity of signaling molecules, including receptor tyrosine kinases (8Bremer E.G. Hakomori S. Bowen-Pope D.F. Raines E. Ross R. J. Biol. Chem. 1984; 259: 6818-6825Abstract Full Text PDF PubMed Google Scholar), Src kinases (9Shu L. Shayman J.A. J. Biol. Chem. 2003; 278: 31419-31425Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and phospholipase C (10Shu L. Lee L. Shayman J.A. J. Biol. Chem. 2002; 277: 18447-18453Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Receptor-mediated endocytosis is regulated by glycosphingolipids as well. However, little if any data exist demonstrating a cross-talk between lysosomes and plasma membrane signaling. Furthermore, with the exception of Niemann-Pick C disease, little data exist demonstrating the accumulation of sphingolipids outside of the lysosome in storage diseases. To evaluate whether the glycosphingolipid content in plasma membrane caveolar fractions is affected in the setting of lysosomal storage disease, aortic endothelial cells from α-Gal A null mice were studied. We report that absence of α-Gal A activity in mouse aortic endothelial cells (MAECs) results in the age-dependent accumulation of globo series glycosphingolipids in plasma membrane caveolin-associated lipid rafts. Reagents—Recombinant human α-Gal A enzyme (Fabrazyme®), produced in a Chinese hamster ovary mammalian cell expression system, was a kind gift from Genzyme Corp. (Cambridge, MA). d-threo-Ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol (d-t-EtDO-P4) was synthesized in our laboratory as previously described (11Lee L. Abe A. Shayman J.A. J. Biol. Chem. 1999; 274: 14662-14669Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Endothelial cell growth supplement was purchased from BD Biosciences. Fetal bovine serum (FBS) was obtained from America Type Culture Collection (ATCC) (Manassas, VA). Collagen type I, collagenase type I, and Percoll were from Sigma-Aldrich. OptiPrep was purchased from Accurate Chemical & Scientific Corp. (Westburg, NY). All of the lipid internal standards were acquired from Matreya (Pleasant Gap, PA). Animals—C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). α-Gal A-knock-out mice with a 129SVJXC57BL/6 cross-breeding background were bred and maintained under standard protocols at the University of Michigan as previously described. The genotype of α-Gal A-deficient mice was confirmed by PCR analysis. The care and euthanasia of the mice was in accordance with the standards in “The Guide for the Care and Use of Laboratory Animals” DHEW publication number (National Institutes of Health) 86-23, revised 1985. Cell Cultures—The primary MAECs were isolated from α-Gal A-knock-out and wild-type C57BL/6J mice, at ages from 1 to 8 months, by utilizing a non-mechanical and non-enzymatic method with minor modifications as described previously (7Shu L. Murphy H.S. Cooling L.L. Shayman J.A. J. Am. Soc. Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar). Penicillin, streptomycin, and fungizone significantly delayed endothelial cells growth from explanted aortic rings and were therefore omitted from RPMI plating medium. The outgrowth of endothelial cells from aortic rings was markedly facilitated within first 72 h in the absence of antibiotics. Aortic rings thus were discarded at culture day 3 to avoid the possible contamination of non-endothelial cell types (12Chen S.F. Fei X. Li S.H. Microvasc. Res. 1995; 50: 119-128Crossref PubMed Scopus (87) Google Scholar). After removing the aortic rings, cells were maintained in completed RPMI medium consisting of 20% FBS, 2 mm l-glutamine, 1× nonessential amino acid, 0.05 mg/ml endothelial cell growth supplement, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.1 mg/ml heparin until confluent. Cell Treatments—Because high concentrations of serum (≥15%) in culture medium induced increased Gb3, the percentage of serum in medium was gradually reduced after cells were serially passaged. MAECs at passages 0 and 1 (P0 and P1) were grown in RPMI medium containing 20% FBS, and MAECs at P2 and P3 were cultured in 15% FBS-RPMI medium. MAECs at P4, only maintained in 10% FBS-RPMI medium in excess of 2 days, were used for experiments unless otherwise indicated. A stock solution of d-t-EtDO-P4 dissolved in 100% Me2SO was diluted with plain RPMI, and recombinant human α-Gal A was freshly reconstituted with plain RPMI medium just before use. Treated MAECs, maintained in 10% FBS-RPMI, were exposed to either 0.3 μm d-t-EtDO-P4 or 10 μg/ml recombinant human α-Gal A for 1 or 2 days before being harvested. Isolation of Caveolae—Caveolae were isolated from cultured MAECs using a detergent-free method that takes advantage of the unique buoyant density of caveolar membranes (13Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar). Briefly, cells grown in 150- × 25-mm dishes were collected by scraping in buffer A (0.25 m sucrose/20 mm Tricine/1 mm EDTA, pH 7.8). Whole cells were fractionated by homogenization, and plasma membrane fractions were separated by centrifugation in buffer A containing 30% Percoll. Caveolar membranes were further purified from plasma membrane fractions by centrifugation in a 20% to 10% OptiPrep linear gradient and concentrated by a second centrifugation in a 5% OptiPrep-Buffer A discontinuous gradient. A distinct opaque band of caveolar membrane in the 5% OptiPrep was collected for lipid extraction and mass analysis. Immunoblotting—The total protein concentration of each sample was determined by the with bovine serum as a of were with sample buffer consisting of mm 10% and separated under were to using a and separated under were for at in sample buffer containing before The membranes were in containing 5% and 5% serum and with mouse 1 and The primary were with and with the system. was with a mouse Lipid extraction of total a as previously described by L. Lee L. Y. E. Shayman J.A. Arch. PubMed Scopus Google Scholar). The caveolar membrane were from caveolar fractions with and A was by the of and to the caveolar membranes at a of After in a for caveolar were from the by centrifugation at × for The was collected with a a and and were by the of of 3 of and of The sample was at × for The was The was and with and at the of to OptiPrep in caveolar The in were under a of and Lipid glycosphingolipids were purified from and caveolar membrane by and as described in (7Shu L. Murphy H.S. Cooling L.L. Shayman J.A. J. Am. Soc. Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar). Briefly, were for of using a total with as a standard J. PubMed Google Scholar). A of the of total was with in at for 1 After with (0.25 the in the were with 0.05 m mm at for The were then purified with mm and The glycosphingolipids Gb3, Gb4, GlcCer, and were with as previously (7Shu L. Murphy H.S. Cooling L.L. Shayman J.A. J. Am. Soc. Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar). and were separated in a consisting of and by to standard of in 100 of for cholesterol and in lipid of total were from by to standards in parallel on the data from three experiments were by the and as A of 0.05 was and aortic endothelial cells are to by enzymatic an was to primary cultures of endothelial an and for the and of aortic endothelial The were and from The outgrowth of aortic endothelial cells was by the of from the plating medium. The contamination of cell including was by of the aortic rings to the at non-endothelial cells to from the aortic rings. The of the endothelial cell was by the of markedly endothelial cells growth by the of endothelial cell growth PubMed Scopus Google Scholar) and induced an on cells by the activity of growth J. Res. Full Text PDF PubMed Scopus Google Scholar). The of the plasma membrane from MAECs obtained from wild-type and α-Gal A-knock-out mouse was and were enriched in the membrane fractions from wild-type and null endothelial 1 and 2 were enriched in wild-type and null The levels of 1 were in the knock-out cells and decreased with The was observed for levels were in the fractions from the knock-out The of serum on the of Gb3 in wild-type cultured cells was by gradually the concentration of serum in the culture medium. demonstrated that total Gb3 was in wild-type endothelial cells (7Shu L. Murphy H.S. Cooling L.L. Shayman J.A. J. Am. Soc. Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar). After days of culture in 10% FBS-RPMI Gb3 was in caveolar from wild-type MAECs as well However, under these the presence of Gb3 in cultured α-Gal A-knock-out MAECs and increased in caveolar membrane as well as in total as a of the age of the The Gb3 accumulation in α-Gal A-knock-out MAECs was in the cultured cells to passages not The Gb3 content at passages By mass it was observed that the accumulation of Gb3 in caveolar membranes isolated from cultured α-Gal A-knock-out MAECs was age-dependent not ages the Gb3 levels in caveolar membranes were to levels as total age however, Gb3 was in caveolar membranes when as The accumulation of Gb3 in caveolar membranes reached a in cells from to mice, whereas the for total Gb3 was observed in cells obtained from The large of Gb3 in caveolar membranes a possibility that the content of caveolar enriched within caveolar as well. and are of Gb3. The levels of in total and caveolar membrane of wild-type and α-Gal A-knock-out MAECs were by with in was in the levels of between in total and in caveolar membrane isolated from cultured wild-type However, the levels of in caveolar membrane from cultured α-Gal A-knock-out MAECs significantly in an age-dependent and not The levels of in total and caveolar membrane of MAECs from Fabry mice months of were markedly with wild-type age the levels of in the caveolar membranes of Fabry MAECs increased By 8 months of the in caveolar of α-Gal A-knock-out MAECs was in of wild-type A for accumulation in caveolar of α-Gal A-knock-out MAECs not documented in cells obtained from The levels of in total and caveolar membranes were using a that of and determined to a standard from of with in caveolar membranes of wild-type MAECs, the levels of in caveolar membranes of α-Gal A-knock-out MAECs increased with age and Gb3 The linear of caveolar in α-Gal A-knock-out MAECs not a at Fabry in globotetraosylceramide a of Gb3, were observed when in the caveolar in cultured Fabry MAECs in to cell The of was that observed with and increased in caveolar of α-Gal A-knock-out MAECs and reached maximal levels in cells from However, the of in the caveolar from MAECs obtained from Fabry mice older 6 months decreased to wild-type the of an for is well established that not only are enriched in in the levels of cholesterol in caveolar membrane and total of Fabry MAECs were with the levels of cholesterol in wild-type progressive deposition of Gb3 in total and caveolar cholesterol levels in lipid decreased gradually as with a containing and 10% 8 months of the of cholesterol in the of α-Gal A-knock-out MAECs was with that in of cells The of in caveolar of cultured MAECs was an 6 and However, most of the was observed in the caveolar of cultured MAECs isolated from Fabry mice months of age 8 and We previously that recombinant human α-Gal A protein and the inhibitor d-t-EtDO-P4, Gb3 from total of cultured α-Gal A-knock-out MAECs with (7Shu L. Murphy H.S. Cooling L.L. Shayman J.A. J. Am. Soc. Nephrol. 2005; 16: 2636-2645Crossref PubMed Scopus (29) Google Scholar). In the of these on the of Gb3 in of α-Gal A-knock-out MAECs were The of excessive Gb3 from by d-t-EtDO-P4 was A with 0.3 μm d-t-EtDO-P4 for h was as effective as 1 or μm d-t-EtDO-P4 for 2 days in caveolar Gb3, and effective when from the culture medium for 2 days By contrast, enzymatic of Gb3 with recombinant human α-Gal A protein was and the concentration of enzyme from 10 to μg/ml for 1 day reduced the caveolar Gb3 levels from to not However, a of Gb3 was observed in caveolar when further enzyme was for 2 days The cholesterol levels in caveolar and total of α-Gal A-knock-out MAECs were α-Gal A-knock-out MAECs were exposed to either recombinant human α-Gal A or d-t-EtDO-P4 for 2 Gb3 by d-t-EtDO-P4 restored cholesterol levels on total and caveolar under However, only of the cholesterol content with α-Gal A was Caveolar cholesterol levels were after a with a of α-Gal A or d-t-EtDO-P4 by with α-Gal and The cells were then cultured for 2 days The caveolar cholesterol levels to in the wild-type cells in Fabry MAECs previously with d-t-EtDO-P4 decreased within 2 days in cells with α-Gal A. lysosomal storage have been of these the accumulation of C. PubMed Scopus Google Scholar) recognized that these from the of lysosomal to it has been recognized that the basis for lysosomal storage result from the or lysosomal lysosomal membrane and the lack of that regulate lysosomal hydrolase activity G. Cell Biol. PubMed Scopus Google Scholar). is the and basis of these result in and is significantly less well it has been to understand the common of accumulation results in such a of phenotypes the disorders. have been to the of lysosomal storage disease. These in lysosomal gene expression with the of and in signaling. that sphingolipids outside of the has less the results from the presence of levels of sphingolipids in membrane to the lysosome the of signaling are on the composition and in to cholesterol are of caveolin-associated lipid rafts. only are these of lipid enriched in and the of these to sphingolipids are to the cell to of lysosomes R. J. Cell Biol. 2001; PubMed Scopus Google Scholar). or in of sphingolipids and E. G. 1995; PubMed Scopus Google Scholar). These and findings that sphingolipids are not to lysosomes the of caveolin-associated that the and lysosomes are and These findings and J. Cell Biol. 10: Full Text Full Text PDF PubMed Scopus Google Scholar) to that the or of for is lipid accumulation in these results in a with These changes of caveolar in lysosomal membrane and and in lysosome and of these the of changes in content within have long been recognized as of cell signaling at the plasma The activity of receptor tyrosine including the receptor and growth is by the content. The of phospholipase and a of Src kinases demonstrated to affected by or of glycosphingolipids (9Shu L. Shayman J.A. J. Biol. Chem. 2003; 278: 31419-31425Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, L. Lee L. Shayman J.A. J. Biol. Chem. 2002; 277: 18447-18453Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). it has been that these changes may by the of to membrane glycosphingolipids as of a a Hakomori J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These an do glycosphingolipids and changes in their concentrations regulate signaling In the study have the plasma membrane caveolin-associated lipid content in endothelial cells of wild-type and α-Gal A null These cells may a useful model of Fabry disease, they retain high levels of Gb3 observed that the caveolar content of Gb3 is increased in the knock-out cells with wild-type The in Gb3 content is increased as a of the age of the changes are observed in the levels of the Gb3 and levels of these glycosphingolipids are of the wild-type cells and with By months of age the levels of these glycolipids are to or in excess of in the wild-type in the levels of cholesterol By contrast, in levels is The caveolar content of glycosphingolipids of Fabry endothelial cells with α-Gal A and d-t-EtDO-P4 were effective in the Gb3 content of the fractions. These data that the of Gb3 is to to changes in or The changes in glycosphingolipid content however, between the The of d-t-EtDO-P4 for a of with α-Gal A of The response to the in potentially α-Gal A only in the of Gb3. By contrast, GlcCer, LacCer, and levels decreased when the inhibitor was the of cholesterol in the was effective α-Gal A in normalizing the cholesterol content and the was these data that the composition and content of in the of the α-Gal A null To the of our is the first report of a in plasma membrane caveolin-associated glycosphingolipids in a lysosomal storage However, the lipid composition in membrane fractions has been in mouse model of lysosomal storage disease, Niemann-Pick C. In a study the cholesterol content of plasma membranes was observed to increased from of The increased cholesterol impaired signaling and was restored cholesterol E. S. Sci. 2005; Scholar). In cholesterol levels have been to or L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. G. Y. Y. PubMed Scopus Google Scholar, Y. J. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. J. Lipid Res. 2002; 43: Full Text Full Text PDF PubMed Google Scholar). In study the age-dependent of Gb3 and globo series glycosphingolipids in the caveolin-associated lipid fractions of plasma membranes from the α-galactosidase A null mouse endothelial The changes are and by that either Gb3 or The of these changes an in the basis of the vascular of Fabry disease and the of lysosomal storage