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An In Vitro Model of Fabry Disease

Liming Shu^*, Hedwig S. Murphy, Laura Cooling and James A. Shayman^*

^* Nephrology Division, Department of Internal Medicine, and Department of Pathology, University of Michigan, Ann Arbor, Michigan

Address correspondence to: Dr. James A. Shayman, Nephrology Division, Department of Internal Medicine, University of Michigan, Box 0676, Room 1560 MSRBII, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0676. Phone: 734-763-0992; Fax: 734-763-0982; E-mail: jshayman{at}umich.edu

Received for publication April 11, 2005. Accepted for publication June 20, 2005.

Fabry disease is an X-linked inherited loss of -galactosidase A (-Gal A). Affected patients experience complications that include neuropathy, renal failure, and cardiovascular disease. Although the genetic and biochemical basis of this sphingolipidosis is well studied, the basis for the vascular disease remains poorly understood. In an attempt to create a suitable in vitro model of this disease, conditions for the growth of primary cultures of aortic endothelial cells from wild-type and -Gal A –/0 mice were established. The cultured cells demonstrated CD-31 expression by flow cytometry and LDL binding by immunofluorescence. The glycolipid expression patterns were compared between wild-type and -Gal A null cells. Importantly, cells from -Gal A –/0 mice but not -Gal A +/0 mice expressed high levels of the globo-series glycosphingolipid globotriaosylceramide (Gb3). The age-dependent elevation in Gb3 was measured. By 4 mo of age, -Gal A –/0 mouse aortic endothelial cells achieved their peak Gb3 levels. The ability to lower Gb3 levels pharmacologically was assessed next. The glucosylceramide synthase inhibitor ethylenedioxyphenyl-P4 significantly lowered but did not eliminate Gb3 levels by 96 h of treatment. Gb3 synthesis was completely blocked as measured by [¹⁴C]galactose labeling. Recombinant -Gal A more significantly lowered Gb3 levels by 48 h but had a more limited effect on de novo synthesis. Together, both agents eliminated detectable Gb3. In summary, primary cultures of aortic endothelial cells from Fabry mice retain the phenotype of elevated globo-series glycosphingolipids. These cells provide a useful model for comparing pharmacologic agents used for glycolipid reduction.

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Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673