Abstract 530: Decreased NSF S-Nitrosylation in the Aorta and an Age-Dependent Endothelial Activation in Mice with Fabry Disease

Abstract

Fabry disease is an X-linked lysosomal storage disorder caused by loss of activity of the enzyme, α-galactosidase A (GLA). The loss of GLA function leads to an age-dependent accumulation of globotriaosylceramide (Gb3) in endothelial cells. Endothelial Gb3 accumulation is associated with endothelial nitric oxide synthase (eNOS) uncoupling and decreased nitric oxide (NO) bioavailability. We hypothesized that GLA deficiency promotes endothelial inflammation. von Willebrand factor (VWF) is the main component of Weibel Palade bodies (WPB) and is secreted upon endothelial inflammation. We observed significantly elevated plasma VWF in GLA null mice (FA) compared to age-matched Wild-Type mice (WT) (p=0.046 at 2 months and p<0.001 at 5 and 17 months), indicating increased endothelial inflammation in FA. Out of the many proteins that regulate WPB exocytosis, N-ethylmaleimide sensitive factor (NSF) is a critical mediator of the exocytic machinery and a major target of NO. NO can reversibly modify NSF cysteine residues via a process called S-nitrosylation (SNO), leading to decreased WPB exocytosis. To evaluate whether GLA deficiency promotes a decrease in SNO-NSF, nitrosylated cysteines of aortic homogenates from male WT and FA were first labeled with biotin through a biotin switch assay. Next, biotinylated proteins were isolated with streptavidin-agarose beads. We observed an approximately 60% decrease in the level of SNO-NSF in the aorta of FA compared to that of WT by Western blot (WT vs. Fabry: 1.0±0.03 vs. 0.4±0.11, p=0.002, n=4-5/group) whereas total protein expression of NSF and GAPDH were not different between groups. The level of thioredoxin-1 (TRX-1) was significantly elevated in FA compared to WT (WT vs. Fabry: 1.0±0.05 vs. 1.4±0.14, p=0.01, n=6-9/group), suggesting high levels of reactive oxygen species and protein denitrosylase activity in Fabry disease. In conclusion, these results provide evidence of endothelial activation in Fabry disease. GLA deficiency resulted in decreased SNO-NSF and increased TRX-1 in parallel with the robust elevation of VWF. Future study is required for further understanding of the mechanistic links between these observations, and to determine whether this is a reversible phenomenon in the setting of Fabry disease.