Abstract 395: Elevated Von Willebran Factor in an in vitro Model of Fabry Disease

Abstract

Fabry disease is an X-linked disorder caused by a defect in the gene encoding the lysosomal hydrolase, alpha-galactosidase A (GLA). The loss in GLA function causes an age-dependent accumulation of globotriaosylceramide in the endothelial cells, which results in decreased nitric oxide bioavailability and premature mortality in the affected patients. Based on this, we hypothesized that GLA deficiency promotes endothelial inflammation. We observed a significant age-dependent increase in the plasma von Willebrand Factor (vWF) in Fabry mice compared to age-matched wild type mice (p=0.046 at 2 months and p=0.0004 at 17 months). To evaluate the molecular mechanisms of increased vWF in Fabry disease, small interfering RNA (siRNA) technique was used to knock-down GLA in EA.hy926 cells. Our initial studies with this cell line demonstrated that thrombin (2U/mL) induced a time-dependent vWF secretion into the cell culture medium with an 80-fold increase in the level of vWF after 90 min stimulation (p=0.001 compared to basal level; n=3 replicates per group), measured by a human antibody against vWF and AlphaLISA. Next, the siRNA studies revealed a greater than 95% knockdown of GLA protein (via western blotting) in the GLA-siRNA group compared to the control group with no effect in the scrambled (SCR) sequence-siRNA group (CON vs. GLA vs. SCR arbitrary units: 1.0±0.03 vs. 0.01±0.01 vs. 1.1±0.08, p<0.001; GLA vs. SCR). Importantly, no difference in GAPDH protein was observed between groups. To determine the direct effect of GLA deficiency on vWF, the confluent cells in each group were incubated in serum-free medium overnight. Consistent with the hypothesis, we observed approximately 60% increase in vWF in the cell culture medium in the GLA siRNA group compared with the untreated and scrambled-sequence control groups. (CON vs. GLA vs. SCR arbitrary units: 38.5±4.7 vs. 61.3±1.1 vs. 31.4±3.8, p=0.02; GLA vs. SCR; n=3 replicates per group). In conclusion, the data provide evidence of endothelial activation in the setting of GLA deficiency, demonstrated by the robust elevation of vWF mediated by GLA gene disruption. Future study is required to further understand the mechanism of the elevated vWF and decreased nitric oxide bioavailability in Fabry disease.