α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease.

Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in in vivo and in vitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both in vitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease.

Endothelial nitric oxide synthase uncoupling and microvascular dysfunction in the mesentery of mice deficient in α-galactosidase A.

A defect in the gene for the lysosomal enzyme α-galactosidase A (Gla) results in globotriaosylceramide (Gb3) accumulation in Fabry disease and leads to premature death from cardiac and cerebrovascular events. However, gastrointestinal symptoms are often first observed during childhood in these patients and are not well understood. In this study, we demonstrate an age-dependent microvasculopathy of the mesenteric artery (MA) in a murine model of Fabry disease (Gla-knockout mice) resulting from dysregulation of the vascular homeostatic enzyme endothelial nitric oxide synthase (eNOS). The progressive accumulation of Gb3 in the MA was confirmed by thin-layer chromatographic analysis. A total absence of endothelium-dependent dilation was observed in MAs from mice at 8 mo of age, while suppression of ACh-mediated vasodilation was evident from 2 mo of age. Endothelium-independent dilation with sodium nitroprusside was normal compared with age-matched wild-type mice. The microvascular defect in MAs from Fabry mice was endothelium-dependent and associated with suppression of the active homodimer of eNOS. Phosphorylation of eNOS at the major activation site (Ser(1179)) was significantly downregulated, while phosphorylation at the major inhibitory site (Thr(495)) was remarkably enhanced in MAs from aged Fabry mice. These profound alterations in eNOS bioavailability at 8 mo of age were observed in parallel with high levels of 3-nitrotyrosine, suggesting increased reactive oxygen species along with eNOS uncoupling in this vascular bed. Overall, the mesenteric microvessels in the setting of Fabry disease were observed to have an early and profound endothelial dysfunction associated with elevated reactive nitrogen species and decreased nitric oxide bioavailability.

Voluntary wheel running activates Akt/AMPK/eNOS signaling cascades without improving profound endothelial dysfunction in mice deficient in α-galactosidase A.

Fabry disease is caused by loss of activity of the lysosomal hydrolase α-galactosidase A (GLA). Premature life-threatening complications in Fabry patients arise from cardiovascular disease, including stroke and myocardial infarction. Exercise training has been shown to improve endothelial dysfunction in various settings including coronary artery disease. However, the effects of exercise training on endothelial dysfunction in Fabry disease have not been investigated. Gla knockout mice were single-housed in a cage equipped with a voluntary wheel (EX) or no wheel (SED) for 12 weeks. Exercised mice ran 10 km/day on average during the voluntary running intervention (VR) period. Despite significantly higher food intake in EX than SED, body weights of EX and SED remained stable during the VR period. After the completion of VR, citrate synthase activity in gastrocnemius muscle was significantly higher in EX than SED. VR resulted in greater phosphorylation of Akt (S473) and AMPK (T172) in the aorta of EX compared to SED measured by western blot. Furthermore, VR significantly enhanced eNOS protein expression and phosphorylation at S1177 by 20% and 50% in the aorta of EX when compared with SED. Similarly, plasma nitrate and nitrite levels were 77% higher in EX than SED. In contrast, measures of anti- and pro-oxidative enzymes (superoxide dismutase and p67phox subunit of NADPH oxidase) and overall oxidative stress (plasma oxidized glutathione) were not different between groups. Although the aortic endothelial relaxation to acetylcholine was slightly increased in EX, it did not reach statistical significance. This study provides the first evidence that VR improves Akt/AMPK/eNOS signaling cascades, but not endothelial function in the aorta of aged Gla deficient mice.