Heat shock protein 70s are modifiers of endothelial function in Fabry disease.

Abstract

INTRODUCTION: Fabry disease (FD) is a systemic disorder with manifestations of heart, vascular, and kidney disease. Identical genetic variants in the α-galactosidase A (GLA) gene exhibit variable clinical phenotypes consistent with the existence of disease modifiers. Prior work in the Gla null mouse identified the vascular endothelium as a primary site for dysfunction associated with FD vasculopathy. METHODS: Endothelial-specific translating ribosome affinity purification followed by RNA sequencing (EC TRAP-seq) was employed to identify differences in the in vivo endothelial cell gene expression profiles between hemizygous null male (Gla) and homozygous null female (Gla) compared to wild type male (Gla) and female (Gla) mice. RESULTS: Significant increases in transcript levels for heat shock protein (Hsp)70s (both Hsp1a and Hsp1b) and associated regulatory proteins were identified in the EC TRAP-seq analysis and verified by both Western blotting and immunohistochemistry of whole kidneys. Increased expression of Hsp70 was also confirmed in CRISPR-Cas9 mediated ablation of GLA in EA.hy926 cells (somatic hybrid used for cardiovascular disease research) and primary mouse glomerular endothelial cells. Decreased ATP-stimulated endothelial nitric oxide synthase activity was observed in GLA-deficient EA.hy926 cells, and this was worsened in the presence of two inhibitors of Hsp70 (VER155008 and PES-Cl). Additionally, increased staining for 3-nitrotyrosine was observed in the Gla knockdown cells; this was also further increased in the presence of Hsp70 inhibition. In lysosomal permeability assays, EA.hy926 GLA knockdown cells were more resistant to lysosomal permeability compared to the wild type cells. The increased resistance to lysosomal injury was reversed with HSP70 inhibition. CONCLUSIONS: EC TRAP-seq identifies Hsp70s as potential modifiers of endothelial function in FD.