Endothelial Cell Dysfunction and Hypoxia as Potential Mediators of Pain in Fabry Disease: A Human-Murine Translational Approach

Abstract

Fabry disease (FD) is caused by α-galactosidase A (AGAL) enzyme deficiency, leading to globotriaosylceramide accumulation (Gb3) in several cell types. Pain is one of the pathophysiologically incompletely understood symptoms in FD patients. Previous data suggest an involvement of hypoxia and mitochondriopathy in FD pain development at dorsal root ganglion (DRG) level. Using immunofluorescence and quantitative real-time polymerase chain reaction (qRT PCR), we investigated patient-derived endothelial cells (EC) and DRG tissue of the GLA knockout (KO) mouse model of FD. We address the question of whether hypoxia and mitochondriopathy contribute to FD pain pathophysiology. In EC of FD patients (P1 with pain and, P2 without pain), we found dysregulated protein expression of hypoxia-inducible factors (HIF) 1a and HIF2 compared to the control EC (p < 0.01). The protein expression of the HIF downstream target vascular endothelial growth factor A (VEGFA, p < 0.01) was reduced and tube formation was hampered in the P1 EC compared to the healthy EC (p < 0.05). Tube formation ability was rescued by applying transforming growth factor beta (TGFβ) inhibitor SB-431542. Additionally, we found dysregulated mitochondrial fusion/fission characteristics in the P1 and P2 EC (p < 0.01) and depolarized mitochondrial membrane potential in P2 compared to control EC (p < 0.05). Complementary to human data, we found upregulated hypoxia-associated genes in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). At protein level, nuclear HIF1a was higher in the DRG neurons of old GLA KO mice compared to WT mice (p < 0.01). Further, the HIF1a downstream target CA9 was upregulated in the DRG of old GLA KO mice compared to WT DRG (p < 0.01). Similar to human EC, we found a reduction in the vascular characteristics in GLA KO DRG compared to WT (p < 0.05). We demonstrate increased hypoxia, impaired vascular properties, and mitochondrial dysfunction in human FD EC and complementarily at the GLA KO mouse DRG level. Our data support the hypothesis that hypoxia and mitochondriopathy in FD EC and GLA KO DRG may contribute to FD pain development.

Molecular Pathogenesis of Central and Peripheral Nervous System Complications in Anderson–Fabry Disease

“…These findings further support the notion that the pathogenesis of globotriaosylceramide deposits in AFD is not yet fully understood [44]. Concerning the pathology of cerebral vessels in AFD, the involvement has been observed [45][46][47][48] of the subarachnoidal arteries of medium size with narrowing of the lumen due to intimal fibrosis mixed with smooth muscle cells (SMC), with membrane abnormalities and stiffening of internal elastic tunica due to the total or partial change of the medial SMC and subsequent fibrosis and adventitial fibrosis. AFD involves smooth muscle too, but it is not clear whether the first step in Fabry vasculopathy involves endothelial cells, with a subsequent prothrombotic state, or if it begins in the smooth muscle cells of the arterial media layer [45][46][47][48].…”

“…Concerning the pathology of cerebral vessels in AFD, the involvement has been observed [ 45 , 46 , 47 , 48 ] of the subarachnoidal arteries of medium size with narrowing of the lumen due to intimal fibrosis mixed with smooth muscle cells (SMC), with membrane abnormalities and stiffening of internal elastic tunica due to the total or partial change of the medial SMC and subsequent fibrosis and adventitial fibrosis. AFD involves smooth muscle too, but it is not clear whether the first step in Fabry vasculopathy involves endothelial cells, with a subsequent prothrombotic state, or if it begins in the smooth muscle cells of the arterial media layer [ 45 , 46 , 47 , 48 ]. It has been reported that lyso-Gb3 plays a crucial role in the pathogenesis of Fabry vasculopathy, and it has been proposed that smooth muscle cells, rather than endothelial cells, represent the main target of cell accumulation.…”

“…It has been reported that lyso-Gb3 plays a crucial role in the pathogenesis of Fabry vasculopathy, and it has been proposed that smooth muscle cells, rather than endothelial cells, represent the main target of cell accumulation. Smooth muscle cells exposed to lyso-Gb3 proliferate, and this proliferation has been reported as linked with the hypertrophy of arterial walls [ 46 , 47 , 48 , 49 , 50 ]. Accumulation of lyso-Gb3 within the media layer of the arteries may also promote cell proliferation, with the fibrotic remodeling of the arterial wall leading to arterial wall stiffness.…”

“…All these cellular and biochemical events seem to represent the candidate step to initiate an inflammatory cascade with prothrombotic and pro-inflammatory effects on leukocytes, endothelial cells, and vascular smooth muscle cells [ 47 ]. Indeed, inflammatory pathogenesis of Anderson–Fabry disease (AFD) complications in the central nervous system (CNS) has been previously documented [ 48 ]. It is important to note that the CNS serves as the primary target for numerous lysosomal storage disorders.…”