Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation

Abstract

Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.

The Endothelial Glycocalyx as a Target of Excess Soluble Fms-like Tyrosine Kinase-1

“…We used the fluorescent eGC markers Ulex europaeus agglutinin-1 (UEA-1) and wheat germ agglutinin (WGA) to quantify changes in the glycocalyx [ 21 , 22 ]. Only a slight but not significant decrease in UEA-1 fluorescence intensity was observed when HUVECs were treated with sFlt-1 (2 µg/mL) for 24 h compared to control cells.…”

“…Numerous studies have shown that the eGC undergoes conformational changes when challenged with multiple biophysical and biochemical factors, including inflammatory factors, high salt (NaCl), and shear and compressive stresses [ 22 , 30 , 34 , 36 ]. A healthy, intact eGC is upright and soft, while a shed eGC is flat and soft.…”

“…After washing with HBSS, the fluorescent signals of UEA-1-FITC and DAPI were monitored using 488/518 nm and 360/460 (Excitation/Emission), respectively, in the presence of 50 µL HBSS using a microplate reader (Tecan infinite microplate reader M200 Pro Tecan, Salzburg, Austria). Results are calculated by dividing the fluorescent signal of UEA-1-FITC by the DAPI signal (number of cells) [ 22 ].…”

Markers of endothelial glycocalyx dysfunction in Clarkson disease

“…BOECs were stained with fluorescein-conjugated Ulex Europaeus (Gorse) Agglutinin I (UEA I, ThermoFisher) (final concentration, 5 μg/mL for 30 min at room temperature) as described previously [ 19 ] and fluorescence was visualized using a Leica DMI4000 microscope. Values were quantified at 459 nm (emission) and 515 nm (excitation) in a plate reader.…”

Endothelial glycocalyx in hepatopulmonary syndrome: An indispensable player mediating vascular changes

“…The interaction of monocytes with endothelial cells is also on the basis of eGCX damage. The products of eGCX degradation including heparan sulfate and chondroitin sulfate may enhance the chemotactic migration of monocytes, although glypican 1 mounted on the membrane of endothelial cells is reported to inhibit monocyte adhesion ( 88 – 90 ). Hence, the destruction of eGCX may be precisely limited to the area where monocytes are recruited most intensively ( 91 , 92 ).…”