Heparanase 2 Is an Important Regulator of Glomerular Glycocalyx and Influences Albuminuria in Zebrafish via Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor (FGF) Signaling

Background: The endothelial glycocalyx (eGCX) is a glycan-rich layer important for vascular homeostasis. Shedding of heparan sulfates (HS) from the eGCX by heparanase leads to endothelial dysfunction. Heparanase 2 (Hpa2) is known to interact with (HS). We investigated the role of Hpa2 for the vascular system in zebrafish to establish a novel vertebrate model to study of eGCX in renal and vascular disease. Methods: We use zebrafish larvae as research model. Expression and localization of Hpa2 was examined by in situ hybridization and immune fluorescence. hpse2 loss-of-function (LOF) was induced by CRISPR-Cas9 or morpholino antisense strategies and eGCX was quantified. Endothelial permeability was analyzed in Tg(l-fabp:VDB-EGFP) fish. EC morphology, and expression profiles were examined in Tg(flia1:eGFP) fish and by transmission electron microscopy. Recombinant Hpa2 was injected into hpse2 LOF fish and its effect on vascular pathology analyzed. Hpa2 was applied in EC cell cultures to study signal transduction of FGF2 and VEGFA. Results: hpse2 was detected in hepatic tissue of zebrafish from 72 hpf onwards and localized the protein in blood vessels. hpse2 LOF caused a vascular phenotype. hpse2 LOF reduces eGCX and causes changes in the endothelial transcriptome characterized by genes involved in ECM-receptor interaction and signal transduction regulation. Recombinant hHpa2 rescued the hpse2 LOF phenotype in zebrafish. In vitro studies showed that Hpa2 competes with heparin-binding growth factors FGF2 and VEGFA for binding on the EC surface and consequently reduces the cellular response these factors cause. Pharmacological inhibition of VEGFR2 and FGFR in alleviated the hpse2 LOF phenotype in zebrafish. Conclusion: We conclude that Hpa2 is necessary to maintain EC homeostasis by preventing HS breakdown. Recombinant Hpa2 prevents endothelial dysfunction and reduces proteinuria. Hpa2 induced maintenance of HS glycans strongly regulates growth factor-dependent signaling in EC. These findings may translate into novel treatment strategies with recombinant Hpa2. Funding: Private Foundation Support