Heparanase 2 regulates endothelial permeability and prevents from proteinuria via VEGFA and FGF signaling

Heparan sulfates (HS) attached to the apical surface of vascular endothelial cells (ECs) play an important role in regulating endothelial permeability and ligand recognition by cell-surface receptors. Recently, heparanase 2 (Hpa2) was described as a regulatory molecule that controls HS shedding. However, the role of Hpa2 in regulating HS physiology in the vascular endothelium is largely unknown. We use zebrafish larvae as our primary animal model. Hpa2 expression and localization was examined by in situ hybridization and immunofluorescence. Hpa2 loss of-function (LOF) was induced by CRISPR-Cas9 or morpholino antisense strategies. We assessed vascular permeability, blood vessel architecture, and EC morphology using transgenic zebrafish and transmission electron microscopy. EC expression profiles were analyzed in Hpa2-LOF larvae. We tested the capacity of recombinant Hpa2 to modulate signaling in ECs by the heparin-binding growth factors fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A165 (VEGFA165) by western blotting. Recombinant Hpa2 was applied in Hpa2-LOF zebrafish larvae and pharmacological inhibition of FGF and VEGFA signaling was conducted. We detected hpse2 expression in hepatic tissue and localized the protein in blood vessels. Hpa2-LOF caused increased vascular permeability, occasional hypersprouting, and altered EC and extracellular matrix (ECM) morphology. Hpa2-LOF reduced HS levels and caused changes in the endothelial transcriptome characterized by dysregulated genes involved in ECM-receptor interaction and signal transduction regulation. Recombinant Hpa2 rescued the Hpa2-LOF phenotype in zebrafish. Hpa2 competes with FGF2 and VEGFA165 for binding on the EC surface and consequently reduces the cellular response these factors elicit. Pharmacological inhibition of these pathways alleviated the Hpa2-LOF phenotype in zebrafish. We conclude that Hpa2 is a circulating molecule that maintains vascular integrity by regulating HS-dependent processes on the EC surface. Therapeutic application of Hpa2 may be a novel treatment strategy for microvascular disease.