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

Abstract

BackgroundHeparan 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. Shedding of heparan sulfate (HS) from the EC surface increases vascular leakage and is associated with vascular diseases. Recently, heparanase 2 (Hpa2) was described as a novel regulatory molecule that controls HS shedding. However, its role in regulating HS physiology in the vascular endothelium is largely unknown. Here, we characterize the role of endogenous Hpa2 in the vertebrate vascular system. MethodsWe 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 and HS quantity were analyzed in Hpa2-LOF larvae. The capacity of recombinant Hpa2 to modulate signaling in ECs by the HS-binding growth factors fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A165 (VEGFA165) was tested by western blotting and immunofluorescence. Attenuation of the Hpa2-LOF phenotype was tested in vivo in zebrafish larvae via use of recombinant Hpa2 and pharmacological inhibition of FGF and VEGFA signaling. ResultsWe detected hpse2 expression in hepatic tissue and localized the protein in blood vessels. Hpa2-LOF larvae exhibited increased vascular permeability, occasional hypersprouting, and altered EC and extracellular matrix (ECM) morphology. Hpa2-LOF also reduced HS levels and caused changes in the endothelial transcriptome characterized by dysregulated genes involved in ECM-receptor interaction and signal transduction regulation. Recombinant hHpa2 rescued the Hpa2-LOF phenotype in zebrafish. We showed in vitro that 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. ConclusionWe conclude that Hpa2 is a circulating molecule that maintains vascular integrity by regulating HS-dependent processes on the EC surface. These results may translate into novel strategies applying recombinant Hpa2 to treat microvascular diseases.