Heparanase 2 Modulates Vascular Permeability via Heparan Sulfate-Dependent Growth Factor Signaling

Heparan sulfate (HS) glycans 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 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 using zebrafish as our primary research model. We detected Hpa2 expression in hepatic tissue and localized the protein in blood vessels. Hpa2 loss-of-function (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 Hpa2 rescued the Hpa2-LOF phenotype in zebrafish. Hpa2 competes with fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A165 (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. These results may translate into novel strategies applying recombinant Hpa2 to treat microvascular diseases. To investigate the effect of heparanase 2 (Hpa2) on endothelial cells in zebrafish larvae, we induced a Hpa2 loss-of-function scenario and isolated endothelial cells from pooled larvae. Bulk RNA sequncing from EC-derived RNA was perfromed comparing the expression profile of Hpa2 loss-of-fuction vs. control larvae.