Effect of heparan sulfate cleavage on homeostatic gene expression in microvascular endothelial cells

Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of HS shedding on mechanisms of vascular injury and repair has not been evaluated. The objective of this work was to characterize the effect of eGC HS shedding on the transcriptional landscape of vascular endothelial cells. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparin lyase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparin lyase III treatment. Pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding. Primary human lung microvascular ECs (HLMVEC) were conditioned under shear stress (15 dynes/cm2) for 48 hours prior to a 6-hour treatment with vehicle (PBS) or heparin lyase III (n = 4 per group). After treatment, flow was discontinued, and cell monolayers were washed thrice with PBS prior to RNA harvest. Total RNA was isolated from HLMVEC, and cell lysate from 2 slides was combined into a single sample to ensure robust RNA quantity, which generated 2 RNA samples per treatment group for bulk sequencing.