Approximately 50% of the world is at risk of dengue virus (DENV) infection, which is characterised by prominent vascular leakage in severe cases. This is initiated through microvascular permeability. Previous work showed DENV-2 Non-structural protein-1 (NS1) to disrupt endothelial-pericyte interactions to increase microvascular permeability, utilising human umbilical vein endothelial cells (HUVECs) and saphenous venous pericytes (SVPs) co-cultures.

This project aimed to determine whether this effect applies across DENV’s four serotypes and elucidate potential mechanisms. Additionally, the current endothelial-pericyte model was modified to include liver pericytes, known as hepatic stellate cells (HSCs), as the liver is a significant site of dengue pathology.

DENV NS1-treated HUVEC-SVP and HUVEC-HSC co-cultures lowered pericyte’s support of endothelial barrier function, when measured using transendothelial electrical resistance (TEER) readings. Moreover, SVPs treated with NS1 reduced vascular structure formation when seeded with endothelial cells in Geltrex matrix. RT-qPCR confirmed reduced pro-angiogenic factor production in NS1-treated SVPs. Secretome analysis of SVPs treated with DENV-2 NS1, identified a potential mediator, Heart Development Protein with EGF-like domains-1 (HEG-1). Its implication in NS1- induced endothelial dysfunction was verified through immunocytochemical analysis of NS1-treated SVPs and through HEG-1 treatment on HUVECs, following TEER and RNA sequencing experiments.

HSCs were shown to proliferate and differentiate in response to DENV NS1 utilising the MTT assay, immunocytochemistry of alpha-smooth muscle actin (α-SMA) and through flow cytometric analysis of α-SMA and proliferative marker Ki67. HSCs were also shown to host DENV replication using TCID50 assays. Furthermore, vascular spheroids developed using magnetic cell culture produced instability in response to NS1 as evidenced through brightfield and immunocytochemical analysis.

Overall, DENV can disrupt endothelial-pericyte interactions which in the context of the liver, could be due to HSC differentiation into myofibroblasts. These results provide insights to the mechanisms involved in DENV’s microvascular effects, to help establish future treatments and biomarkers.