Endoglin Knockdown inhibits Proliferation and NO Production in murine Sinusoidal Endothelial Cells in vitro

Introduction: Endoglin (ENG) is a 180 kDA homodimeric membrane protein which acts as an auxiliary TGF-β Receptor modulating TGF-β downstream signalling. It plays an important role in various cellular processes like proliferation and differentiation. ENG is also involved in VEGF/eNOS signalling and nitric oxide (NO) production thereby fine-tuning angiogenetic processes. However, the interplay between ENG, eNOS and VEGF has still not been clarified and is an object of current research.

Aim: Here we investigate the influence of ENG on NO production and VEGF-induced proliferation in primary murine liver sinusoidal endothelial cells (LSECs).

Methods:

Endothelial cells were isolated from C57BL/6 mice and purified by a magnetic separation with CD146 MACS beads. The purified cells were allowed get adherent overnight on 6-well plates. Thereafter cells were transfected with an anti-ENG or non-target siRNA (5 nmol/well with 5 µl lipofectamine) for 48h followed by stimulation with recombinant murine VEGF (5 ng/ml) or the eNOS activator calcium ionophore A32187 (Ca-I, 1µM) for 48 hours. Supernatant aliquots were taken every 24h for determination of NO production. A scratch was made with a sterile 1000 µl pipette tip in each well after the cell were adherent and the cell proliferation was monitored by photographic measurement every 24h after stimulation. The NO production was determined by measuring the nitrite and nitrate concentration of the supernatant (Griess Test). At the end of the stimulation step cell lysates were used for western blot analysis of ENG-, VEGF-, eNOS- and β-actin expression.

Results: We observed an ENG knockdown efficiency of > 70%. Furthermore the ENG knockdown samples showed a 29% ( ± 7%) and 52% ( ± 12%), p < 0.05, decrease in NO production in VEGF and Ca-I stimulated samples respectively compared to the ENG wt controls. The proliferation of ENG deficient LSECs decreased by 28% ( ± 9%) in the photographically analyzed scratch assays compared to wt controls. Western blots revealed a 34% ( ± 8%) decrease in eNOS expression in ENG deficient LSECs compared to wt cells.

Conclusion: This study shows the interaction between ENG and the VEGF-eNOS pathway and its influence on LSEC proliferation. The results bring new insights into the role of endoglin in LSEC pathophysiology and liver perfusion.

Corresponding author: Scholten, David

E-Mail: [e-mail]