Abstract
Roundabout (Robo) receptors 1 and 4 and their ligand Slit2, are novel regulators of endothelial cell (EC) function. Robo1/4 can have co-operative and/or opposing effects on cell function. Expression levels vary between EC and it is likely that functional consequences of Slit2 are determined by the predominant Robo receptor. A better understanding of the Slit/Robo pathway in pulmonary artery (PA) EC could be relevant to conditions such as pulmonary artery hypertension.
Generally, Slit2-Robo4 interaction protects EC cells during inflammation. We hypothesised that greater Robo4 expression in EC, compared with Robo1, was necessary for an anti-inflammatory effect of Slit2. Our aim was to measure Robo1/4 on human PAEC, in comparison to human umbilical vein (HUV) EC; and to measure responses to Slit2. Our objectives were: (1) to activate EC with TNF (10ng/ml TNF, 23h) and measure Robo-1/4; (2) to assess effect of Slit2 (20nM) on GM-CSF release in PAEC and HUVEC.
Robo expression in PAEC and HUVEC were determined by FACS; GM-CSF release, by ELISA.
Under basal conditions, 95±1% of HUVEC expressed Robo4; whereas, only 10±3% of cells expressed Robo1 (n=5). TNF significantly (P<0.036) decreased Robo4 (64±12%), but not Robo1 expression. By contrast, only 55±5% of PAEC expressed Robo4, whereas 75±5% of cells expressed Robo1. As with HUVEC, TNF only decreased Robo4 expression in PAEC. In functional experiments, Slit2 decreased GM-CSF release from TNF-activated HUVEC (25%); but not from PAEC.
In PAEC, expressing high levels of Robo1 and less Robo4, Slit2 did not reduce GM-CSF. These findings suggest that PAEC are more resistant to the anti-inflammatory effects of Slit2.
- Copyright ©the authors 2016