Abstract 20870: Insulin Induces the Release of Endothelial Barrier Stabilizing Factor From Platelets by a Nitric Oxide-G Kinase-Dependent Pathway
Introduction: Recent findings suggest that besides its metabolic effects, insulin also modulates the cardiovascular functions. Recently, it has been shown by us and others that insulin also acts as an anti-inflammatory agent. The failure of endothelial barrier function is one of the earliest manifestations of inflammation and atherosclerosis. The importance of insulin for the endothelial barrier function in these (patho-) physiological conditions has not been well described. Here the hypothesis was tested whether insulin can induce the release of endothelial barrier stabilizing factor from platelets.
Methods: The study was performed on well established model of isolated human platelets and cultured human umbilical vein endothelial cells (HUVEC). Thrombin was used as an inflammatory agent.
Results: Exposure of supernatant from freshly isolated platelets (3 × 108/ml) to HUVEC monolayer reduced basal permeability (albumin flux) by 10±5% (n=5, P < 0.05 for all further parameters). On the other hand thrombin (0.2 IU/ml) caused a 3-fold increase in permeability. Supernatant from insulin stimulated platelets (1 IU/ml; 10 min) resulted in a further 0.50-fold decrease in basal permeability and completely antagonized thrombin-induced hyperpermeability. This barrier protective effect was completely abolished when platelets were pre-incubated with HNMP(AM)3 (insulin receptor inhbitor; 1 μM), wortmannin (PI3K inhibitor; 1 μM), L-NAME and L-NNA (NO-synthase inhibitors; 100 μM), NS2028 (sGC inhibitor; 100 μM) and KT5823 (PKG inhibitor; 100 μM). Thrombin caused a 3-fold increase in myosin light chain phosphorylation in HUVEC, 0.5-fold reduction in Rac1 activity, 3-fold increase in RhoA activity (important regulators of endothelial barrier; pulldown assay) and loss of VE-cadherin from cell-cell adhesions (confocal microscopy). These thrombin effects on HUVEC were abrogated by pre-incubation with insulin stimulated platelet-supernatant.
Conclusions: We show here for the first time that insulin mediates the release of endothelial barrier stabilizing factor from platelets via PI3K/Akt-NO pathway. This platelet factor stabilizes endothelial barrier via strenghthening of cell-cell adhesions and inactivating endothelial contractile machinery.
- © 2010 by American Heart Association, Inc.