Abstract 5258: Insulin Stabilizes Coronary Microvascular Endothelial Barrier via PI3K/Rac1-mediated Stabilization of cell-cell adhesions
Recent findings, both in animal and clinical studies show that insulin, besides its metabolic effects, has protective effects under inflammatory conditions as well as in reducing major cardiovascular events in patients undergoing vascular surgery. Loss of endothelial barrier is one of the earliest manifestations of inflammation and atherosclerosis. The role of insulin in the regulation of endothelial barrier function is not well described. The integrity of the endothelial barrier is highly dependent on acto-myosin based endothelial contractile machinery and adherens junctions. Rho-GTPases (Rac1 and RhoA) are well described as important regulators of actin cytoskeleton and adherens junctions and maintain integrity of endothelial barrier. Here the hypothesis was tested whether insulin stabilizes endothelial barrier via Rac1-mediated stabilization of adherens junctions.
Methods and Results: In cultured coronary microvascular endothelial cells from rat heart (RCEC) insulin reduced the macromolecular permeability (albumin flux) from 4.9±0.2 × 10−6 cm2/s to 3.4±0.3 × 10−6 cm2/s (n5, P<0.05 for all further parameters) in a concentration-dependent manner with a maximum effect with 1U/ml and caused 3-fold increase in Akt phosphorylation (Western blot). This protective effect on permeability could be blocked by HNMP(AM)3 (1μM, a specific insulin receptor inhibitor), and Wortmannin (1 μM; PI3K inhibitor), suggesting that insulin reduces permeability via receptor-mediated activation of Akt. Furthermore, insulin induced a 3-fold activation of Rac1 (pull down assay), increased cortical actin and VE-cadherin at cell-cell adhesions (confocal microscopy). These insulin effects could be blocked by HNMP(AM)3, and wortmannin. Furthermore, inhibition of Rac1 with NSC23766 (100 μM; pharmacological inhibitor of Rac1) abrogated insulin mediated barrier stabilization.
Conclusion: We show here for the first time that insulin enhances endothelial barrier function in RCEC monolayers via PI3K/Akt-mediated activation of Rac1, which stabilizes endothelial cell adherens junctions. Further understanding of this mechanism could be beneficial in the development of therapeutic strategies for vessel protection during pathological conditions.