Abstract
BackgroundVascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway.MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively.ResultsWe found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell–cell contact regions and the increase of actin stress fibers induced by TNF-α.ConclusionsThe present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.25PWBVc4Kdr4QaTem7i6UUVideo abstract
Highlights
Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis
We found that S1PC, a major sulfur-containing constituent of aged garlic extract (AGE), improved Tumor-necrosis factor-α (TNF-α)-induced endothelial barrier dysfunction by inhibiting both RhoA and Rac pathways through the suppression of the common upstream molecule, guanine nucleotide exchange factor (GEF)-H1, in the TNF-α signaling
To identify the responsible constituents of AGE, we examined the protective effects of three sulfur-containing constituents, S1PC (Fig. 1a), SAC and SAMC (Additional file 1: Figure S1b), against the endothelial permeability in Human umbilical vein endothelial cells (HUVECs)
Summary
Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Chronic inflammation even at low-grade induces disruption of barrier function and enhanced hyperpermeability of vascular endothelium, which leads to the breakdown of vascular homeostasis [2, 3] This dyshomeostasis causes the invasion of macrophages and neutrophils, inflammation within the artery wall and thereby promotes several vascular diseases including atherosclerosis [4, 5]. VE-cadherin interacts with actin cytoskeleton via p120, β-catenin and several actin-binding proteins [8, 9] In this way, endothelial barrier function is maintained by controlling these junctional proteins and actin remodeling
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