ABSTRACT
AIMS: The inflammatory response to injurious agents is tightly regulated to avoid adverse consequences of inappropriate leucocyte accumulation or failed resolution. Lipopolysaccharide (LPS)-activated endothelium recruits leucocytes to the inflamed tissue through controlled expression of membrane-associated adhesion molecules. LPS responses in macrophages are known to be regulated by integrin-linked kinase (ILK); in this study, we investigated the role of ILK in the regulation of the LPS-elicited inflammatory response in endothelium. METHODS AND RESULTS: This study was performed on immortalized mouse endothelial cells (EC) isolated from lung and coronary vasculature. Cells were thoroughly characterized and the role of ILK in the regulation of the LPS response was investigated by suppressing ILK expression using siRNA and shRNA technologies. Phenotypic and functional analyses confirmed that the immortalized cells behaved as true EC. LPS induced the expression of the inflammatory genes E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). ILK knockdown impaired LPS-mediated endothelial activation by preventing the induction of ICAM-1 and VCAM-1. Blockade of the LPS-induced response inhibited the inflammatory-related processes of firm adhesion and trans-endothelial migration of leucocytes. CONCLUSION: ILK is involved in the expression of cell adhesion molecules by EC activated with the inflammatory stimulus LPS. This reduced expression modulates leucocyte adhesion to the endothelium and the extravasation process. This finding suggests ILK as a potential anti-inflammatory target for the development of vascular-specific treatments for inflammation-related diseases.
Subject(s)
Cell Adhesion Molecules/metabolism , Endothelial Cells/drug effects , Inflammation Mediators/metabolism , Inflammation/enzymology , Leukocyte Rolling/drug effects , Leukocytes/drug effects , Lipopolysaccharides/pharmacology , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Adhesion/drug effects , Cell Adhesion Molecules/genetics , Coculture Techniques , E-Selectin/metabolism , Endothelial Cells/enzymology , Endothelial Cells/immunology , Humans , Inflammation/genetics , Inflammation/immunology , Intercellular Adhesion Molecule-1/metabolism , Jurkat Cells , Leukocytes/immunology , Mice , Mice, Inbred C57BL , Phenotype , Poly I-C/pharmacology , Protein Serine-Threonine Kinases/genetics , RNA Interference , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Epidemiologic, genetic, and biochemical studies support an antiatherogenic role for paraoxonase (PON) 1. While the precise mechanism by which PON1 protects against the development of atherosclerosis is unclear, in vitro studies and the results from PON1 knockout and transgenic mice suggest that this protective effect may be attributed to PON1's ability to attenuate the oxidative modification of lipoprotein particles. The two other members of the PON gene family, namely, PON2 and PON3, have also been reported to possess antioxidant properties and may exhibit antiatherogenic capacities as well. Previous studies have demonstrated that PON1 expression is downregulated by oxidative stress. In contrast, more recent studies have shown that PON2 expression is upregulated in response to oxidative stress-inducing agents, while PON3 expression remains unchanged. While the physiological function of these proteins is unknown, studies currently underway using PON2 and PON3 knockout and transgenic mice should enable us to tease out the apparently redundant functions of these three proteins and yield clues as to their physiological function as well as their role in atherogenesis.
