Abstract 4888: A Novel Role of Kruppel-like Factor 2 (KLF2) in Regulating Endothelial Barrier Function

Abstract

Background : Selective permeability is a life sustaining function of endothelial cells. Defects in permeability are implicated in several diseases such as sepsis, ischemia reperfusion injury and diabetes. Previous studies by our group and others have identified KLF2 as a molecular switch regulating endothelial gene expression and function. However the role of KLF2 in vascular permeability remains unknown. Methods and Results : Adenoviral overexpression of KLF2 in HUVECs strongly attenuated the increase of endothelial permeability by thrombin (1U/ml), histamine (200 μ M) and hydrogen peroxide (500 μ M) as measured by two independent assays – transendothelial electrical resistance (TER) and FITC-Dextran passage using a transwell system. Conversely, KLF2 deficiency in HUVECs (by siRNA knockdown) and primary mouse endothelial cells (derived from KLF2 +/+ and KLF2+/− mice) exhibited a marked increase in thrombin-induced permeability. To assess the effect of KLF2 in vascular permeability in vivo, we measured the leakage of Evans Blue dye into interstitial tissues of the mouse ear (expressed as μ g of dye/mg of ear tissue) after treatment with mustard oil. Consistent with our in vitro observation, by comparison to KLF2+/+ mice, KLF2 +/− mice exhibited a significantly higher degree of vascular leak (53.4±2.68 μ g/mg in KLF2+/+ versus 72.36±4.71 μ g/mg in KLF2+/−, p<0.05). Mechanistically, our gain and loss-of-function studies indicate that KLF2 differentially affects key endothelial targets that regulate barrier function. Specifically, KLF2 markedly attenuated agonist- induced increase in Myosin Light Chain (MLC) phosphorylation (a key signaling molecule that promotes endothelial barrier permeability), increased Rac1 activation (a small GTPase that has been shown to confer barrier protection properties), and increased the expression of occludin and zona occludens-1 (tight junction proteins that promote barrier function). Conclusions : These observations identify KLF2 as an essential regulator of endothelial barrier function. Targeting of KLF2 function may be beneficial in disorders characterized by excessive vascular permeability such as sepsis. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).