Kruppel-like factor 2 (KLF2) is a central regulator of endothelial and monocyte/macrophage gene expression and function in vitro. While the composite effects of KLF2 in these two cell types predict that it likely inhibits vascular inflammation, the role of KLF2 in this process in vivo is uncharacterized. In this study, we provide evidence that hemizygous deficiency of KLF2 increased diet-induced atherosclerosis in apolipoprotein E (ApoE) deficient mice. Our studies highlight an important role for KLF2 in primary macrophage foam cell formation via the potential regulation of the key lipid binding protein adipocyte Protein 2 (aP2)/fatty-acid binding protein 4 (FABP4). These novel observations establish that KLF2 is an atheroprotective factor.
Objective A central function of the endothelium is to serve as a selective barrier that regulates fluid and solute exchange. While perturbation of barrier function can contribute to numerous disease states, our understanding of the molecular mechanisms regulating this aspect of endothelial biology remains incompletely understood. Accumulating evidence implicates the Kruppel-like factor 2 (KLF2) as a key regulator of endothelial function. However, its role in vascular barrier function is unknown. Methods and Results To assess the role of KLF2 in vascular barrier function in vivo, we measured the leakage of Evans Blue dye into interstitial tissues of the mouse ear after treatment with mustard oil. By comparison to KLF2+/+ mice, KLF2+/− mice exhibited a significantly higher degree of vascular leak. In accordance with our in vivo observation, adenoviral overexpression of KLF2 in HUVECs strongly attenuated the increase of endothelial leakage by thrombin and H2O2 as measured by FITC-Dextran passage. Conversely, KLF2 deficiency in HUVECs and primary endothelial cells derived from KLF2+/− mice exhibited a marked increase in thrombin and H2O2-induced permeability. Mechanistically, our studies indicate that KLF2 confers barrier-protection via differential effects on the expression of key junction protein occludin and modification of a signaling molecule (myosin light chain) that regulate endothelial barrier integrity. Conclusions These observations identify KLF2 as a novel transcriptional regulator of vascular barrier function.
The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. The molecular mechanisms regulating endothelial homeostasis, however, remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. The function of CCN3 in endothelial cells has yet to be elucidated. Immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, and veins. We found that CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) and HMG CoAreductase inhibitors (statins). Promoter analyses identified the transcription factor Kruppel-like factor 2 (KLF2) as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines reduced CCN3 expression. Adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of vascular adhesion molecule-1 (VCAM-1). Consistent with this observation, CCN3 significantly reduced monocyte adhesion. Conversely, CCN3 knockdown in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Gain and loss-offunction mechanistic studies demonstrated that CCN3 negatively regulates nuclear factor kappaB (NF-κB) activity by reducing its translocation into the nucleus and subsequent binding to the VCAM-1 promoter, suggesting that CCN3's anti-inflammatory effects occur secondary to inhibition of NF-κB nuclear accumulation. This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.
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