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The transcription factor Kruppel-like factor 2 (KLF2) is a critical anti-inflammatory and anti-atherogenic molecule in vascular endothelium. Enhancing KLF2 expression and activity improves endothelial function and prevents atherosclerosis. However, the pharmacological and molecular regulators for KLF2 are scarce. Using high-throughput luciferase reporter assay to screen for KLF2 activators, we have identified tannic acid (TA), a polyphenolic compound, as a potent KLF2 activator that attenuates endothelial inflammation. Mechanistic studies suggested that TA induced KLF2 expression in part through the ERK5/MEF2 pathway. Functionally, TA markedly decreased monocyte adhesion to ECs by reducing expression of adhesion molecule VCAM1. Using lung ECs isolated from Klf2+/+ and Klf2+/− mice, we showed that the anti-inflammatory effect of TA is dependent on KLF2. Collectively, our results demonstrate that TA is a potent KLF2 activator and TA attenuated endothelial inflammation through upregulation of KLF2. Our findings provide a novel mechanism for the well-established beneficial cardiovascular effects of TA and suggest that KLF2 could be a novel therapeutic target for atherosclerotic vascular disease.

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 CoA-reductase 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-of-function 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.

Vascular endothelium–derived nitric oxide (NO), originally identified as endothelium-derived relaxing factor,1–3 plays a pivotal role in regulation of vascular homeostasis.4 NO is a major regulator of vascular tone and blood pressure, and has multiple antiatherogenic roles including antiinflammatory, antithrombotic, antiproliferative, and antioxidant effects.4–6 Loss of the bioavailability of endothelium-derived NO is the hallmark of endothelial dysfunction and is implicated in the pathogenesis of cardiovascular disease such as hypertension and atherosclerosis.7,8 Therefore, it is of great interest to understand the molecular mechanisms regulating NO production by endothelium, which is likely to provide new insight into endothelial function in health and disease. See page 1015 Endothelial NO synthase (eNOS) is a highly regulated, calcium (Ca2+)/calmodulin (CaM)-dependent enzyme responsible for the physiological production of NO in the vasculature.9,10 In endothelial cells (ECs), NO is formed from its precursor L-arginine via the enzymatic activation of eNOS with cofactors such as tetrahydrobiopterin (BH4). eNOS activation and subsequent NO production is stimulated by a variety of physical stimuli such as fluid shear stress generated by blood flow and by many humoral factors including acetylcholine, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin.10–12 Increasing evidence suggest that eNOS is regulated by subcellular localization,9,13,14 posttranslational modifications such as phosphorylation at serine 1179 (S1179) by Akt,15–17 and interactions with several regulatory proteins such as heat shock protein 90 (HSP90) and caveolin-1.18,19 In particular, subcellular localization of eNOS is critical for optimal coupling of extracellular stimulation to NO production.10 In ECs, eNOS appears to localize at peripheral aspects of the Golgi complex and cholesterol-rich microdomains of the plasma membrane (PM) such as caveolae. Cotranslational N-myristoylation and posttranslational cysteine palmitoylation of eNOS determine its membrane targeting.9,13 It has been proposed that eNOS membrane localization may bring eNOS …