Background-Although 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to modulate endothelial function, the transcriptional mechanisms underlying these effects are incompletely understood. We hypothesized that Lung-Kruppel-like factor (LKLF/KLF2), a novel and potent regulator of endothelial gene expression, may mediate the downstream effects of statins. Here we report that statin-induced expression of endothelial NO synthase (eNOS) and thrombomodulin is KLF2 dependent. Methods and Results-KLF2 mRNA was induced by treatment with multiple statins in a concentration-dependent manner.Multiple lines of evidence suggest that this induction is dependent on inhibition of the Rho pathway and requires de novo transcription. Furthermore, promoter deletion and mutational analyses suggest that mevastatin induced KLF2 promoter activity through a single myocyte enhancer factor binding site. Finally, small-interfering RNA-mediated knockdown of KLF2 strongly attenuated the ability of mevastatin to increase eNOS and thrombomodulin accumulation in endothelial cells. Conclusions-Taken

The Kruppel-like factor KLF2 was recently identified as a novel regulator of endothelial pro-inflammatory and pro-thrombotic function. Here it is shown that overexpression of KLF2 potently inhibits vascular permeability factor/vascular endothelial growth factor (VEGF-A)-mediated angiogenesis and tissue edema in the nude ear mouse model of angiogenesis. In vitro, KLF2 expression retards VEGF-mediated calcium flux, proliferation and induction of pro-inflammatory factors in endothelial cells. This effect is due to a potent inhibition of VEGFR2/KDR expression and promoter activity. These observations identify KLF2 as a regulator of VEGFR2/ KDR and provide a foundation for novel approaches to regulate angiogenesis.Angiogenesis, the outgrowth of new vessels from preexisting blood vessels, is an important feature of both normal physiology and pathologic states including chronic inflammatory diseases and tumor development (1-3). The growth factor VEGF-A 1 is a key regulator of physiologic and pathologic angiogenesis (4). Originally identified on the basis of its ability to induce vascular permeability (5), VEGF-A is now recognized as a potent inducer of endothelial proliferation, migration, and survival. Furthermore, VEGF-A also acts as a proinflammatory cytokine and induces the expression of a number of molecules implicated in regulating angiogenesis such specific enzymes (e.g. cyclooxygenase-2 (COX-2)), adhesion molecules (e.g. E-selectin, VCAM-1) (6), and pro-coagulant factors (e.g. tissue factor) (7). The effects of VEGF-A and its family members are mediated by three structurally related receptor tyrosine kinases termed VEGFR1/Flt-1, VEGFR2/KDR/Flk-1, and VEGFR3/Flt4 (8 -13). Among these three receptors, VEGFR2 has emerged as the main receptor mediating VEGF-A effects related to angiogenesis such as endothelial cell proliferation, migration, and proinflammatory activation. In contrast, VEGFR1 is thought to mediate inhibitory and/or decoy effects (14) (15) in vascular endothelial cells. Finally, VEGFR3 is mainly expressed in lymphatics and regulates aspects of lymphatic endothelial cell biology (13). The importance of VEGF-A/VEGFR2 axis is further underscored by the fact that both ligand and receptor levels is increased in pathologic states such as tumor beds (16 -20). It follows that identification of mechanisms that may reduce the expression of either ligand or receptor may serve as the basis for inhibiting angiogenesis in pathologic states.The Sp/Kruppel-like factor (KLF) family of transcription factors is a subclass of the zinc-finger family of transcriptional regulators implicated in the regulation of cellular growth and differentiation (21). To date 20 members have been identified that include 4 Sp factors (Sp1-4) and 16 KLF factors (KLF1-16) (22). Members of this family can bind with varying affinities to the same DNA sequences (termed GC-box or CACCC element) and varying transcriptional activities. Furthermore, members of this family can modulate each other's function through a number of distinct mechanisms s...