G. Brandon Atkins

KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1β and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element–binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

Kruppel-Like Factor 2 as a Novel Mediator of Statin Effects in Endothelial Cells

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 together, these observations indicate that statin-dependent induction of eNOS and thrombomodulin requires KLF2 and thereby provides a novel molecular target for modulating endothelial function in vascular disease.

Hemizygous deficiency of Krüppel-like factor 2 augments experimental atherosclerosis.

Krüppel-like factor (KLF)2 is a central regulator of endothelial and monocyte/macrophage gene expression and function in vitro. Although the composite effects of KLF2 in these 2 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–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/fatty acid–binding protein 4. These novel observations establish that KLF2 is an atheroprotective factor.

Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal.

Kruppel-like Factor 2 Inhibits Hypoxia-inducible Factor 1α Expression and Function in the Endothelium

Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key proangiogenic factors to promote angiogenesis. Recent studies have identified Kruppel-like factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating HIF-1 expression and function has not been evaluated. KLF2 expression was induced acutely by hypoxia in endothelial cells. Adenoviral overexpression of KLF2 inhibited hypoxia-induced expression of HIF-1α and its target genes such as interleukin 8, angiopoietin-2, and vascular endothelial growth factor in endothelial cells. Conversely, knockdown of KLF2 increased expression of HIF-1α and its targets. Furthermore, KLF2 inhibited hypoxia-induced endothelial tube formation, whereas endothelial cells from mice with haploinsufficiency of KLF2 showed increased tube formation in response to hypoxia. Consistent with this ex vivo observation, KLF2 heterozygous mice showed increased microvessel density in the brain. Mechanistically, KLF2 promoted HIF-1α degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1α and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1α by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1α expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

Kruppel-Like Factor 2 Regulates Endothelial Barrier Function

Objective—A central function of the endothelium is to serve as a selective barrier that regulates fluid and solute exchange. Although 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 with KLF2+/+ mice, KLF2+/− mice exhibited a significantly higher degree of vascular leak. In accordance with our in vivo observation, adenoviral overexpression of KLF2 in human umbilical vein endothelial cells strongly attenuated the increase of endothelial leakage by thrombin and H2O2 as measured by fluorescein isothiocyanate dextrans (FITC-dextran) passage. Conversely, KLF2 deficiency in human umbilical vein endothelial cells 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. Conclusion—These observations identify KLF2 as a novel transcriptional regulator of vascular barrier function.

ENDOTHELIAL DIFFERENTIATION: MOLECULAR MECHANISMS OF SPECIFICATION AND HETEROGENEITY

A complex and diverse vascular system is requisite for the survival of higher organisms. The process of vascular development is highly regulated, involving the de novo formation of vessels (vasculogenesis), followed by expansion and remodeling of the primitive vasculature (angiogenesis), culminating in differentiation of endothelial phenotypes, as found in the mature vascular system. Over the last decade, significant advances have been made in understanding the molecular regulation of endothelial cell development and differentiation. Endothelial development, in particular the mechanisms in play during vasculogenesis and angiogenesis, is discussed in a sister review to this article. This review highlights the key pathways governing in endothelial differentiation, with a focus on the major molecular mechanisms of endothelial specification and heterogeneity.

Kruppel-like factor 2 protects against ischemic stroke by regulating endothelial blood brain barrier function

During an ischemic stroke normal brain endothelial function is perturbed, resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells (ECs), and serves as a key molecular switch regulating endothelial function and promoting vascular health. In this study we sought to determine the role of KLF2 in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion was performed in KLF2-deficient (KLF2(-/-)), KLF2 overexpressing (KLF2(tg)), and control mice, and stroke volume was analyzed. BBB function was assessed in vivo by real-time neuroimaging using positron emission tomography and Evans blue dye assay. KLF2(-/-) mice exhibited significantly larger strokes and impairment in BBB function. In contrast, KLF2(tg) mice were protected against ischemic stroke and demonstrated preserved BBB function. In concordance, gain- and loss-of-function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective. Mechanistically, KLF2 was demonstrated, both in vitro and in vivo, to regulate the critical BBB tight junction factor occludin. These data are first to identify endothelial KLF2 as a key regulator of the BBB and a novel neuroprotective factor in ischemic stroke.

A novel role of CCN3 in regulating endothelial inflammation

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.

Circadian control of bile acid synthesis by a KLF15- Fgf15 axis

Recent studies have shown that starburst dwarf galaxies have steeply rising rotation curves in their inner parts, pointing to a close link between the intense star formation and a centrally concentrated mass distribution (baryons and dark matter). More quiescent dwarf irregulars typically have slowly rising rotation curves, although some “compact” irregulars with steep, inner rotation curves exist. We analyze archival Hubble Space Telescope images of two nearby “compact” irregular galaxies (NGC 4190 and NGC 5204), which were selected solely on the basis of their dynamical properties and their proximity. We derive their recent star-formation histories by fitting colormagnitude diagrams of resolved stellar populations, and find that the star-formation properties of both galaxies are consistent with those of known starburst dwarfs. Despite the small sample, this strongly reinforces the notion that the starburst activity is closely related to the inner shape of the potential well.Circadian control of nutrient availability is critical to efficiently meet the energetic demands of an organism. Production of bile acids (BAs), which facilitate digestion and absorption of nutrients, is a major regulator of this process. Here we identify a KLF15-Fgf15 signalling axis that regulates circadian BA production. Systemic Klf15 deficiency disrupted circadian expression of key BA synthetic enzymes, tissue BA levels and triglyceride/cholesterol absorption. Studies in liver-specific Klf15-knockout mice suggested a non-hepatic basis for regulation of BA production. Ileal Fgf15 is a potent inhibitor of BA synthesis. Using a combination of biochemical, molecular and functional assays (including ileectomy and bile duct catheterization), we identify KLF15 as the first endogenous negative regulator of circadian Fgf15 expression. Elucidation of this novel pathway controlling circadian BA production has important implications for physiologic control of nutrient availability and metabolic homeostasis.