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

The role of ERK5 in endothelial cell function

Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function

Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and Attenuates Lung and Liver Inflammation during Murine Endotoxemia

Background: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings: Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness

Cytoprotective pathways in the vascular endothelium. Do they represent a viable therapeutic target?

The vascular endothelium is a critical interface, which separates the organs from the blood and its contents. The endothelium has a wide variety of functions and maintenance of endothelial homeostasis is a multi-dimensional active process, disruption of which has potentially deleterious consequences if not reversed. Vascular injury predisposes to endothelial apoptosis, dysfunction and development of atherosclerosis. Endothelial dysfunction is an end-point, a central feature of which is increased ROS generation, a reduction in endothelial nitric oxide synthase and increased nitric oxide consumption. A dysfunctional endothelium is a common feature of diseases including rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus and chronic renal impairment. The endothelium is endowed with a variety of constitutive and inducible mechanisms that act to minimise injury and facilitate repair. Endothelial cytoprotection can be enhanced by exogenous factors such as vascular endothelial growth factor, prostacyclin and laminar shear stress. Target genes include endothelial nitric oxide synthase, heme oxygenase-1, A20 and anti-apoptotic members of the B cell lymphoma protein-2 family. In light of the importance of endothelial function, and the link between its disruption and the risk of atherothrombosis, interest has focused on therapeutic conditioning and reversal of endothelial dysfunction. A detailed understanding of cytoprotective signalling pathways, their regulation and target genes is now required to identify novel therapeutic targets. The ultimate aim is to add vasculoprotection to current therapeutic strategies for systemic inflammatory diseases, in an attempt to reduce vascular injury and prevent or retard atherogenesis

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

INTRODUCTION AND OBJECTIVES: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. METHODS AND RESULTS: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. CONCLUSIONS: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development

Tumor Necrosis Factor Alpha-Mediated Reduction of KLF2 Is Due to Inhibition of MEF2 by NF-κB and Histone Deacetylases

Activation of the endothelium by inflammatory cytokines is a key event in the pathogenesis of vascular disease states. Proinflammatory cytokines repress the expression of KLF2, a recently identified transcriptional inhibitor of the cytokine-mediated activation of endothelial cells. In this study the molecular basis for the cytokine-mediated inhibition of KLF2 is elucidated. Tumor necrosis factor alpha (TNF-α) potently inhibited KLF2 expression. This effect was completely abrogated by a constitutively active form of IκBα, as well as treatment with trichostatin A, implicating a role for the NF-κB pathway and histone deacetylases. Overexpression studies coupled with observations with p50/p65 null cells support an essential role for p65. A combination of promoter deletion and mutational analyses, chromatin immunoprecipitation assays, and coimmunoprecipitation studies indicates that p65 and histone deacetylases 4 cooperate to inhibit the ability of MEF2 factors to induce the KLF2 promoter. These studies identify a novel mechanism by which TNF-α can inhibit endothelial gene expression. Furthermore, the inhibition of MEF2 function by p65 and HDAC4 has implications for other cellular systems where these factors are operative