Abstract
AbstractThe flow-responsive transcription factor KLF2 is acquiring a leading role in the regulation of endothelial cell gene expression. A genome-wide microarray expression profiling is described employing lentivirus-mediated, 7-day overexpression of human KLF2 at levels observed under prolonged flow. KLF2 is not involved in lineage typing, as 42 endothelial-specific markers were unaffected. Rather, KLF2 generates a gene transcription profile (> 1000 genes) affecting key functional pathways such as cell migration, vasomotor function, inflammation, and hemostasis and induces a morphology change typical for shear exposure including stress fiber formation. Protein levels for thrombomodulin, endothelial nitric oxide synthase, and plasminogen activator inhibitor type-1 are altered to atheroprotective levels, even in the presence of the inflammatory cytokine TNF-α. KLF2 attenuates cell migration by affecting multiple genes including VEGFR2 and the potent antimigratory SEMA3F. The distribution of Weibel-Palade bodies in cultured cell populations is normalized at the single-cell level without interfering with their regulated, RalA-dependent release. In contrast, thrombin-induced release of Weibel-Palade bodies is significantly attenuated, consistent with the proposed role of VWF release at low–shear stress regions of the vasculature in atherosclerosis. These results establish that KLF2 acts as a central transcriptional switch point between the quiescent and activated states of the adult endothelial cell.
Highlights
We previously described a lentivirus-driven overexpression approach, where human KLF2 is expressed using the relatively moderate phosphoglycerate kinase (PGK) promoter, thereby increasing KLF2 expression to levels similar to those observed in Human umbilical vein endothelial cell (HUVEC) that are exposed to prolonged pulsatile flow.[10]
In order to determine the complete repertoire of KLF2 downstream genes in endothelial cells, gene expression analysis was performed on these 5 independent overexpression experiments using oligonucleotide-based microarrays containing specific 60mer oligonucleotides for 18 600 human genes
KLF2-null mice die during embryogenesis due to a defective vasculature,[11] but the exact stage of endothelial differentiation that is critically affected by the absence of KLF2 remains to be identified
Summary
The zinc finger transcription factor lung Kruppel-like factor (LKLF/KLF2) has emerged as a prime and pivotal candidate for directly relaying biomechanical shear forces into a gene transcription profile that might determine endothelial phenotype in response to flow.[6,7,8,9,10] We previously demonstrated that KLF2 mRNA and protein levels are substantially and uniquely induced by prolonged steady or pulsatile laminar flow both in vitro and in vivo.[6,10] we have demonstrated that KLF2 is expressed in endothelial cells of the adult vasculature, but its expression drops to low levels near sites of flow disturbance, coinciding with regions that show pathologic changes of the vascular wall.[10]. The primary bioinformatics analysis is complemented by real-time reverse transcriptase–polymerase chain reaction (RTPCR) and protein and cell biologic analysis, most illustrated by KLF2 effects on von Willebrand Factor (VWF), a key endothelial marker protein. These data reveal that KLF2 systematically controls the expression of many genes that are involved in the functionally mature differentiated endothelium but is not involved in determining endothelial lineage typing
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