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Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium.

ABSTRACT: Endothelial transcription factors Krüppel-like factor 4 (KLF4) and KLF2 are implicated in protection against atherogenesis. Steady-state microRNA (miR) regulation of KLFs in vivo is accessible by screening region-specific endothelial miRs and their targets.A subset of differentially expressed endothelial miRs was identified in atherosusceptible versus protected regions of normal swine aorta. In silico analyses predicted highly conserved binding sites in the 3'-untranslated region (3'UTR) of KLF4 for 5 miRs of the subset (miR-26a, -26b, -29a, -92a, and -103) and a single binding site for a miR-92a complex in the 3'UTR of KLF2. Of these, only miR-92a knockdown and knock-in resulted in responses of KLF4 and KLF2 expression in human arterial endothelial cells. Dual luciferase reporter assays demonstrated functional interactions of miR-92a with full-length 3'UTR sequences of both KLFs and with the specific binding elements therein. Two evolutionarily conserved miR-92a sites in KLF4 3'UTR and 1 site in KLF2 3'UTR were functionally validated. Knockdown of miR-92a in vitro resulted in partial rescue from cytokine-induced proinflammatory marker expression (monocyte chemotactic protein 1, vascular cell adhesion molecule-1, E-selectin, and endothelial nitric oxide synthase) that was attributable to enhanced KLF4 expression. Leukocyte-human arterial endothelial cell adhesion experiments supported this conclusion. In swine aortic arch endothelium, a site of atherosusceptibility where miR-92a expression was elevated, both KLFs were expressed at low levels relative to protected thoracic aorta.miR-92a coregulates KLF4 and KLF2 expression in arterial endothelium and contributes to phenotype heterogeneity associated with regional atherosusceptibility and protection in vivo.

SUBMITTER: Fang Y

PROVIDER: S-EPMC3306477 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium.

Fang Yun Y Davies Peter F PF

Arteriosclerosis, thrombosis, and vascular biology 20120119 4

<h4>Objective</h4>Endothelial transcription factors Krüppel-like factor 4 (KLF4) and KLF2 are implicated in protection against atherogenesis. Steady-state microRNA (miR) regulation of KLFs in vivo is accessible by screening region-specific endothelial miRs and their targets.<h4>Methods and results</h4>A subset of differentially expressed endothelial miRs was identified in atherosusceptible versus protected regions of normal swine aorta. In silico analyses predicted highly conserved binding sites ...[more]

PMID: 22267480

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Project description:ObjectiveUnderstanding message delivery among vascular cells is essential for deciphering the intercellular communications in cardiovascular diseases. MicroRNA (miR)-92a is enriched in endothelial cells (ECs) and circulation under atheroprone conditions. Macrophages are the primary immune cells in atherosclerotic lesions that modulate lesion development. Therefore, we hypothesize that, in response to atheroprone stimuli, ECs export miR-92a to macrophages to regulate their functions and enhance atherosclerotic progression. Approach and Results: We investigated the macrophage functions that are regulated by EC miR-92a under atheroprone microenvironments. We first determined the distributions of functional extracellular miR-92a by fractionating the intravesicular and extravesicular compartments from endothelial conditioned media and mice serum. The results indicate that extracellular vesicles are the primary vehicles for EC miR-92a transportation. Overexpression of miR-92a in ECs enhanced the proinflammatory responses and low-density lipoprotein uptake, while impaired the migration, of cocultured macrophage. Opposite effects were found in macrophages cocultured with ECs with miR-92a knockdown. Further analyses demonstrated that intravesicular miR-92a suppressed the expression of target gene KLF4 (Krüppel-like factor 4) in macrophages, suggesting a mechanism by which intravesicular miR-92a regulates recipient cell functions. Indeed, the overexpression of KLF4 rescued the EC miR-92a-induced macrophage atheroprone phenotypes. Furthermore, an inverse correlation of intravesicular miR-92a in blood serum and KLF4 expression in lesions was observed in atherosclerotic animals, indicating the potential function of extracellular miR-92a in regulating vascular diseases.ConclusionsEC miR-92a can be transported to macrophages through extracellular vesicles to regulate KLF4 levels, thus leading to the atheroprone phenotypes of macrophage and, hence, atherosclerotic lesion formation.

Project description:A chronic proinflammatory state precedes pathological change in arterial endothelial cells located within regions of susceptibility to atherosclerosis. The potential contributions of regulatory microRNAs to this disequilibrium were investigated by artery site-specific profiling in normal adult swine. Expression of endothelial microRNA10a (miR-10a) was lower in the athero-susceptible regions of the inner aortic arch and aorto-renal branches than elsewhere. Expression of Homeobox A1 (HOXA1), a known miR-10a target, was up-regulated in the same locations. Endothelial transcriptome microarray analysis of miR-10a knockdown in cultured human aortic endothelial cells (HAEC) identified IkappaB/NF-kappaB-mediated inflammation as the top category of up-regulated biological processes. Phosphorylation of IkappaBalpha, a prerequisite for IkappaBalpha proteolysis and NF-kappaB activation, was significantly up-regulated in miR-10a knockdown HAEC and was accompanied by increased nuclear expression of NF-kappaB p65. The inflammatory biomarkers monocyte chemotactic protein 1 (MCP-1), IL-6, IL-8, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were elevated following miR-10a knockdown. Conversely, knockin of miR-10a (a conservative 25-fold increase) inhibited the basal expression of VCAM-1 and E-selectin in HAEC. Two key regulators of IkappaBalpha degradation--mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and beta-transducin repeat-containing gene (betaTRC)--contain a highly conserved miR-10a binding site in the 3' UTR. Both molecules were up-regulated by miR-10a knockdown and suppressed by miR-10a knockin, and evidence of direct miR-10a binding to the 3' UTR was demonstrated by luciferase assay. Comparative expression studies of endothelium located in athero-susceptible aortic arch and athero-protected descending thoracic aorta identified significantly up-regulated MAP3K7, betaTRC, phopho-IkappaBalpha, and nuclear p65 expression suggesting that the differential expression of miR-10a contributes to the regulation of proinflammatory endothelial phenotypes in athero-susceptible regions in vivo.

Dataset Information

Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium.

Publications

Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium.

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