Shear stress-induced long non-coding RNA, NIMBUSS, is required for the KLF2-mediated gene expression profile associated with vascular homeostasis II
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ABSTRACT: To investigate the regulation of the long non-coding RNA (lncRNA) transcriptome of endothelial cells in response to shear stress, gene expression profiling was performed using a microarray for both protein-coding genes and lncRNAs. The expression of known flow-induced protein coding genes, eNOS, KLF2, and KLF4, were quantified using real-time PCR. Identifying lncRNAs that are up-regulated or down-regulated in the presence or absence of laminar flow will provide insight into gene regulatory mechanisms that contribute to vascular homeostasis or disease.
Project description:The endothelial cell (EC) response to shear stress is partly mediated by the transcription factor KLF2, which is preferentially activated by laminar flow and promotes an atheroprotective phenotype. The up-regulation of KLF2 elicits anti-inflammmatory, anti-coagulant, and pro-vasodilatory processes. To investigate the function of the flow-induced long non-coding RNA (lncRNA) NIMBUSS, we performed loss-of-function studies using siRNA for NIMBUSS in ECs overexpressing KLF2.
Project description:Endothelial cell (EC)-enriched protein coding genes, such as endothelial nitric oxide synthase (eNOS), define quintessential EC-specific physiologic functions. It is not clear whether long noncoding RNAs (lncRNAs) also define cardiovascular cell-type specific phenotypes, especially in the vascular endothelium. Here, we report the existence of a set of EC-enriched lncRNAs and define a role for STEEL (spliced transcript – endothelial enriched lncRNA) in angiogenic potential, macrovascular/microvascular identity and shear stress responsiveness. STEEL is expressed from the terminus of the HOXD locus and is transcribed antisense to HOXD transcription factors. STEEL RNA increases the number and integrity of de novo perfused microvessels in an in vivo model and augments angiogenesis in vitro. The STEEL RNA is polyadenylated, nuclear-enriched and has microvascular predominance. Functionally, STEEL regulates a number of genes in diverse endothelial cells. Of interest, STEEL upregulates both eNOS and the transcription factor Kruppel-like factor 2 (KLF2), and is subject to feedback inhibition by both eNOS and shear-augmented KLF2. Mechanistically, STEEL upregulation of eNOS and KLF2 is transcriptionally mediated, in part, via interaction of chromatin-associated STEEL with the poly-ADP ribosylase, PARP1. For instance, STEEL recruits PARP1 to the KLF2 promoter. This work identifies a role for EC-enriched lncRNAs in the phenotypic adaptation of ECs to both body position and hemodynamic forces, and establishes a newer role for lncRNAs in the transcriptional regulation of EC identity.
Project description:The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers anti-inflammatory properties to endothelial cells through inhibition of activator protein 1, presumably by interfering with MAPK cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA micro-array analysis. No gross changes in MAPKs were detected, rather phosphorylation of actin cytoskeleton-associated proteins, including Focal Adhesion Kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH2-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, we term shear fibers, which are distinct from thrombin- or TNF-α-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers. Tramscriptome profiling: Three independent isolates of Human Umbilical Vein Endothelial cells were transduced with lentiviral vectors expressing Kruppel Like Factor 2 (KLF2) or no protein (mock), and at time after transduction 24 h, 48 h, 72 h , RNA was isolated and hybridized to GPL4868 microarrays using dye swap procedure Kinome profiling: Two independent isolates of Human Umbilical Vein Endothelial cells were transduced with lentiviral vectors expressing Kruppel Like Factor 2 (KLF2) or no protein (mock), and at time after transduction 72 h , total cellular protein was isolated and hybridized to Kinexus KAM-1.1 phosphoprotein (kinexus) microarrays using dual color procedure in duplicate
Project description:Renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in the immortalized renal proximal tubular epithelial cell line. Cells were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device and compared to static controls.
Project description:The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers anti-inflammatory properties to endothelial cells through inhibition of activator protein 1, presumably by interfering with MAPK cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA micro-array analysis. No gross changes in MAPKs were detected, rather phosphorylation of actin cytoskeleton-associated proteins, including Focal Adhesion Kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH2-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, we term shear fibers, which are distinct from thrombin- or TNF-α-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.
Project description:Pkd1-/- renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in the immortalized Pkd1-/- renal proximal tubular epithelial cell line. Cells were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device and compared to static controls.
Project description:Many studies have characterised the effect of normal laminar shear stress (LSS) on endothelial responses, however elevated shear stress, as would be experienced overlying a stenotic plaque, has not been studied in depth. Therefore we used transcriptomics and related functional analyses to compare cells exposed to laminar shear stress at 15 or 75 dynes/cm2 for 24 hours (LSS15-normal or LSS75-high shear stress). Human umbilical vein endothelial cells (HUVEC n=4 per flow condition from different batches of pooled donor HUVEC p2) were cultured for 24 hours on gelatin coated slides under laminar shear stress of either 15 dynes/cm2 or 75 dynes/cm2 (LSS15 or LSS75) to assess the effect of high shear stress on endothelial cells. Total RNA was obtained using Qiagen kit and array analysis performed by Service XS (Leiden, Netherlands).
Project description:Laminar shear stress due to constant blood flow is known to play a critical role in maintaining vascular health. In contrast, endothelial cell senescence appears to be closely associated with the incidence of vascular disorder. In an attempt to identify functional biomarkers for age-related vascular health/disease, the present study investigated differential gene expression of young and senescent human umbilical vein endothelial cells (HUVECs) under static and laminar shear stress. We used a cDNA microarray method to compare gene expression profiles of young and senescent HUVECs under static and laminar shear stress conditions. Experiment Overall Design: Senescent cells were prepared by continuous subculture in vitro, and a cone-and-plate device was used to impose laminar shear stress onto cells. Young and senescent cells were exposed to laminar shear stress or maintained under static conditions. Total mRNA was extracted and gene expression profiles were analyzed by cDNA microarray.
Project description:The goal of this study was to identify gene signatures in HUVECs exposed to atheroprone low laminar shear stress (LSS) or atheroprotective high laminar shear stress (HSS). The obtained data was used to verify that HSS and LSS application induces gene expression patterns similar to more complex pulsatile and oscillatory flow, respectively.
Project description:To determine the role of STEEL in endothelial cell (EC) gene regulation, gene expression analysis was conducted on control and STEEL siRNA-treated human dermal microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs). A total of 225 protein-coding genes were downregulated and 80 were upregulated when both EC types were grouped for analysis. In HMVEC alone, 544 protein-coding genes were downregulated and 218 were upregulated. In HUVEC alone, 177 protein-coding genes were downregulated and 125 were upregulated. Prominently, STEEL siRNA depletion results in the downregulation of two notable protein-coding genes, eNOS and KLF2, which are modulated in ECs subjected to continuous laminar shear stress.