Dataset Information

Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing.

ABSTRACT: sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5-1% O?) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O? concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3'-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3'-end processing.

SUBMITTER: Ikeda T

PROVIDER: S-EPMC3133880 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing.

Ikeda Takayuki T Sun Li L Tsuruoka Naoki N Ishigaki Yasuhito Y Yoshitomi Yasuo Y Yoshitake Yoshino Y Yonekura Hideto H

The Biochemical journal 20110601 2

sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5-1% O₂) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O₂ concentration fell, whereas mFlt-1 (m ...[more]

PMID: 21382012

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Project description:BackgroundAdaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells.Methodology/principal findingsHuman umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max).Conclusions/significanceFor the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.

Project description:Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8-12 weeks of gestation, n?=?6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5-20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both physiological and pathological conditions.

Dataset Information

Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing.

Publications

Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing.

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