Hypoxic Regulation of Vascular Endothelial Growth Factor mRNA Stability Requires the Cooperation of Multiple RNA Elements

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3′ untranslated region (3′UTR), but also contains destabilizing elements in the 5′UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5′UTR, coding region, and 3′UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.

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All-Trans Retinoic Acid Inhibits Vascular Endothelial Growth Factor Expression in a Cell Model of Neutrophil Activation

Endocrinology ◽

10.1210/en.2005-0854 ◽

2006 ◽

Vol 147 (3) ◽

pp. 1264-1270 ◽

Cited By ~ 23

Author(s):

Meng Kian Tee ◽

Jean-Louis Vigne ◽

Robert N. Taylor

Keyword(s):

Vascular Endothelial Growth Factor ◽

Retinoic Acid ◽

Growth Factor ◽

Vascular Endothelial ◽

Vegf Gene ◽

Vegf Mrna ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Neutrophil Differentiation ◽

Endothelial Growth Factor

Infiltrating neutrophil granulocytes are a particularly rich source of vascular endothelial growth factor (VEGF) in the endometrium and may contribute to the angiogenesis of endometriosis lesions. The objective of this study is to evaluate the expression and regulation of VEGF in endometrial neutrophils and in a model of neutrophil differentiation relevant to endometriosis. Immunohistochemistry was performed on endometriosis patient biopsies and cultured neutrophil-like HL-60 cells were assessed. The study was set in a reproductive biology division within an academic medical center. Endometrial biopsies were performed on women with endometriosis and HL-60 cells were treated with all-trans retinoic acid (atRA) and dimethyl sulfoxide in vitro. Immunofluorescence histochemistry, VEGF mRNA and protein quantification, and transfection studies of VEGF gene promoter-luciferase constructs were all main outcome measures. Immunofluorescence studies verified the presence of neutrophils in eutopic endometrium from women with endometriosis. Examination of the regulation of VEGF using differentiated HL-60 cells as a model, revealed that atRA induced a dose- and time-dependent suppression of VEGF mRNA and protein. Transient transfection, truncation, EMSA, and site-directed mutagenesis of human VEGF promoter-luciferase constructs in HL-60 cells indicated that atRA repressed VEGF gene transcription via a direct repeat 1 element located between −443 and −431 bp relative to the transcription initiation site. Because retinoic acid is synthesized de novo in endometrial cells under the influence of progesterone, our findings suggest that the up-regulated VEGF and angiogenesis in tissue from women with endometriosis may reflect failure of neutrophil differentiation in these cases, and provide a rationale for retinoid therapy in this condition.

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Lentivirus-Mediated RNA Interference Targeting Vascular Endothelial Growth Factor Gene Enhances the Sensitivity of K562 Cells to Imatinib

Blood ◽

10.1182/blood.v112.11.4632.4632 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4632-4632

Author(s):

Li Li ◽

Ri Zhang ◽

Jiannong Cen ◽

Ziling Zhu

Keyword(s):

Vascular Endothelial Growth Factor ◽

Rna Interference ◽

Growth Factor ◽

Cellular Proliferation ◽

K562 Cells ◽

Imatinib Treatment ◽

Vascular Endothelial ◽

Vegf Gene ◽

Vegf Mrna ◽

Endothelial Growth Factor

Abstract Background: Vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis, plays an important role in tumor cell proliferation and migration. Elevated VEGF levels are found in both the plasma and the bone marrow of patients with chronic myelogeneous leukemia(CML). Moreover, the CML-associated oncogene Bcr-Abl induces endogenous secretion of VEGF. The aim of this study was to construct a lentiviral vector-mediated RNA interference (RNAi) of vascular endothelial growth factor (VEGF) gene and to investigate the effects of RNAi inhibiting VEGF gene alone or in combination with imatinib on the proliferation and apoptosis of K562 leukemic cell line. Methods: A lentiviral expression vector containing short hairpin RNA (shRNA ) targeting VEGF was constructed and cotransfected with the pPACKH1 packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blot and ELISA. Cellular proliferation was determined by trypan blue exclusion and MTT assay. imatinib -induced apoptosis was analyzed by flow cytometry. Results: The lentiviral shRNA vector targeting VEGF has been constructed and transfected into K562 cells successfully. The expression of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562–con cells (mock transfection). The proliferation rate of K562-shVEGF cells slowed down. After imatinib treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562–con cells. Conclusion: Suppression of VEGF by lentivirus-mediated RNAi could effectively inhibit proliferation and increase the sensitivity of K562 cells to imatinib. These results indicate that Bcr/Abl-targeting compound in combination with anti-angiogenic therapies may provide a potent therapeutic strategy to CML.

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BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1α, through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin

Blood ◽

10.1182/blood-2002-01-0109 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3767-3775 ◽

Cited By ~ 208

Author(s):

Matthias Mayerhofer ◽

Peter Valent ◽

Wolfgang R. Sperr ◽

James D. Griffin ◽

Christian Sillaber

Keyword(s):

Gene Expression ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Mammalian Target Of Rapamycin ◽

Hypoxia Inducible Factor ◽

Vascular Endothelial ◽

Vegf Gene ◽

Vegf Mrna ◽

Phosphoinositide 3 Kinase ◽

Endothelial Growth Factor

Recent data suggest that vascular endothelial growth factor (VEGF), a cytokine involved in autocrine growth of tumor cells and tumor angiogenesis, is up-regulated and plays a potential role in myelogenous leukemias. In chronic myelogenous leukemia (CML), VEGF is expressed at high levels in the bone marrow and peripheral blood. We show here that the CML-associated oncogene BCR/ABL induces VEGF gene expression in growth factor–dependent Ba/F3 cells. Whereas starved cells were found to contain only baseline levels of VEGF mRNA, Ba/F3 cells induced to express BCR/ABL exhibited substantial amounts of VEGF mRNA. BCR/ABL also induced VEGF promoter activity and increased VEGF protein levels in Ba/F3 cells. Moreover, BCR/ABL was found to promote the expression of functionally active hypoxia-inducible factor-1 (HIF-1), a major transcriptional regulator of VEGF gene expression. BCR/ABL-induced VEGF gene expression was counteracted by the phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 and rapamycin, an antagonist of mammalian target of rapamycin (mTOR), but not by inhibition of the mitogen-activated protein kinase pathway. Similarly, BCR/ABL-dependent HIF-1α expression was inhibited by the addition of LY294002 and rapamycin. Together, our data show that BCR/ABL induces VEGF- and HIF-1α gene expression through a pathway involving PI3-kinase and mTOR. BCR/ABL-induced VEGF expression may contribute to the pathogenesis and increased angiogenesis in CML.

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Enhanced Vascular Endothelial Growth Factor Gene Expression in Ischaemic Skin of Critical Limb Ischaemia Patients

International Journal of Vascular Medicine ◽

10.1155/2012/691528 ◽

2012 ◽

Vol 2012 ◽

pp. 1-4 ◽

Cited By ~ 1

Author(s):

Silvia Bleda ◽

Joaquín de Haro ◽

Francisco Acin ◽

César Varela ◽

Leticia Esparza

Keyword(s):

Gene Expression ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Critical Limb Ischaemia ◽

Control Group ◽

Venous Disease ◽

Vascular Endothelial ◽

Vegf Gene ◽

Vegf Expression ◽

Endothelial Growth Factor

Objectives. To perform a quantitative analysis of the vascular endothelial growth factor (VEGF) gene transcription in the skin of ischemic legs and provide information for VEGF in the pathogenesis in critical limb ischemia (CLI).Methods. Skin biopsies were obtained from 40 patients with CLI. Control samples came from 44 patients with chronic venous disease. VEGF gene expression was analysed using quantitative polymerase chain reaction.Results. Patients with CLI had higher skin VEGF expression than control group (RQ: 1.3 ± 0.1 versus 1,P=0.04).Conclusions. We found an association between ischemic skin and an elevated VEGF expression in legs from patients with CLI. These data support that the mechanism for VEGF upregulation in hypoxia conditions is intact and acts appropriately in the ischaemic limbs from patients with CLI.

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Effects of high glucose on vascular endothelial growth factor expression in vascular smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.5.h2224 ◽

1997 ◽

Vol 273 (5) ◽

pp. H2224-H2231 ◽

Cited By ~ 23

Author(s):

Rama Natarajan ◽

Wei Bai ◽

Linda Lanting ◽

Noe Gonzales ◽

Jerry Nadler

Keyword(s):

Vascular Endothelial Growth Factor ◽

Smooth Muscle ◽

Growth Factor ◽

Vascular Smooth Muscle Cells ◽

Vascular Endothelial ◽

Ang Ii ◽

Vegf Expression ◽

Vegf Mrna ◽

Mrna And Protein Expression ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF), in addition to its growth-promoting effects on endothelial cells, can also increase vascular permeability and monocyte migration. It has therefore been implicated in the pathogenic neovascularization associated with diabetic retinopathy and atherosclerosis. However, the factors regulating VEGF expression in the vascular wall are not fully understood. In this study, we examined the regulation of VEGF expression in vascular smooth muscle cells (VSMC) by hyperglycemia as well as by angiotensin II (ANG II). We also examined whether the 12-lipoxygenase (12-LO) product 12-hydroxyeicosatetraenoic acid (12-HETE) can alter VEGF expression, since 12-LO products of arachidonic acid have angiogenic properties, and ANG II as well as high glucose (HG, 25 mM) can increase 12-LO activity and expression in VSMC. Studies were carried out in human (HSMC) or porcine VSMC (PSMC), which were cultured for at least two passages under normal glucose (NG, 5.5 mM) or HG conditions. HG culture alone increased the expression of VEGF mRNA and protein in both HSMC and PSMC. Furthermore, ANG II treatment significantly induced VEGF mRNA and protein expression only in VSMC cultured in HG and not NG. In addition, 12-HETE significantly increased VEGF mRNA and protein expression in HSMC cultured in NG as well as in HG. Cells cultured in HG also secreted significantly greater amounts of VEGF into the culture medium. These results suggest that elevated VEGF production under HG conditions may play a role in the accelerated vascular disease observed in diabetes.

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Expression of Vascular Endothelial Growth Factor in Human Monocyte/Macrophages Stimulated with Lipopolysaccharide

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612921 ◽

2001 ◽

Vol 85 (01) ◽

pp. 171-176 ◽

Cited By ~ 48

Author(s):

Hiroyuki Itaya ◽

Hidemi Yoshida ◽

Masayuki Koyama ◽

Sohei Suzuki ◽

Kei Satoh ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Map Kinase ◽

Human Monocyte ◽

P38 Map Kinase ◽

Vascular Endothelial ◽

Vegf Expression ◽

Vegf Mrna ◽

Endothelial Growth Factor

SummaryVascular endothelial growth factor (VEGF) is a mitogen for endothelial cells. We have studied the production of VEGF by human macrophages in response to lipopolysaccharide (LPS). Macrophages stimulated with LPS expressed VEGF mRNA and protein in concentration- and time-dependent manners. The LPS-induced expression of VEGF was inhibited by cycloheximide pretreatment, which suggested that synthesis of certain factor(s) is required for the LPS activity. The induction of VEGF was also suppressed by SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase. These results suggest that the LPS-induced VEGF expression depends on the p38-mediated expression of c-Jun, which constitutes the AP-1 complex and binds to the AP-1 site in the VEGF promoter. Pretreatment of the cells with dexamethasone did not affect the LPS-induced upregulation of VEGF mRNA but strongly inhibited VEGF protein production, and the involvement of posttranscriptional regulation on VEGF expression by dexamethasone was suggested. The conditioned medium of LPS-stimulated macrophages enhanced the growth of cultured endothelial cells and it was inhibited by an antibody against VEGF. We conclude that macrophages produce VEGF in response to the stimulation with LPS, which may be partly mediated by the p38 MAP kinase pathway.

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Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion

Reproductive Sciences ◽

10.1177/1933719118765979 ◽

2018 ◽

Vol 26 (10) ◽

pp. 1351-1359 ◽

Cited By ~ 1

Author(s):

Cecilia Y. Cheung ◽

Debra F. Anderson ◽

Marion Rouzaire ◽

Loïc Blanchon ◽

Vincent Sapin ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Retinoic Acid ◽

Growth Factor ◽

Mrna Levels ◽

Vascular Endothelial ◽

Gene Promoters ◽

Vegf Expression ◽

Vegf Mrna ◽

Pregnant Sheep ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) has been proposed as an important regulator of amniotic fluid absorption across the amnion into the fetal vasculature on the surface of the placenta. However, the activators of VEGF expression and action in the amnion have not been identified. Using the pregnant sheep model, we aimed to investigate the presence of the retinoic acid (RA) pathway in ovine amnion and to determine its effect on VEGF expression. Further, we explored relationships between RA receptors and VEGF and tested the hypothesis that RA modulates intramembranous absorption (IMA) through induction of amnion VEGF in sheep fetuses subjected to altered IMA rates. Our study showed that RA receptor isoforms were expressed in sheep amnion, and RA response elements (RAREs) were identified in ovine RARβ and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARβ messenger RNA (mRNA) and increased VEGF transcript levels. In sheep fetuses, increases in IMA rate was associated with elevated VEGF mRNA levels in the amnion but not in the chorion. Further, RARβ mRNA was positively correlated with VEGF mRNA levels in the amnion and not chorion. We conclude that an RA pathway is present in ovine fetal membranes and that RA is capable of inducing VEGF. The finding of a positive relationship between amnion VEGF and RARβ during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.

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Tristetraprolin Inhibits Ras-dependent Tumor Vascularization by Inducing Vascular Endothelial Growth Factor mRNA Degradation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0570 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4648-4658 ◽

Cited By ~ 72

Author(s):

Khadija Essafi-Benkhadir ◽

Cercina Onesto ◽

Emmanuelle Stebe ◽

Christoph Moroni ◽

Gilles Pagès

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Antitumor Agent ◽

Mrna Degradation ◽

Vascular Endothelial ◽

Vegf Expression ◽

Vegf Mrna ◽

Angiogenic Potential ◽

Angiogenic Switch ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and overexpression of VEGF are common denominators of tumors from different origins. We have established a new link between these two fundamental observations converging on VEGF mRNA stability. In this complex phenomenon, tristetraprolin (TTP), an adenylate and uridylate-rich element-associated protein that binds to VEGF mRNA 3′-untranslated region, plays a key role by inducing VEGF mRNA degradation, thus maintaining basal VEGF mRNA amounts in normal cells. ERKs activation results in the accumulation of TTP mRNA. However, ERKs reduce the VEGF mRNA-destabilizing effect of TTP, leading to an increase in VEGF expression that favors the angiogenic switch. Moreover, TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice. As a consequence, TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on VEGF mRNA. Determination of TTP and ERKs status would provide useful information for the evaluation of the angiogenic potential in human tumors.

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Statins Augment Vascular Endothelial Growth Factor Expression in Osteoblastic Cells via Inhibition of Protein Prenylation

Endocrinology ◽

10.1210/en.2002-220682 ◽

2003 ◽

Vol 144 (2) ◽

pp. 681-692 ◽

Cited By ~ 145

Author(s):

Toyonobu Maeda ◽

Tetsuya Kawane ◽

Noboru Horiuchi

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Mrna Expression ◽

Osteoblastic Cells ◽

Vascular Endothelial ◽

Protein Prenylation ◽

Vegf Expression ◽

Vegf Mrna ◽

Endothelial Growth Factor ◽

Vegf Mrna Expression

Statins such as simvastatin are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that inhibit cholesterol synthesis. We presently investigated statin effects on vascular endothelial growth factor (VEGF) expression in osteoblastic cells. Hydrophobic statins including simvastatin, atorvastatin, and cerivastatin–but not a hydrophilic statin, pravastatin–markedly increased VEGF mRNA abundance in nontransformed osteoblastic cells (MC3T3-E1). Simvastatin (10−6m) time-dependently augmented VEGF mRNA expression in MC3T3-E1 cells, mouse stromal cells (ST2), and rat osteosarcoma cells (UMR-106). According to heterogeneous nuclear RNA and Northern analyses, 10−6m simvastatin stimulated gene expression for VEGF in MC3T3-E1 cells without altering mRNA stability. Transcriptional activation of a VEGF promoter-luciferase construct (−1128 to +827), significantly increased by simvastatin administration. As demonstrated by gel mobility shift assay, simvastatin markedly enhanced the binding of hypoxia-responsive element-protein complexes. These results indicate that the stimulation of the VEGF gene by simvastatin in MC3T3-E1 cells is transcriptional in nature. VEGF secretion into medium was increased in MC3T3-E1 by 10−6m simvastatin. Pretreating MC3T3-E1 cells with mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced VEGF mRNA expression; manumycin A, a protein prenylation inhibitor, mimicked statin effects on VEGF expression. The effect of simvastatin was blocked by pretreatment with wortmannin and LY294002, specific phosphatidylinositide-3 kinase inhibitors. Simvastatin enhanced mineralized nodule formation in culture, whereas coincubation with mevalonate, geranylgeranyl pyrophosphate, LY294002, or VEGF receptor 2 inhibitor (SU1498) abrogated statin-induced mineralization. Thus, statins stimulate VEGF expression in osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation.

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Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3713 ◽

2003 ◽

Vol 50 (1) ◽

pp. 49-59 ◽

Cited By ~ 151

Author(s):

Hideo Kimura ◽

Hiroyasu Esumi

Keyword(s):

Nitric Oxide ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Heme Oxygenase 1 ◽

No Production ◽

Vascular Endothelial ◽

Vegf Gene ◽

Vegf Expression ◽

Endothelial Growth Factor

Physiologically, angiogenesis is tightly regulated, or otherwise it leads to pathological processes, such as tumors, inflammatory diseases, gynecological diseases and diabetic retinopathy. The vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis. The VEGF gene expression is regulated by a variety of stimuli. Hypoxia is one of the most potent inducers of the VEGF expression. The hypoxia inducible factor 1 (HIF-1) plays as a key transcription factor in hypoxia-mediated VEGF gene upregulation. Nitric oxide (NO) as well as hypoxia is reported to upregulate the VEGF gene by enhancing HIF-1 activity. The Akt/protein kinase B (PKB) pathway may be involved in NO-mediated HIF-1 activation in limited cell lines. There are some reports of negative effects of NO on HIF-1 and VEGF activity. These conflicting data of NO effects may be attributed mainly to the amount of released NO. Indeed, NO can be a positive or negative modulator of the VEGF gene under the same conditions simply by changing its amounts. The VEGF-mediated angiogenesis requires NO production from activated endothelial NO synthase (eNOS). Activation of eNOS by VEGF involves several pathways including Akt/PKB, Ca(2+)/calmodulin, and protein kinase C. The NO-mediated VEGF expression can be regulated by HIF-1 and heme oxygenase 1 (HO-1) activity, and the VEGF-mediated NO production by eNOS can be also modulated by HIF-1 and HO-1 activity, depending upon the amount of produced NO. These reciprocal relations between NO and VEGF may contribute to regulated angiogenesis in normal tissues.

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