Vascular endothelial growth factor mRNA levels as a biomarker for short-termN-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinogenesis bioassay

Embryonic losses are high in mammals, with more than 30% of fertilized eggs not resulting in an offspring. The development of the placenta is critical for normal fetal growth and development as it provides for exchange of respiratory gases, nutrients, and wastes between the fetal and maternal systems. Placental vascular development determines the rate of placental blood flow, which is a primary determinant of placental function. Recent studies suggest that vascular endothelial growth factor (VEGF), its receptors (VEGFR), along with angiopoietins (Ang-1 and Ang-2) and their common receptor Tie-2, are major placental angiogenic factors, along with fibroblast growth factor-2 (FGF-2) and its receptor (FGFR). To evaluate the patterns of placental expression of these factors during early placental development, gravid uteri were obtained from ewes (n = 6 per day) on Days 12, 18, 24, 30, and 40 of gestation (day of mating = Day 0). At slaughter the uterine and embryonic tissues were weighed and representative samples of utero-placenta (CAR - caruncle, maternal placenta; ICAR - intracarunclar, endometrium; FM, fetal membranes) were snap frozen on dry ice and analyzed for relative mRNA levels by real-time RT-PCR (ABI Prism 7000, Sequence Detection System, Applied Biosystems, Monza, Italy) of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-1 (VEGFR-1), vascular endothelial growth factor receptor-2 (VEGFR-2), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), receptor for both angiopoietins (Tie-2), fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor (FGFR). The data were analyzed by nonlinear procedures using proc reg of SAS (SAS Institute, Inc., Cary, NC, USA). In CAR, the data showed the exponential increase from Days 12 to 40 in mRNA expression for VEGFR-1 (P < 0.0004; 0.04398e0.08794�day), VEGFR-2 (P < 0.01; 0.119208e0.06537�day), Ang-1 (P < 0.005; 0.00488e0.10881�day), Ang-2 (P < 0.0001; 0.01591e0.07864�day), Tie-2 (P < 0.03; 0.00488e0.06852�day), and FGFR (P < 0.08; 0.24577e0.04721�day). In the CAR, we also observed an exponential decrease in mRNA concentration for VEGF (P < 0.05; 28.193e-1.0719�day). In ICAR, we observed an exponential increase in mRNA concentration for VEGF (P < 0.05; 1.11685e0.06865�day), VEGFR-1 (P < 0.07; 0.09853e0.0383�day), Ang-1 (P < 0.09; 0.009318e0.05711�day), and Ang-2 (P < 0.004; 0.012647e0.09973�day). For FM, no changes in mRNA levels were observed from Days 12 to 40, but levels of all mRNAs were similar to those in CAR and ICAR. Based on the patterns of mRNA expression, these data indicate that these angiogenic factors may play an important role in early placental angiogenesis in sheep. This work was supported by NIH grant HL64141 to LPR and DAR.

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Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A2AReceptor Agonists and Endotoxin Involves Transcriptional Regulation via the Hypoxia Response Element in the VEGF Promoter

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0596 ◽

2007 ◽

Vol 18 (1) ◽

pp. 14-23 ◽

Cited By ~ 84

Author(s):

Madhuri Ramanathan ◽

Grace Pinhal-Enfield ◽

Irene Hao ◽

Samuel Joseph Leibovich

Keyword(s):

Vascular Endothelial Growth Factor ◽

Transcriptional Regulation ◽

Growth Factor ◽

Promoter Activity ◽

Luciferase Reporter ◽

Mrna Levels ◽

Dependent Manner ◽

Vascular Endothelial ◽

Vegf Expression ◽

Endothelial Growth Factor

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2Areceptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1α expression. HIF-1α mRNA levels were increased in LPS-treated macrophages in an NF-κB–dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1α promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1α promoter activity, indicating that the A2AR-dependent increase in HIF-1α mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1α protein and DNA binding levels. Deletion of putative NF-κB–binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-κB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1α through the HRE. HIF-1α is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.

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Up-regulation of vascular endothelial growth factor receptor Flt-1 after endothelial denudation: role of transcription factor Egr-1

Blood ◽

10.1182/blood.v95.11.3387.011k09_3387_3395 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3387-3395

Author(s):

Felipe Vidal ◽

Julián Aragonés ◽

Arántzazu Alfranca ◽

Manuel O. de Landázuri

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Tyrosine Kinase ◽

Binding Site ◽

Vascular Injury ◽

Mrna Levels ◽

Vascular Endothelial ◽

Transcription Analysis ◽

Endothelial Denudation ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is highly expressed in vascular remodeling processes and accelerates reendothelialization after mechanical denudation. Two VEGF tyrosine kinase receptors have been reported—fms-like–tyrosine kinase-1 (Flt-1) and kinase domain region (KDR). Little is known about the regulation of the expression of these receptors after vascular injury. Herein, we have analyzed the expression of Flt-1 after mechanical denudation of primary cultures of endothelial cells, which has been considered a useful in vitro model to study endothelium responses to vascular injury. After denudation, the Flt-1 protein and mRNA levels are clearly up-regulated, and transient transfection experiments showed a strong induction of theflt-1 promoter-dependent transcription. Analysis of the flt-1 promoter sequence revealed the presence of a putative binding site for the early growth response factor-1 (Egr-1) at positions −24 to −16. Electrophoretic mobility shift and supershift assays showed that Egr-1 was able to bind to this DNA sequence, and cotransfection of the flt-1 promoter reporter plasmid with an Egr-1 expression vector resulted in enhancement of its transcriptional activity. Furthermore, the mutation of the Egr-1 binding site markedly reduced the denudation-induced flt-1promoter activity. These data demonstrate that Flt-1 is up-regulated after endothelial denudation and that Egr-1 plays a relevant role in this process.

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Egr-1 gene is induced by the systemic administration of the vascular endothelial growth factor and the epidermal growth factor

Blood ◽

10.1182/blood.v96.5.1772.h8001772_1772_1781 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1772-1781

Author(s):

Lixin Liu ◽

Jo C. Tsai ◽

William C. Aird

Keyword(s):

Skeletal Muscle ◽

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factors ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Mrna Levels ◽

Vascular Endothelial ◽

Epidermal Growth ◽

Endothelial Growth Factor

Egr-1 is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. In cultured endothelial cells, the Egr-1 gene has been shown to respond to a variety of extracellular signals. However, the physiological relevance of these findings remains unclear. To address this question, the growth factor-mediated response of the Egr-1 gene under in vivo conditions was analyzed. To that end, either vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) was injected into the intraperitoneal cavity of mice. Growth factors were delivered to all tissues examined, as evidenced by the widespread distribution of I125-labeled growth factors and the phosphorylation of their respective receptors. In Western blot analyses of whole-tissue extracts, Egr-1 protein levels were shown to be induced in the heart, brain, liver, and spleen of VEGF-treated mice, and in the heart, lung, brain, liver and skeletal muscle of EGF-treated animals. Changes in Egr-1 levels did not correlate with changes in receptor phosphorylation or ERK1/2 phosphorylation. In Northern blot analyses, VEGF induced Egr-1 mRNA levels in all tissues examined except lung and kidney, whereas EGF led to increased transcripts in all tissues except kidney. In immunofluorescence studies, VEGF induced Egr-1 in microvascular endothelial cells of the heart and liver, and EGF induced Egr-1 in the microvascular bed of skeletal muscle. Taken together, these results suggest that the Egr-1 gene is differentially regulated in response to systemically administered VEGF and EGF.

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Vascular Endothelial Growth Factor, Basic Fibroblast Growth Factor, and Pigment Epithelium-Derived Factor Expression in the Neovascular Iris in Retinal Diseases

Journal of Ophthalmology ◽

10.1155/2018/8025951 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Heng Miao ◽

Xianru Hou ◽

De-Kuang Hwang ◽

Yong Tao

Keyword(s):

Vascular Endothelial Growth Factor ◽

Surgical Treatment ◽

Growth Factor ◽

Pigment Epithelium ◽

Mrna Levels ◽

Vascular Endothelial ◽

Fibroblast Growth ◽

Protein Levels ◽

Pigment Epithelium Derived Factor ◽

Endothelial Growth Factor

Objective. To determine the expression of cytokines in the iris of patients with neovascular glaucoma (NVG). Methods. Patients with NVG associated with proliferative diabetic retinopathy (PDR, group 1) or central retinal vein occlusion (CRVO, group 2) who had undergone surgical treatment were enrolled. Patients with primary open-angle glaucoma requiring surgical treatment were included in the control group (group 3). All iris specimens were obtained during trabeculectomy, 7 days after intravitreal injections of ranibizumab. The messenger RNA (mRNA) and protein levels of three target cytokines—vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and pigment epithelium-derived factor (PEDF)—in the iris were analyzed and compared. Results. We included 39 eyes from 39 patients (12, 15, and 12 in groups 1, 2, and 3, resp.). The protein and mRNA levels of PEDF were higher in two NVG groups. The protein levels, but not mRNA level, of bFGF were higher in the two NVG groups. The protein and mRNA levels of VEGF were similar in the three groups. Conclusions. The protein level of bFGF increased in the irises of the NVG patients was not expressed by the iris itself, whereas PEDF may be expressed by the iris tissue in these patients.

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Expression of vascular endothelial growth factor in the growth plate is stimulated by estradiol and increases during pubertal development

Journal of Endocrinology ◽

10.1677/joe-09-0337 ◽

2010 ◽

Vol 205 (1) ◽

pp. 61-68 ◽

Cited By ~ 10

Author(s):

Joyce Emons ◽

Andrei S Chagin ◽

Torun Malmlöf ◽

Magnus Lekman ◽

Åsa Tivesten ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Growth Plate ◽

Bone Growth ◽

Pubertal Development ◽

Mrna Levels ◽

Vascular Endothelial ◽

Epiphyseal Growth Plate ◽

Vegf Expression ◽

Endothelial Growth Factor

Longitudinal bone growth is regulated in the growth plate. At the end of puberty, growth velocity diminishes and eventually ceases with the fusion of the growth plate through mechanisms that are not yet completely understood. Vascular endothelial growth factor (VEGF) has an important role in angiogenesis, but also in chondrocyte differentiation, chondrocyte survival, and the final stages of endochondral ossification. Estrogens have been shown to up-regulate VEGF expression in the uterus and bone of rats. In this study, we investigated the relation between estrogens and VEGF production in growth plate chondrocytes both in vivo and in vitro. The expression of VEGF protein was down-regulated upon ovariectomy and was restored upon estradiol (E2) supplementation in rat growth plates. In cultured rat chondrocyte cell line RCJ3.1C5.18, E2 dose dependently stimulated 121 and 189 kDa isoforms of VEGF, but not the 164 kDa isoform. Finally, VEGF expression was observed at both protein and mRNA levels in human growth plate specimens. The protein level increased during pubertal development, supporting a link between estrogens and local VEGF production in the growth plate. We conclude that estrogens regulate VEGF expression in the epiphyseal growth plate, although the precise role of VEGF in estrogen-mediated growth plate fusion remains to be clarified.

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Egr-1 gene is induced by the systemic administration of the vascular endothelial growth factor and the epidermal growth factor

Blood ◽

10.1182/blood.v96.5.1772 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1772-1781 ◽

Cited By ~ 48

Author(s):

Lixin Liu ◽

Jo C. Tsai ◽

William C. Aird

Keyword(s):

Skeletal Muscle ◽

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factors ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Mrna Levels ◽

Vascular Endothelial ◽

Epidermal Growth ◽

Endothelial Growth Factor

Abstract Egr-1 is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. In cultured endothelial cells, the Egr-1 gene has been shown to respond to a variety of extracellular signals. However, the physiological relevance of these findings remains unclear. To address this question, the growth factor-mediated response of the Egr-1 gene under in vivo conditions was analyzed. To that end, either vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) was injected into the intraperitoneal cavity of mice. Growth factors were delivered to all tissues examined, as evidenced by the widespread distribution of I125-labeled growth factors and the phosphorylation of their respective receptors. In Western blot analyses of whole-tissue extracts, Egr-1 protein levels were shown to be induced in the heart, brain, liver, and spleen of VEGF-treated mice, and in the heart, lung, brain, liver and skeletal muscle of EGF-treated animals. Changes in Egr-1 levels did not correlate with changes in receptor phosphorylation or ERK1/2 phosphorylation. In Northern blot analyses, VEGF induced Egr-1 mRNA levels in all tissues examined except lung and kidney, whereas EGF led to increased transcripts in all tissues except kidney. In immunofluorescence studies, VEGF induced Egr-1 in microvascular endothelial cells of the heart and liver, and EGF induced Egr-1 in the microvascular bed of skeletal muscle. Taken together, these results suggest that the Egr-1 gene is differentially regulated in response to systemically administered VEGF and EGF.

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Prostaglandin E2 regulation of amnion cell vascular endothelial growth factor expression: relationship with intramembranous absorption rate in fetal sheep

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00070.2014 ◽

2014 ◽

Vol 307 (3) ◽

pp. R354-R360 ◽

Cited By ~ 6

Author(s):

Cecilia Y. Cheung ◽

Michael K. Beardall ◽

Debra F. Anderson ◽

Robert A. Brace

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Amniotic Fluid ◽

Absorption Rate ◽

Mrna Levels ◽

Vascular Endothelial ◽

Fetal Sheep ◽

Fluid Infusion ◽

Caveolin 1 ◽

Endothelial Growth Factor

We hypothesized that prostaglandin E2 (PGE2) stimulates amniotic fluid transport across the amnion by upregulating vascular endothelial growth factor (VEGF) expression in amnion cells and that amniotic PGE2 concentration correlates positively with intramembranous (IM) absorption rate in fetal sheep. The effects of PGE2 at a range of concentrations on VEGF164 and caveolin-1 gene expressions were analyzed in cultured ovine amnion cells. IM absorption rate, amniotic fluid (AF) volume, and PGE2 concentration in AF were determined in late-gestation fetal sheep during control conditions, isovolumic fetal urine replacement (low IM absorption rate), or intra-amniotic fluid infusion (high IM absorption rate). In ovine amnion cells, PGE2 induced dose- and time-dependent increases in VEGF164 mRNA levels and reduced caveolin-1 mRNA and protein levels. VEGF receptor blockade abolished the caveolin-1 response, while minimally affecting the VEGF response to PGE2. In sheep fetuses, urine replacement reduced amniotic PGE2 concentration by 58%, decreased IM absorption rate by half, and doubled AF volume ( P < 0.01). Intra-amniotic fluid infusion increased IM absorption rate and AF volume ( P < 0.01), while amniotic PGE2 concentration was unchanged. Neither IM absorption rate nor AF volume correlated with amniotic PGE2 concentration under each experimental condition. Although PGE2 at micromolar concentrations induced dose-dependent responses in VEGF and caveolin-1 gene expression in cultured amnion cells consistent with a role of PGE2 in activating VEGF to mediate AF transport across the amnion, amniotic PGE2 at physiological nanomolar concentrations does not appear to regulate IM absorption rate or AF volume.

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Antagonistic Functions of Tetradecanoyl Phorbol Acetate-Inducible-Sequence 11b and HuR in the Hormonal Regulation of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Stability by Adrenocorticotropin

Molecular Endocrinology ◽

10.1210/me.2005-0121 ◽

2006 ◽

Vol 20 (4) ◽

pp. 916-930 ◽

Cited By ~ 27

Author(s):

Nadia Cherradi ◽

Cyrille Lejczak ◽

Agnes Desroches-Castan ◽

Jean-Jacques Feige

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Nuclear Export ◽

Mrna Levels ◽

Vascular Endothelial ◽

Vegf Mrna ◽

Adrenocortical Cells ◽

Tetradecanoyl Phorbol Acetate ◽

Endothelial Growth Factor ◽

Vegf Mrna Expression

Abstract Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3′-untranslated region (3′-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3′-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3′-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

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