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

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.

Download Full-text

The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.

Molecular Biology of the Cell ◽

10.1091/mbc.4.12.1317 ◽

1993 ◽

Vol 4 (12) ◽

pp. 1317-1326 ◽

Cited By ~ 749

Author(s):

J E Park ◽

G A Keller ◽

N Ferrara

Keyword(s):

Extracellular Matrix ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Vascular Endothelial ◽

Vegf Mrna ◽

Angiogenic Potential ◽

Molecular Masses ◽

Matrix Bound ◽

Endothelial Growth Factor ◽

Vegf Isoforms

Vascular endothelial growth factor (VEGF)mRNA undergoes alternative splicing events that generate four different homodimeric isoforms, VEGF121, VEGF165, VEGF189, or VEGF206. VEGF121 is a nonheparin-binding acidic protein, which is freely diffusible. The longer forms, VEGF189 or VEGF206, are highly basic proteins tightly bound to extracellular heparin-containing proteoglycans. VEGF165 has intermediate properties. To determine the localization of VEGF isoforms, transfected human embryonic kidney CEN4 cells expressing VEGF165, VEGF189, or VEGF206 were stained by immunofluorescence with a specific monoclonal antibody. The staining was found in patches and streaks suggestive of extracellular matrix (ECM). VEGF165 was observed largely in Golgi apparatus-like structures. Immunogold labeling of cells expressing VEGF189 or VEGF206 revealed that the staining was localized to the subepithelial ECM. VEGF associated with the ECM was bioactive, because endothelial cells cultured on ECM derived from cells expressing VEGF189 or VEGF206 were markedly stimulated to proliferate. In addition, ECM-bound VEGF can be released into a soluble and bioactive form by heparin or plasmin. ECM-bound VEGF189 and VEGF206 have molecular masses consistent with the intact polypeptides. The ECM may represent an important source of VEGF and angiogenic potential.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.10.4.907 ◽

1999 ◽

Vol 10 (4) ◽

pp. 907-919 ◽

Cited By ~ 119

Author(s):

J. A. Dibbens ◽

D. L. Miller ◽

A. Damert ◽

W. Risau ◽

M. A. Vadas ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Vascular Endothelial ◽

Coding Region ◽

Vegf Gene ◽

Vegf Expression ◽

Vegf Mrna ◽

Rapid Degradation ◽

Multiple Regions ◽

Endothelial Growth Factor

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Glomerular endothelial cells in culture express and secrete vascular endothelial growth factor

AJP Renal Physiology ◽

10.1152/ajprenal.1994.266.1.f81 ◽

1994 ◽

Vol 266 (1) ◽

pp. F81-F88 ◽

Cited By ~ 3

Author(s):

K. Uchida ◽

S. Uchida ◽

K. Nitta ◽

W. Yumura ◽

F. Marumo ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Protein Kinase ◽

Mrna Abundance ◽

Vascular Endothelial ◽

Vegf Expression ◽

Vegf Mrna ◽

Glomerular Endothelial Cells ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a specific growth factor for endothelial cells, and its abundant expression has been reported in kidney glomeruli. In this study, we focused on glomerular endothelial cells (GEN) as a possible source of VEGF secretion and sought to uncover a potential autocrine role of VEGF for GEN. Ribonuclease protection assay demonstrated VEGF mRNA expression in cultured GEN, and 46-kDa VEGF protein was detected in the conditioned medium by immunoblot analysis using polyclonal antibody raised against the NH2-terminal portion of VEGF. Removal of fetal bovine serum (FBS) from the culture medium for 2 h decreased VEGF mRNA abundance, which was restored by the readdition of FBS (10%) within 2 h. The effect of FBS was completely abolished by protein kinase inhibitor H-7 (10 microM), suggesting that FBS-stimulated VEGF mRNA induction involves activation of protein kinases. The treatment of GEN with 10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the VEGF mRNA abundance fivefold, supporting the idea that VEGF expression is regulated by protein kinase C. [3H]thymidine incorporation into GEN treated with TPA (10(-7) M) was inhibited by neutralizing antibody for VEGF. Thus VEGF was identified as an autocrine growth factor for GEN in vitro. Its physiological role might be the regulation of GEN proliferation, and the induction of VEGF expression by FBS and TPA suggests its involvement in the response of glomerular capillary endothelial cells to injury in certain pathophysiological states.

Download Full-text

Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis

Biochemical Journal ◽

10.1042/bj3280179 ◽

1997 ◽

Vol 328 (1) ◽

pp. 179-183 ◽

Cited By ~ 59

Author(s):

Atsushi ASANO ◽

Masami MORIMATSU ◽

Hideki NIKAMI ◽

Toshihide YOSHIDA ◽

Masayuki SAITO

Keyword(s):

Vascular Endothelial Growth Factor ◽

Adipose Tissue ◽

Growth Factor ◽

Brown Adipose Tissue ◽

Cold Exposure ◽

Vascular Endothelial ◽

Vegf Expression ◽

Vegf Mrna ◽

Brown Adipose ◽

Endothelial Growth Factor

Cold exposure produces adaptive hyperplasia and growth of brown adipose tissue (BAT), the major site of non-shivering thermogenesis in rodents, associated with increased angiogenesis in this tissue. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, was found to be expressed abundantly in BAT of the rat. When rats were exposed to cold at 4 °C, the VEGF mRNA level in BAT was increased by 2-3-fold in 1-4 h, but returned to the basal level within 24 h. VEGF expression in other tissues such as heart, kidney and lung did not change after cold exposure. The cold-induced increase in VEGF mRNA was abolished by surgical sympathetic denervation, but mimicked by administration of noradrenaline or a β3-adrenoceptor agonist CL316,243, indicating the critical role of the β-adrenergic pathway in VEGF expression in BAT. Among three isoforms of VEGF, the mRNA of a short form (VEGF120) lacking heparin-binding activity was preferentially increased after cold exposure and treatment with the adrenergic agonists. These results suggest that cold exposure activates the sympathetic nerves and leads to a rapid increase in synthesis of VEGF in BAT, which in turn stimulates the proliferation of surrounding vascular endothelial cells.

Download Full-text

Alteration in angiogenic potential of granulosa-lutein cells and follicular fluid contributes to luteal defects in polycystic ovary syndrome

Human Reproduction ◽

10.1093/humrep/deaa351 ◽

2020 ◽

Author(s):

Krutika Patil ◽

Indira Hinduja ◽

Srabani Mukherjee

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cell ◽

Growth Factor ◽

Polycystic Ovary ◽

Tube Formation ◽

Vascular Endothelial ◽

Transcript Levels ◽

Angiogenic Potential ◽

Angiogenic Genes ◽

Endothelial Growth Factor

Abstract STUDY QUESTION Is angiogenic potential of follicular fluid (FF) and granulosa-lutein cells (GLCs) altered in polycystic ovary syndrome (PCOS) and does it play a role in corpus luteum (CL) defect observed in them? SUMMARY ANSWER FF and GLCs of women with PCOS show reduced expression of pro-angiogenic factors compared to controls and exhibit a diminished capacity to induce angiogenesis. WHAT IS KNOWN ALREADY In women with PCOS, CL insufficiency and frequent miscarriage are reported, which may be due to defect in CL. The development of new blood vessels is essential to promote ovarian folliculogenesis and functional CL formation. The vasculature formation in CL which is important for its function is still unexplored in these women. STUDY DESIGN, SIZE, DURATION This case-control study was conducted in 30 healthy control women and 30 women with PCOS undergoing controlled ovarian hyperstimulation for IVF. The FF, GLCs and serum were collected from all participants during ovum pick up. PARTICIPANTS/MATERIALS, SETTING, METHODS The capacity of FF to induce angiogenesis was assessed by measuring levels of pro-angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) and its tube formation and wound healing potential using human umbilical vein endothelial cells (HUVECs). We investigated the angiogenic potential and endothelial cell-like nature of GLCs using several approaches such as the expression of angiogenic genes by quantitative PCR, DiI-conjugated acetylated low-density lipoproteins (Dil-Ac-LDL) internalization assay, tube formation assay, expression of endothelial cell markers by immunofluorescence analysis. In addition, correlation of transcript levels of angiogenic genes with oocyte parameters was studied. MAIN RESULTS AND THE ROLE OF CHANCE FF and serum levels of VEGF and FGF2 were significantly higher and lower, respectively, in PCOS compared to controls. The tube formation and wound healing capacity of HUVECs was found to be reduced when measured after supplementation with FF of women with PCOS compared to controls. This suggests a decreased angiogenic capacity of FF in women with PCOS. Tube formation (P = 0.003) and Dil-Ac-LDL internalization (P = 0.03) ability of GLCs were significantly reduced in women with PCOS compared to controls. Protein expression levels of endothelial markers, vascular endothelial growth factor A (VEGFA) (P = 0.004), vascular endothelial growth factor receptor 2 (VEGFR2) (P = 0.011), TEK Receptor Tyrosine Kinase (Tie-2) (P = 0.026), fibroblast growth factor receptor 1 (FGFR1) (P = 0.026) and CD31 (P = 0.035) and transcript levels of angiogenic genes VEGFA (P = 0.042), hypoxia inducing factor 1A (HIF1A) (P = 0.025), FGF2 (P = 0.038), angiopoietin 1 (ANGPT1) (P = 0.028), heparin sulfate proteoglycan 2 (HSPG2) (P = 0.016), ADAM metallopeptidase with thrombospondin type1 motif, 1 (ADAMTS1) (P = 0.027) and fibronectin 1 (FN1) (P = 0.016) were found to be low in GLCs of PCOS compared to controls. Thus, the findings of this study indicate that endothelial cell-like characteristics of GLCs were significantly decreased in PCOS. Furthermore, transcript levels of VEGFA (r = 0.46, P = 0.009), ADAMTS1 (r = 0.55, P = 0.001), FGF2 (r = 0.42, P = 0.022) and ANGPT2 (r = 0.47, P = 0.008) showed a positive correlation with oocyte fertilization rate. LIMITATIONS, REASONS FOR CAUTION The vasculature formation in CL is not possible to study in women, but we explored the angiogenic characteristics of FF and GLC obtained from women with PCOS to speculate any vascularization defect of CL in these women. The FF and GLCs were obtained from the stimulated cycle during oocyte retrieval, which may not exactly mimic the in-vivo condition. The small sample size is another limitation of this study. Larger sample size and support by color Doppler studies on CL blood flow would help to strengthen our findings. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that the altered angiogenic potential of FF and GLCs may affect vasculature development required for CL formation and function in PCOS. These findings pave the way to devise therapeutic strategies to support angiogenesis process in follicle of women with PCOS, which may improve CL insufficiency, progesterone levels and prevent frequent miscarriages in these women. Furthermore, our study also hypothesizes that the vascularization around the ovarian follicles is also compromised which may lead to the growth arrest of the follicles in PCOS, however, this needs thorough investigations. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Grant BT/PR16524/MED/97/346/2016 from the Department of Biotechnology, Government of India. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A

Download Full-text