Vascular Endothelial Growth Factor mRNA Stability Research Articles

AMP-Activated Protein Kinase Signaling Stimulates VEGF Expression and Angiogenesis in Skeletal Muscle

AMP-activated protein kinase (AMPK) is regulated by various cellular stresses. Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is also upregulated by several stress-inducible factors such as hypoxia and stimulation by cytokines and growth factors. Here, we investigated whether AMPK signaling in muscle has a role in regulating VEGF-mediated angiogenic processes. AICAR stimulated VEGF mRNA and protein levels in C2C12 myotube cultures. Transduction with dominant-negative AMPK abolished AICAR-induced VEGF expression at both steady state mRNA and protein levels. AICAR increased VEGF mRNA stability without affecting VEGF promoter activity. AICAR also stimulated p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Activation of p38 MAPK was suppressed by transduction with dominant-negative AMPK, suggesting that AMPK is upstream of p38 MAPK. The p38 MAPK inhibitor SB203580 blocked AICAR-induced increase in VEGF mRNA and protein levels, indicating that AICAR-mediated VEGF induction is dependent on p38 MAPK signaling. AICAR treatment increased VEGF expression and accelerated angiogenic repair of ischemic hindlimbs in mice in an AMPK-dependent manner. These data indicate that AMPK-p38 MAPK signaling cascade can increase VEGF production in muscle and promote angiogenesis in response to ischemic injury.

Oncostatin M causes VEGF release from human airway smooth muscle: synergy with IL-1β

Vascular endothelial growth factor (VEGF), a potent angiogenesis factor, likely contributes to airway remodeling in asthma. We sought to examine the effects and mechanism of action of IL-6 family cytokines on VEGF release from human airway smooth muscle (HASM) cells. Oncostatin M (OSM), but not other IL-6 family cytokines, increased VEGF release, and IL-1beta enhanced OSM-induced VEGF release. OSM increased VEGF mRNA expression and VEGF promoter activity, whereas IL-1beta had no effect. IL-1beta did not augment the effects of OSM on VEGF promoter activity but did augment OSM-induced VEGF mRNA expression and mRNA stability. The STAT3 inhibitor piceatannol decreased both OSM-induced VEGF release and synergy between OSM and IL-1beta, without affecting responses to IL-1beta alone. Piceatannol also inhibited OSM-induced VEGF mRNA expression. In contrast, inhibitors of MAPK pathway had no effect on OSM or OSM plus IL-1beta-induced VEGF release. OSM increased type 1 IL-1 receptor (IL-1R1) mRNA expression, as measured by real-time PCR, and piceatannol attenuated this response. Consistent with the increase in IL-1R1 expression, OSM markedly augmented IL-1beta-induced VEGF, MCP-1, and IL-6 release. In summary, our data indicate OSM causes VEGF expression in HASM cells by a transcriptional mechanism involving STAT3. IL-1beta also synergizes with OSM to increase VEGF release, likely as a result of effects of IL-1beta on VEGF mRNA stability as well as effects of OSM on IL-1R1 expression. This is the first description of a role for OSM on IL-1R1 expression in any cell type. OSM may contribute to airway remodeling observed in chronic airway disease.

New horizons for VEGF. Is there a role for nuclear localization?

Angiogenesis, or new blood vessel formation, is a physiological response of tissues to hypoxia or ischemia. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that is up-regulated by hypoxia. The mechanisms responsible for hypoxic induction of VEGF are still not completely understood, though both transcriptional and post-transcriptional mechanisms are involved. In recent years, we have investigated cis-regulatory sequences and trans-acting factors which mediate the hypoxia-induced increase in VEGF mRNA stability. In particular, we have identified a 40 bp sequence motif in the 3'-untranslated region of VEGF mRNA, which is critical for the increase in VEGF mRNA stability with hypoxia and have shown that the RNA-binding protein, HuR, binds to this region. By means of indirect immunofluorescence experiments using monoclonal antibodies against HuR, we demonstrated that HuR localizes to the nucleus under hypoxia. As HuR binds to VEGF mRNA and appears to mediate stabilization of VEGF mRNA, it was of interest to show whether a fraction of VEGF protein localizes similarly to the nucleus. Double-labeling immunofluorescence showed that VEGF protein colocalizes with HuR in discrete nuclear compartments and nuclear VEGF protein was increased in hypoxia. These results indicate that VEGF may have a nuclear function, especially during hypoxia.

Translational regulation of vascular endothelial growth factor expression in renal epithelial cells by angiotensin II

ANG II regulates growth factor expression in the kidney. We investigated whether ANG II regulated vascular endothelial growth factor (VEGF) synthesis in proximal tubular epithelial (MCT) cells. ANG II (1 nM) increased VEGF protein expression within 5 min, the effect lasting for 30 min. There was no change in VEGF mRNA levels or mRNA stability, and transcription inhibitors did not affect ANG II-induced VEGF expression. Regulation of VEGF translation was investigated. Polyribosomal analysis revealed selective enrichment of heavy ribosomes (polysomes) with VEGF mRNA transcripts compared with light ribosomes in ANG II-treated cells, although distribution of GAPDH was unaltered. In vitro translation of total RNA from polysomal fractions showed selective increase in VEGF protein synthesis in ANG II-treated cells. Preincubation with LY-294002, a PI 3-kinase inhibitor, or expression of dominant-negative Akt prevented ANG II-stimulated increase in VEGF translation. ANG II increased phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1, critical events that regulate the initiation phase of protein translation. ANG II failed to increase VEGF mRNA translation in cells stably expressing the phosphorylation mutant of 4E-BP1. Our data illustrate that a rapid increase in VEGF protein expression by ANG II is regulated at the initiation phase of translation of VEGF mRNA in renal epithelial cells. Regulation of VEGF translation by ANG II represents a novel pathway of renal response to injury.

Poly(A)-binding Protein-interacting Protein 2, a Strong Regulator of Vascular Endothelial Growth Factor mRNA

Expression of vascular endothelial growth factor (VEGF) is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3'-untranslated region (3'-UTR) of VEGF mRNA. To identify new protein partners regulating VEGF mRNA stability, we screened a cDNA expression library with an RNA probe corresponding to the entire VEGF mRNA 3'-UTR. We identified the "poly(A)-binding protein-interacting protein 2" (PAIP2) as a new VEGF mRNA 3'-UTR interacting protein. By RNA electromobility shift assays, we showed that PAIP2 binds to two distinct regions of a domain encompassing base 1 to 1280 of the VEGF 3'-UTR. Such in vitro interaction was confirmed using cell extracts in which PAIP2 expression is induced by tetracycline (Tet-on cells). Moreover, we demonstrated by RNA affinity purification as well as by ribonucleoprotein complexes immunoprecipitation, that PAIP2 interacts with VEGF mRNA in vivo. Using an in vitro RNA degradation assay, the half-life of VEGF 3'-UTR was found to be increased by overexpressing PAIP2. PAIP2 stabilizes endogenous VEGF mRNA in Tet-on cells, leading to increased VEGF secretion. Moreover, RNAi-mediated knock-down of PAIP2 significantly reduces the steady-state levels of endogenous VEGF mRNA. We also showed, by in vitro protein-protein interactions and co-immunoprecipitation experiments, that PAIP2 interacts with HuR, an already known VEGF mRNA-binding protein, suggesting cooperation of both proteins for VEGF mRNA stabilization. Hence, PAIP2 appears to be a crucial regulator of VEGF mRNA and as a consequence, any variation in its expression could modulate angiogenesis.

Bucillamine induces the synthesis of vascular endothelial growth factor dose-dependently in systemic sclerosis fibroblasts via nuclear factor-kappaB and simian virus 40 promoter factor 1 pathways.

The pathogenesis of systemic sclerosis (SSc) is characterized by activation of the immune system, impaired angiogenesis, and activated dermal fibroblasts. The effects of the immunosuppressive agent bucillamine (SA 96) on fibroblasts and angiogenic factors have not been examined. SA 96, and particularly its metabolite SA 981, increased the levels of vascular endothelial growth factor (VEGF) mRNA and protein dose-dependently in dermal fibroblasts from patients with SSc and healthy control subjects without influencing cell viability. SSc fibroblast cultures showed consistently a higher inducibility of VEGF than cultures from healthy control subjects. Preincubation with the SP-1 inhibitor mithramycin as well as blockade of nuclear factor (NF)-kappaB signaling with pyrrolidine dithiocarbamate treatment and IkappaB transfection reduced significantly the transcription of VEGF, indicating that both transcription factors contribute to the activation of VEGF by SA 981. Specific binding of NF-kappaB protein to its binding site after treatment with SA 981 was confirmed by electrophoretic mobility shift assay. In contrast, SA 981 did not influence the stability of VEGF mRNA as analyzed with actinomycin D assays. The study provides evidence for a role of NF-kappaB in the transcriptional regulation of the VEGF gene. SA 96 might have positive aspects on the impaired angiogenesis in patients with SSc.

A multi-protein complex containing cold shock domain (Y-box) and polypyrimidine tract binding proteins forms on the vascular endothelial growth factor mRNA. Potential role in mRNA stabilization.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and post-transcriptional regulation plays a major role in VEGF expression. Both the 5'- and 3'-UTR are required for VEGF post-transcriptional regulation but factors binding to functional sequences within the 5'-UTR have not been fully characterized. We report here the identification of complexes, binding to the VEGFmRNA 5'- and 3'-UTR, that contain cold shock domain (CSD) and polypyrimidine tract binding (PTB) RNA binding proteins. Analysis of the CSD/PTB binding sites revealed a potential role in VEGF mRNA stability, in both noninduced and induced conditions, demonstrating a general stabilizing function. Such a stabilizing mechanism had not been reported previously for the VEGF gene. We further found that the CSD/PTB-containing complexes are large multiprotein complexes that are most likely preformed in solution and we demonstrate that PTB is associated with the VEGF mRNA in vivo. Complex formation between CSD proteins and PTB has not been reported previously. Analysis of the CSD/PTB RNA binding sites revealed a novel CSD protein RNA recognition site and also demonstrated that CSD proteins may direct the binding of CSD/PTB complexes. We found the same complexes binding to an RNA-stabilizing element of another growth factor gene, suggesting a broader functional role for the CSD/PTB complexes. Finally, as the VEGF gene is also regulated at the transcriptional level by CSD proteins, we propose a combined transcriptional/post-transcriptional role for these proteins in VEGF and other growth factor gene regulation.

Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity.

We have recently reported that bcl-2 overexpression and hypoxia synergistically interact to modulate vascular endothelial growth factor (VEGF) and in vivo angiogenesis in tumour cells through VEGF mRNA stabilization and hypoxia-inducible factor 1-mediated transcriptional activity. Bcl-2 antisense treatment has shown promising clinical results in patients with malignant melanoma. In the present study, we demonstrated that the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 inhibits bcl-2 expression and angiogenesis in two bcl-2 overexpressing clones derived from the M14 human melanoma cell line. The antiangiogenic effect was determined in in vitro and in vivo angiogenesis assays. In particular, a reduction of hypoxia-induced VEGF secretion was observed after 4625 treatment, and the conditioned medium (CM) of bcl-2 overexpressing clones treated with 4625 and exposed to hypoxic conditions resulted in decreased endothelial cell proliferation when compared to CM of untreated control cells. In addition, we found that CM of 4625 antisense-treated bcl-2 transfectants inhibited in vivo vessel formation in matrigel plugs implanted subcutaneously in C57/B16 mice. Our findings confirm that bcl-2 plays a crucial role in melanoma angiogenesis and demonstrate for the first time that downregulation of bcl-2 by antisense treatment has potential to inhibit angiogenesis independent of its effect on cell survival. The use of 4625 in cancer therapy is suggested as an approach to facilitate simultaneously tumour cell apoptosis and inhibit tumour angiogenesis.

Post‐transcriptional regulation of VEGF expression by oxidised LDL in human macrophages

Accumulation of oxidised low density lipoproteins (oxLDL) in macrophages and their subsequent transformation into lipid-filled foam cells is generally considered an early event in atherosclerosis. Vascular Endothelial Growth Factor (VEGF) may contribute to atherogenesis through increased vascular permeability, chemoattraction towards monocytes and intraplaque vessel formation. In this study we investigate the effect and regulation of VEGF expression in human macrophages stimulated with oxLDL. Vascular Endothelial Growth Factor mRNA and protein expression was assayed using RT-PCR and ELISA, respectively. The activity of mitogen-activated protein kinases (MAPKs) was investigated using Western blots. In human monocyte-derived macrophages, oxLDL significantly increased VEGF mRNA expression and subsequent protein secretion in a concentration-dependent manner after 6 h and 18 h, respectively. Using an in vitro mRNA decay assay, we show that this oxLDL-induced VEGF expression partly is regulated through increased stability of the VEGF mRNA. Involvement of MAPKs has previously been implicated in the stabilisation of VEGF mRNA. Activity of the p38 MAPK, but not the c-jun-N-terminal kinase (JNK), increased in macrophages stimulated with oxLDL (50 micro g mL-1) for 5-15 min. Preincubation with SB202190 (20 micro M), a specific inhibitor of p38 MAPK, significantly decreased the oxLDL-induced VEGF mRNA expression by 40%. The prolonged half-life of VEGF mRNA, induced by oxLDL, was not inhibited by SB202190. OxLDL increases VEGF expression and p38 MAPK activity in human macrophages. The increased VEGF mRNA expression by oxLDL is mediated through at least two intracellular pathways, one involving p38 MAPK and another that independently of p38 MAPK activity increases VEGF mRNA stability.

Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity.

The aim of this paper was to study the molecular mechanisms by which bcl-2 increases hypoxia-induced vascular endothelial growth factor (VEGF) expression. We demonstrated that bcl-2 overexpression in M14 human melanoma cell line enhances hypoxia-induced VEGF mRNA stability and promoter activation. In particular, the half-life of the message was longer in bcl-2 transfectants (approximately 330 min) than in control cells (approximately 180 min). In addition, bcl-2 overexpression increased VEGF promoter activity through the hypoxia-inducible factor-1 (HIF-1) transcription factor. Increased HIF-1a protein expression and DNA binding activity were detected in bcl-2 overexpressing cells compared with control cells. An enhanced functional activity of secreted VEGF was found both in in vitro and in vivo angiogenic assays, and the use of VEGF specific antibodies validated the role of VEGF on bcl-2-induced angiogenesis. Taken together our results indicate that bcl-2 plays an important role in melanoma angiogenesis, and that VEGF mRNA stabilization and HIF-1-mediated transcriptional activity are two important control points in bcl-2/hypoxia-induced VEGF expression.

Stabilization of Vascular Endothelial Growth Factor mRNA by Hypoxia-Inducible Factor 1

Hypoxia regulates expression of vascular endothelial growth factor (VEGF) by increasing its transcription and by stabilizing its mRNA. Despite the pivotal role of hypoxia-inducible factor 1 (HIF-1) in transcriptional activation of hypoxia-responsive genes, it is not known whether HIF-1 mediates hypoxia-induced stabilization of VEGF mRNA. We constructed adenoviral vectors expressing either the wild-type HIF-1α (Ad2/HIF-1α/FL), a constitutively stable hybrid form of HIF-1α (Ad2/HIF-1α/VP16), or no transgene (Ad2/CMVEV). In rat glioma (C6) cells and human cardiac, vascular smooth muscle, and endothelial cells, infection with Ad2/HIF-1α/VP16 or Ad2/HIF-1α/FL increased VEGF expression at both the mRNA and protein levels. Under normoxic conditions, the half-life of VEGF mRNA was 42 min in C6 cells. Hypoxia and Ad2/HIF-1α/VP16 increased the half-life of VEGF mRNA to 3.3 and 2.7 h, respectively, while Ad2/CMVEV had no effect. These studies are the first to demonstrate that overexpression of HIF-1α increases VEGF mRNA stability. Our results also suggest that stabilization of VEGF mRNA by hypoxia is mediated, at least in part, by HIF-1.

Thrombin Induces Increased Expression and Secretion of VEGF from Human FS4 Fibroblasts, DU145 Prostate Cells and CHRF Megakaryocytes

Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angiogenesis. We therefore examined the possibility that thrombin could be inducing the expression of vascular endothelial growth factor (VEGF), which promotes endothelial growth. Primary human FS4 fibroblasts as well as tumor cell lines: prostate DU145 and megakaryocyte CHRF were incubated with thrombin (0.25-1 unit/ml) for 1-8 hrs and then examined for mRNA by Northern Analysis. Enhanced mRNA (approximately 3-4 fold over base line) was noted at 2-4 hrs, with 0.5 u/ml thrombin. The effect was specific for thrombin activity on its PAR-1 receptor, since equal units of hirudin completely inhibited the response and the thrombin effect could be mimicked with the 14 mer thrombin receptor activation peptide (TRAP). Upregulation of mRNA was associated with enhanced VEGF protein synthesis and secretion as assayed by immunoblot. Enhanced expression of VEGF mRNA was not secondary to enhanced transcription (nuclear run on experiments), but due to an >3 fold stabilization of mRNA (Actinomycin D chase experiment). Enhanced VEGF mRNA stabilization is promoted by the PI3Kinase and serine/threonine kinase pathways, since thrombin-induced mRNA expression is inhibited by Wortmanin and H7. No effect was noted with the MAPKinase inhibitor, PD98059. Thus, thrombin-induced tumorigenesis and metastasis is associated with enhanced VEGF protein synthesis and secretion via the stabilization of VEGF mRNA promoted by the PI3Kinase and serine/threonine kinase pathways. This could help explain how thrombin promotes angiogenesis.

Age-dependent Defect in Vascular Endothelial Growth Factor Expression Is Associated with Reduced Hypoxia-inducible Factor 1 Activity

Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6-8 months) or old rabbits (ages 4-5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in old versus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1 alpha-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1 alpha protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia.

Transforming growth factor-beta1 modulates the expression of vascular endothelial growth factor by osteoblasts.

Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-beta1 (TGF-beta1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-beta1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-beta1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-beta1 treatment. Actinomycin D inhibited the TGF-beta1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-beta1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-beta1 induction of VEGF mRNA. Additionally, TGF-beta1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-beta1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-beta1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.

Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells

Osteogenic protein-1 (OP-1 or BMP-7) stimulates new bone formation in vivo and induces cell proliferation and differentiation of osteoblasts in vitro. In the present study, we examined effects of OP-1 on the expression of vascular endothelial growth factor (VEGF) in primary cultures of fetal rat calvaria (FRC) cells. OP-1 increased the steady-state level of VEGF mRNA by about 3-fold in an OP-1 concentration- and time-dependent manner. The increase in VEGF mRNA level depended on transcription and was sensitive to cell replication. The VEGF mRNA stability was unaffected. The mRNA levels for both types of VEGF receptors, Flk-1 and Flt-1 were low but detectable in FRC cells by RT-PCR and were not changed by OP-1. Inhibition of VEGF synthesis and function by antisense oligonucleotide and by suramin, respectively arrested the OP-1-induced alkaline phosphatase activity and mineralized bone nodule formation. Together with published studies of VEGF on vascular endothelial cells which are usually found in close proximity to osteoblastic cells in vivo, these results suggest that VEGF participates in the OP-1-induced osteogenesis by taking part in bone cell differentiation and by promoting angiogenesis at the site of bone formation.

Vascular endothelial growth factor production in peritoneal macrophages of cirrhotic patients: regulation by cytokines and bacterial lipopolysaccharide.

Vascular endothelial growth factor (VEGF) is an angiogenic peptide with vascular permeability and relaxing properties. This study assessed whether peritoneal macrophages of cirrhotic patients can be up-regulated to produce VEGF under proper stimulatory conditions. Macrophages were isolated from ascites. VEGF protein secretion and mRNA expression were measured in basal conditions and after stimulation with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), and interleukin-1 (IL-1). These substances induced a time- and dose-dependent increase in both VEGF production and transcript expression. Assays with actinomycin D showed that VEGF mRNA induction is secondary to both higher VEGF gene transcription and mRNA stability. Ascites and plasma concentration of VEGF was also measured in cirrhotic patients with (n = 15) and without (n = 10) spontaneous bacterial peritonitis (SBP). Plasma values did not differ between both groups of patients. However, ascites VEGF levels were higher in SBP patients than in noninfected cirrhotic patients (710 +/- 183 vs. 94 +/- 15 pg/mL; P <.025). These results indicate that cytokines and LPS markedly increase VEGF protein secretion and mRNA expression in macrophages of cirrhotic patients, and suggest that this substance could be an important mediator of the pronounced arterial vasodilation frequently occurring in SBP patients.

Androgens induce the expression of vascular endothelial growth factor in human fetal prostatic fibroblasts.

Androgens are known to directly stimulate prostate cancer cell growth. We have previously reported that LNCaP prostate cancer cells were dependent upon stromal coinoculation for growth in nude mice and that the stromal cells secreted a potent angiogenic factor, vascular endothelial growth factor (VEGF), which stimulated tumor angiogenesis. Immunohistochemical staining localized VEGF expression primarily to the stromal cells of human fetal and adult hyperplastic prostates, with both stromal and epithelial cell VEGF expression in prostate cancer. In the present studies, we test the hypothesis that androgens, in addition to their direct effects on prostate epithelial cells, have indirect effects on these cells via up-regulation of stromal VEGF production and angiogenesis. Primary cultures of human prostate fetal fibroblasts were treated with dihydrotestosterone (DHT), and the effects on VEGF messenger RNA (mRNA) expression were determined by Northern blotting. DHT (10 nM) increased VEGF mRNA levels maximally after 2 h. Nuclear run-on transcription assays demonstrated a 2-fold increase in the VEGF transcription rate 2 h after the addition of DHT. VEGF mRNA stability was unaffected by DHT addition. VEGF protein levels were determined by enzyme-linked immunosorbent assay and were increased 2-fold 4 h after DHT addition. These data indicate that androgens increase VEGF transcription and secretion of biologically active VEGF from human prostatic stroma. Androgens, therefore, may indirectly enhance prostate growth via up-regulation of VEGF from the surrounding stroma.

Barbiturates decrease the expression of vascular endothelial growth factor in hypoxic cultures of porcine brain derived microvascular endothelial cells

Vascular endothelial growth factor (VEGF) is known to be produced in higher amounts during hypoxia by a variety of cell types and has been shown to increase the permeability of brain derived microvascular endothelial cells (BMEC) during hypoxia by an autocrine mechanism. Because the barbiturates, methohexital (MH) and thiopental (TP), induced a dose-dependent reduction in hypoxia-induced permeability changes of BMEC, the effect of both barbiturates on the VEGF expression during hypoxia was investigated. Both barbiturates decreased the hypoxia-induced expression of VEGF in BMEC in a concentration-dependent manner. This effect is partly caused by the impairment of the hypoxia-induced VEGF mRNA stabilization. VEGF-induced permeability changes during normoxia were unaffected by the barbiturates suggesting that MH and TP are directly reducing hypoxia-induced VEGF synthesis. In conclusion, the inhibiting effect of these barbiturates on the hypoxia-induced VEGF expression results in the decreased permeability of the BMEC monolayer during hypoxia, which may contribute to the described neuroprotective action of barbiturates by reduction of brain edema formation.

Hypoxic Regulation of VEGF mRNA Stability by RNA-binding Proteins

Vascular endothelial growth factor (VEGF), is a potent angiogenic factor whose expression is dramatically induced by hypoxia. We have previously demonstrated that the induction of VEGF by hypoxia is in large part the result of an increased stability of VEGF mRNA. The stabilization of VEGF mRNA by hypoxia is mediated by the binding of sequence-specific RNA-binding proteins. This review focuses on one such protein, HuR, an RNA-binding protein which we have recently shown is critical for the hypoxic stabilization of VEGF mRNA.

Hypoxic Stabilization of Vascular Endothelial Growth Factor mRNA by the RNA-binding Protein HuR

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is dramatically induced by hypoxia due in large part to an increase in the stability of its mRNA. Here we show that HuR binds with high affinity and specificity to the element that regulates VEGF mRNA stability by hypoxia. Inhibition of HuR expression abrogates the hypoxia-mediated increase in VEGF mRNA stability. Overexpression of HuR increases the stability of VEGF mRNA. However, this only occurs efficiently in hypoxic cells. We further show that the stabilization of VEGF mRNA can be recapitulated in vitro. Using an S-100 extract, we show that the addition of recombinant HuR stabilizes VEGF mRNA markedly. These data support the critical role of HuR in mediating the hypoxic stabilization of VEGF mRNA by hypoxia.