Vascular Endothelial Growth Factor Translation Research Articles

Suppression of microRNA-205-5p in human mesenchymal stem cells improves their therapeutic potential in treating diabetic foot disease.

Diabetes is a prevalent disease endangering human health, while diabetic foot disease (DF) is one of the most severe complications of diabetes. Mesenchymal stem cells (MSCs) have been used in DF treatment, taking advantage of the differentiation potential of MSCs into endothelial cells and their production and secretion of trophic factors like vascular endothelial growth factor (VEGF). Molecular modification of MSCs to improve their therapeutic effects has been recently applied in treating other diseases, but not yet in DF. Here, we found that micoRNA-205-5p (miR-205-5p) is expressed in human MSCs, and miR-205-5p inhibits protein translation of VEGF through its interaction with 3′-UTR of the VEGF mRNA. Expression of antisense of miR-205-5p (as-miR-205-5p) significantly increased both cellular and secreted VEGF by MSCs, which significantly improved the therapeutic effects of MSCs on DF-associated wound healing in diabetic NOD/SCID mice. Together, our data suggest that miR-205-5p suppression in MSCs may improve their therapeutic effects on DF, seemingly through augmentation of VEGF-mediated vascularization.

Gossypol has anti-cancer effects by dual-targeting MDM2 and VEGF in human breast cancer

BackgroundMouse double minute 2 (MDM2) and vascular endothelial growth factor (VEGF) are important molecules involved in tumor progression. We researched potential inhibitors that simultaneously target MDM2 and VEGF. In our recent study involving the performance of high-throughput screening with a fluorescence polarization assay, gossypol was identified as one of the top hits that inhibit protein-RNA binding activity. Because MDM2 is an RNA-binding protein and its targets include VEGF mRNA, we investigated whether gossypol has an inhibitory effect on MDM2-VEGF.MethodsUV cross-linking and RNA binding assay, isothermal titration calorimetry assay, and ubiquitination assay were performed to determine mechanisms by which gossypol functions as a dual inhibitor of MDM2 and VEGF. The effect of gossypol on MDM2 and VEGF expression, cancer cell apoptosis, tumor growth and VEGF-mediated angiogenesis were studied in vitro and in vivo in different human breast cancer models with a different p53 status.ResultsWe observed that gossypol inhibited expression of both MDM2 and VEGF in human breast cancer cells with either wild-type or mutant p53. A nechanistic study further demonstrated that, through disrupting the interaction between MDM2 protein and VEGF mRNA, gossypol induced MDM2 self-ubiquitination and decreased VEGF translation simultaneously, which resulted in both apoptosis and anti-angiogenesis effects. In vitro, regardless of p53 status, gossypol induced cancer cell apoptosis. In nude mouse xenograft in vivo models, gossypol suppressed tumor growth and VEGF-mediated angiogenesis.ConclusionGossypol has anti-cancer effects by dual-targeting MDM2 and VEGF in human breast cancer. Our study reveals a novel mechanism by which gossypol functions as an anticancer agent. We believe that MDM2-VEGF targeting represents a novel strategy for improving cancer outcome.

VEGF: From Discovery to Therapy: The Champalimaud Award Lecture.

PurposeIntraocular vascular diseases are leading causes of adult vision loss, and in the mid-1900s, I. C. Michaelson postulated that the retina releases a soluble, diffusible factor that causes abnormal vascular growth and leakage. What became known as “Factor X” eluded investigators for decades.MethodsThe field of cancer research, where Judah Folkman pioneered the concept of angiogenesis, provided the inspiration for the work honored by the 2014 Champalimaud Vision Award. Recognizing that tumors recruit their own blood supply to achieve critical mass, Dr Folkman proposed that angiogenic factors could be therapeutic targets in cancer. Napoleone Ferrara identified vascular endothelial growth factor (VEGF) as such an angiogenic agent: stimulated by hypoxic tumor tissue, secreted, and able to induce neovascularization. VEGF also was a candidate for Factor X, and the 2014 Champalimaud Laureates and colleagues worked individually and collaboratively to identify the role of VEGF in ocular disease.ResultsThe Champalimaud Laureates correlated VEGF with ocular neovascularization in animal models and in patients. Moreover, they showed that VEGF not only was sufficient, but it also was required to induce neovascularization in normal animal eyes, as VEGF inhibition abolished ocular neovascularization in key animal models.ConclusionsThe identification of VEGF as Factor X altered the therapeutic paradigms for age-related macular degeneration (AMD), diabetic retinopathy, retinal vein occlusion, and other retinal disorders.Translational RelevanceThe translation of VEGF from discovery to therapy resulted in the most successful applications of antiangiogenic therapy to date. Annually, over one million patients with eye disease are treated with anti-VEGF agents.

Dopamine agonist inhibits vascular endothelial growth factor protein production and secretion in granulosa cells.

BackgroundDopamine receptor 2 agonists (D2-ags) inhibit vascular endothelial growth factor (VEGF) secretion in luteinized granulosa cells (LGCs) both in vitro and in vivo. However, the mechanism of D2 regulation of the VEGF/VEGF Receptor 2 (VEGFR-2) pathway remains to be elucidated. We sought to determine the effects of D2 signaling on VEGF transcription and translation in LGCs, with the expectation of identifying potential D2-ag-based therapies for ovarian hyperstimulation syndrome (OHSS).FindingsLGCs from egg donors were cultured with chorionic gonadotropin (hCG) in the presence of Actinomycin-D (ActD) or Brefeldin-A (BFA) to evaluate the effects of a D2-ag, cabergoline (Cb2), on VEGF secretion. The contribution of the conventional Gi/Go, Gz and AKT/β-Arrestin pathways in the VEGF regulation was assessed by adding pertussis toxin (PTX), phorbol 12-myristate 13-acetate (PMA), or wortmannin (WT). While Cb2 inhibited VEGF secretion by interfering with VEGF peptide translation and secretion, inhibition of conventional D2 transduction pathways did not reverse Cb2-mediated inhibition of VEGF secretion.ConclusionsThe effects of D2-ag on VEGF translation and secretion are mediated by D2 signaling pathways that have yet to be described. We found that D2-ag inhibits VEGF secretion at the post-transcriptional level, suggesting that D2-ag treatment should be combined with therapies that inhibit VEGF transcription, such as the employment of LH or GnRH for triggering ovulation, to improve the efficacy of OHSS prevention.

Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas

The anaplastic lymphoma kinase (ALK), tyrosine kinase oncogene is implicated in a wide variety of cancers. In this study we used conditional onco-ALK (NPM-ALK and TPM3-ALK) mouse MEF cell lines (ALK+ fibroblasts) and transgenic models (ALK+ B-lymphoma) to investigate the involvement and regulation of angiogenesis in ALK tumor development. First, we observed that ALK expression leads to downregulation of miR-16 and increased Vascular Endothelial Growth Factor (VEGF) levels. Second, we found that modification of miR-16 levels in TPM3-ALK MEF cells greatly affected VEGF levels. Third, we demonstrated that miR-16 directly interacts with VEGF mRNA at the 3'-untranslated region and that the regulation of VEGF by miR-16 occurs at the translational level. Fourth, we showed that expression of both the ALK oncogene and hypoxia-induced factor 1α (HIF1α) is a prerequisite for miR-16 downregulation. Fifth, in vivo, miR-16 gain resulted in reduced angiogenesis and tumor growth. Finally, we highlighted an inverse correlation between the levels of miR-16 and VEGF in human NPM-ALK+ Anaplastic Large Cell Lymphomas (ALCL). Altogether, our results demonstrate, for the first time, the involvement of angiogenesis in ALK+ ALCL and strongly suggest an important role for hypoxia-miR-16 in regulating VEGF translation.

AKT activity regulates the ability of mTOR inhibitors to prevent angiogenesis and VEGF expression in multiple myeloma cells

We recently demonstrated that the mammalian target of rapamycin (mTOR) inhibitor, CCI-779, curtailed the growth of a subcutaneous challenge of multiple myeloma (MM) cells in immunodeficient mice. This antitumor effect was associated with prevention of cell proliferation, induction of apoptosis and inhibition of angiogenesis. Interestingly, myeloma tumors with heightened AKT activation were particularly sensitive to a CCI-779-induced antitumor response. To investigate whether part of the differential sensitivity was due to an AKT-regulated effect on angiogenesis, we compared the effects of mTOR inhibitors against isogenic MM cell lines that only differ by their degree of AKT activity. In this model, heightened AKT activity significantly sensitized MM cells to the following inhibitory effects of mTOR inhibition: angiogenesis in vivo, vascular endothelial growth factor (VEGF) expression in vitro and in vivo and VEGF translation (but not transcription). Assessment of p70S6 kinase activity indicated that rapamycin induced comparable mTOR inhibition in both cell lines suggesting that an adverse effect on VEGF cap-dependent translation would be comparable. Internal ribosome entry site (IRES)-mediated cap-independent translation is a salvage pathway for protein expression when mTOR is inhibited, so we analyzed a possible regulatory role of AKT on VEGF IRES activity. We found that elevated AKT activity inhibited VEGF IRES function. These results support a mechanism whereby AKT prevents VEGF IRES activity in myeloma cells during mTOR inhibition resulting in a more complete abrogation of VEGF translation, and ultimately, angiogenesis.

Epstein-Barr Virus Lytic Infection Is Required for Efficient Production of the Angiogenesis Factor Vascular Endothelial Growth Factor in Lymphoblastoid Cell Lines

Although Epstein-Barr virus (EBV)-associated malignancies are primarily composed of cells with one of the latent forms of EBV infection, a small subset of tumor cells containing the lytic form of infection is often observed. Whether the rare lytically infected tumor cells contribute to the growth of the latently infected tumor cells is unclear. Here we have investigated whether the lytically infected subset of early-passage lymphoblastoid cell lines (LCLs) could potentially contribute to tumor growth through the production of angiogenesis factors. We demonstrate that supernatants from early-passage LCLs infected with BZLF1-deleted virus (Z-KO LCLs) are highly impaired in promoting endothelial cell tube formation in vitro compared to wild-type (WT) LCL supernatants. Furthermore, expression of the BZLF1 gene product in trans in Z-KO LCLs restored angiogenic capacity. The supernatants of Z-KO LCLs, as well as supernatants from LCLs derived with a BRLF1-deleted virus (R-KO LCLs), contained much less vascular endothelial growth factor (VEGF) in comparison to WT LCLs. BZLF1 gene expression in Z-KO LCLs restored the VEGF level in the supernatant. However, the cellular level of VEGF mRNA was similar in Z-KO, R-KO, and WT LCLs, suggesting that lytic infection may enhance VEGF translation or secretion. Interestingly, a portion of the vasculature in LCL tumors in SCID mice was derived from the human LCLs. These results suggest that lytically infected cells may contribute to the growth of EBV-associated malignancies by enhancing angiogenesis. In addition, as VEGF is a pleiotropic factor with effects other than angiogenesis, lytically induced VEGF secretion may potentially contribute to viral pathogenesis.

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.

Akt mediates an angiogenic switch in transformed keratinocytes.

Akt signaling is involved in tumorigenesis via a number of different mechanisms that result in increased proliferation and decreased apoptosis. Previous data have demonstrated that Akt-mediated signaling is functionally involved in keratinocyte transformation. This work investigates the involvement of angiogenesis as a mediator of tumorigenesis in Akt-transformed keratinocytes. Tumors produced by subcutaneous injection of the latter showed increased angiogenic profiles associated with increased vascular endothelial growth factor (VEGF) protein levels. However, in contrast to v-ras(Ha)-transformed keratinocytes, VEGF mRNA levels were not increased. The induction of VEGF protein by Akt is associated with increased phosphorylation and thus activation of p70S6K and eIF4E-binding protein 1, leading to increased VEGF translation. In addition, we observed increased metaloproteinases 2 and 9 expression, but not thrombospondin 1, in tumors derived from Akt-transformed keratinocytes. Collectively, these results demonstrate that Akt is an important mediator of angiogenesis in malignant keratinocytes through a post-transcriptional mechanism.

Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells.

We define a novel mechanism by which integrins regulate growth factor expression and the survival of carcinoma cells. Specifically, we demonstrate that the α6β4 integrin enhances vascular endothelial growth factor (VEGF) translation in breast carcinoma cells. The mechanism involves the ability of this integrin to stimulate the phosphorylation and inactivation of 4E-binding protein (4E-BP1), a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (eIF-4E). The regulation of 4E-BP1 phosphorylation by α6β4 derives from the ability of this integrin to activate the PI-3K–Akt pathway and, consequently, the rapamycin-sensitive kinase mTOR that can phosphorylate 4E-BP1. Importantly, we show that this α6β4-dependent regulation of VEGF translation plays an important role in the survival of metastatic breast carcinoma cells by sustaining a VEGF autocrine signaling pathway that involves activation of PI-3K and Akt. These findings reveal that integrin-mediated activation of PI-3K–Akt is amplified by integrin-stimulated VEGF expression and they provide a mechanism that substantiates the reported role of α6β4 in carcinoma progression.

Angiogenesis and the placental environment

Rapid growth and vascularization of the human placenta are characteristic of early pregnancy and are accomplished in an unusually hypoxic environment. Stimulation of placental growth through hypoxia-induced angiogenesis may therefore be of particular importance. We have previously found that several varieties of vascular endothelial growth factor (VEGF) mRNA, including VEGF165, are present in cultured placental fibroblasts. We hypothesized that hypoxia would increase the transcription and translation of VEGF by these cells and provide one mechanism linking placental development with its environment. Placental fibroblasts were grown in aerobic or anaerobic atmospheric conditions for 72 h. By 24 h the oxygen tension of the anaerobic culture media was significantly less than that of the aerobic cultures. RNA was extracted from the cells at 24, 48 and 72 h. Following reverse transcription polymerase chain reaction (RT-PCR) stronger signals for VEGF were always found in the anaerobic cultures and this was confirmed by competitive PCR. mRNA for VEGF 165 was represented most strongly but the anaerobic cultures also showed clearly mRNA for VEGF 121, VEGF 189 and VEGF 206. The VEGF protein was also measured in the aerobic and anaerobic culture medium. By 72 h the average concentration of VEGF was significantly higher ( P=0.01) in the anaerobic culture medium. VEGF production is one mechanism through which oxygen supply may influence placental development. Examples of this may include the compensatory placental hypertrophy associated with maternal anaemia and with reproduction at high altitude.