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1.  Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1 
PLoS Medicine  2007;4(6):e186.
While vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial.
Methods and Findings
We investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope.
This study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.
Shalom Avraham and colleagues' study provides evidence of a survival system in breast cancer cells by which VEGF acts as an internal autocrine survival factor through its binding to VEGFR1.
Editors' Summary
One woman in eight will develop breast cancer during her lifetime. Most of these women live for many years after their diagnosis and many are cured of their cancer. However, sometimes the cancer grows inexorably and spreads (metastasizes) around the body despite the efforts of oncologists. Characteristics of the tumor known as prognostic factors can indicate whether this spreading is likely to happen. Large tumors that have metastasized have a poorer prognosis than small tumors that are confined to the breast. The expression of specific proteins within the tumor also provides prognostic information. One protein whose expression is associated with a poor prognosis is vascular endothelial growth factor (VEGF). VEGF stimulates angiogenesis—the growth of new blood vessels. Small tumors get the nutrients needed for their growth from existing blood vessels but large tumors need to organize their own blood supply. They do this, in part, by secreting VEGF. This compound binds to proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels), which then send a signal into the cell instructing it to make new blood vessels. Angiogenesis inhibitors, including molecules that block the activity of VEGF receptors, are being developed for the treatment of cancer.
Why Was This Study Done?
Some breast cancer cell lines (cells isolated from breast cancers and grown in the laboratory) make VEGF and VEGF receptors (VEGFR1, VEGFR2, and neuropilin 1 [NRP1]). But, although some studies have reported an association between VEGFR1 expression in breast tumors and a poor prognosis, other studies have found no expression of VEGFR1 in breast tumors. Consequently, the role of VEGF receptors in breast cancer is unclear. In this study, the researchers analyzed the expression and function of VEGF and its receptors in breast cancer cells to investigate whether and how VEGF helps these cells to survive.
What Did the Researchers Do and Find?
The researchers first examined the expression of VEGF receptors in several human breast cancer cell lines. All of them expressed VEGFR1, some expressed NRP1, but VEGFR2 expression was universally low. They then investigated the function of VEGF and its receptors in two human breast cancer cell lines (MDA-MB-231 and MCF-7). In both cell lines, blocking the expression of VEGF or of VEGFR1 (but not of the other two receptors) reduced cell survival by stimulating a specific process of cell death called apoptosis. Unexpectedly, adding VEGF to the cultures did not reverse the effect of blocking VEGF expression, a result that suggests that VEGF and VEGFR1 do not affect breast cancer cell survival by acting at the cell surface. Accordingly, when the researchers examined where VEGFR1 occurs in the cell, they found it on the membranes around the nucleus of the breast cancer cell lines and not on the cell surface; several primary breast tumors and normal breast tissue had the same localization pattern. Finally, the researchers showed that inhibitors of VEGF action that act at the cell surface did not affect the survival of the breast cancer cell lines.
What Do These Findings Mean?
These findings suggest that VEGF helps breast cancer cells to survive in a unique way: by binding to VEGFR1 inside the cell. In other words, whereas VEGF normally acts as a paracrine growth factor (it is released by one cell and affects another cell), in breast cancer cells it might act as an internal autocrine (intracrine) survival factor, a factor that affects the cells in which it is produced. These findings need confirming in more cell lines and in primary breast cancers but could have important implications for the treatment of breast cancer. Inhibitors of VEGF and VEGFR1 that act inside the cell (small molecule drugs) might block breast cancer growth more effectively than inhibitors that act at the cell surface (for example, proteins that bind to the receptor), because internally acting inhibitors might both kill the tumor directly and have antiangiogenic effects, whereas externally acting inhibitors could only have the second effect.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute information for patients and professionals on breast cancer (in English and Spanish) and on angiogenesis (in English and Spanish)
MedlinePlus Encyclopedia information for patients on breast cancer (in English and Spanish)
CancerQuest, information from Emory University on cancer biology and on angiogenesis and angiogenesis inhibitors (in several languages)
Wikipedia pages on VEGF (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC1885450  PMID: 17550303
2.  A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA 
PLoS Medicine  2008;5(5):e94.
Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.
Methods and Findings
Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.
These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Shigetada Teshima-Kondo and colleagues find that cancer cells have a survival system that is regulated by vegf mRNA and that vegf mRNA and its protein may synergistically promote the malignancy of tumor cells.
Editors' Summary
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. When a cancer is small, it uses the body's existing blood supply to get the oxygen and nutrients it needs for its growth and survival. But, when it gets bigger, it has to develop its own blood supply. This process is called angiogenesis. It involves the release by the cancer cells of proteins called growth factors that bind to other proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels). The receptors then send signals into the endothelial cells that tell them to make new blood vessels. One important angiogenic growth factor is “vascular endothelial growth factor” (VEGF). Tumors that make large amounts of VEGF tend to be more abnormal and more aggressive than those that make less VEGF. In addition, high levels of VEGF in the blood are often associated with poor responses to chemotherapy, drug regimens designed to kill cancer cells.
Why Was This Study Done?
Because VEGF is a key regulator of tumor development, several anti-VEGF therapies—drugs that target VEGF and its receptors—have been developed. These therapies strongly suppress the growth of tumor cells in the laboratory and in animals but, when used alone, are no better at increasing the survival times of patients with cancer than standard chemotherapy. Scientists are now looking for an explanation for this disappointing result. Like all proteins, cells make VEGF by “transcribing” its DNA blueprint into an mRNA copy (vegf mRNA), the coding region of which is “translated” into the VEGF protein. Other, “noncoding” regions of vegf mRNA control when and where VEGF is made. Scientists have recently discovered that the noncoding regions of some mRNAs suppress tumor development. In this study, therefore, the researchers investigate whether vegf mRNA has an unrecognized function in tumor cells that could explain the disappointing clinical results of anti-VEGF therapeutics.
What Did the Researchers Do and Find?
The researchers first used a technique called small interfering (si) RNA knockdown to stop VEGF expression in human colon cancer cells growing in dishes. siRNAs are short RNAs that bind to and destroy specific mRNAs in cells, thereby preventing the translation of those mRNAs into proteins. The treatment of human colon cancer cells with vegf-targeting siRNAs made the cells more sensitive to chemotherapy-induced apoptosis (a type of cell death). This sensitivity was only partly reversed by adding VEGF to the cells. By contrast, cancer cells engineered to make more vegf mRNA had increased resistance to chemotherapy-induced apoptosis. Treatment of these cells with an antibody that inhibited VEGF function did not completely block this resistance. Together, these results suggest that both vegf mRNA and VEGF protein have anti-apoptotic effects. The researchers show that the anti-apoptotic activity of vegf mRNA requires a noncoding part of the mRNA called the 5′ UTR, and that whereas human colon cancer cells expressing this 5′ UTR form tumors in mice, cells expressing a mutated 5′ UTR do not. Finally, they report that the expression of several pro-apoptotic genes and of an anti-tumor pathway known as the interferon/STAT1 tumor suppression pathway is down-regulated in tumors that express the vegf 5′ UTR.
What Do These Findings Mean?
These findings suggest that some cancer cells have a survival system that is regulated by vegf mRNA and are the first to show that a 5′UTR of mRNA can promote tumor growth. They indicate that VEGF and its mRNA work together to promote their development and to increase their resistance to chemotherapy drugs. They suggest that combining therapies that prevent the production of vegf mRNA (for example, siRNA-based gene silencing) with therapies that block the function of VEGF might improve survival times for patients whose tumors overexpress VEGF.
Additional Information.
Please access these Web sites via the online version of this summary at
This study is discussed further in a PLoS Medicine Perspective by Hughes and Jones
The US National Cancer Institute provides information about all aspects of cancer, including information on angiogenesis, and on bevacizumab, an anti-VEGF therapeutic (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer, including angiogenesis (in several languages)
Cancer Research UK also provides basic information about what causes cancers and how they develop, grow, and spread, including information about angiogenesis
Wikipedia has pages on VEGF and on siRNA (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2386836  PMID: 18494554
3.  Breast cancer cells stimulate osteoprotegerin (OPG) production by endothelial cells through direct cell contact 
Molecular Cancer  2009;8:49.
Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG) is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment.
It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on ανβ3 ligation and NFκB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFβ had no effect on HuDMEC OPG levels.
These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve ανβ3 ligation and NFκB activation.
PMCID: PMC2719583  PMID: 19604388
4.  56Fe ion irradiation enhances angiogenesis and other inter-cellular determinants of carcinogenesis risk 
Journal of Radiation Research  2014;55(Suppl 1):i124-i126.
In the assessment of radiogenic cancer risk from space flight, it is imperative to consider effects not only on the creation of cancer cells (initiation) but also on cell–cell interactions that play an important and often decisive role in the promotion and progression phases. Autopsy results confirm that most adults carry fully malignant tumors that are held in check at a small size and will never become symptomatic [ 1, 2]. This introduces the possibility that cosmic radiation may significantly influence cancer risk through alteration of the bottleneck inter-tissue interactions responsible for maintaining this dormant state. One such bottleneck is the growth limitation imposed by the failure of the tumor to induce blood vessels (angiogenesis). Other deciding events are the ability of a tumor to proliferate and invade. We have previously shown that proton radiation, the most prevalent radiation in space, has a suppressive effect on all three of these functional responses. It down-regulates angiogenic genes like VEGF and HIF-1α and impairs cell invasion and tumor growth [ 3]. We decided to test these responses after 56Fe irradiation, an HZE radiation type present in the cosmic environment with presumably high carcinogenic potential [ 4].
Human microvascular endothelial cells (HMVEC) and normal human dermal fibroblast (NHDF) cells were irradiated with different doses of 56Fe ion radiation (1 GeV/n) at Brookhaven National Laboratory and RNA was extracted 6 h later. Genomic-wide array analysis was done on the isolated RNA through the Agilent Platform. It was observed that several pro-angiogenic genes like VEGF, IL-6 and HIF-1α were significantly up-regulated after treatment with 56Fe ion radiation (Fig.  1). These results were also confirmed at the mRNA and protein levels with the human and murine lung cancer lines, A549 and LLC, respectively. Additional verification of modulation of these key genes was also observed when lungs of C57BL/6 mice treated with 56Fe ion radiation showed an increase in VEGF and MMP9 mRNA and protein expression 6 h post-irradiation (Fig.  2). Cell invasion was shown to be increased by 56Fe ion radiation in various cell types, including fibroblast, tumor and endothelial progenitor cells. 56Fe ion irradiation also modulated functional processes crucial to angiogenesis. It enhanced the ability of untargeted (bystander) endothelial cells to invade and proliferate in response to factors produced by targeted fibroblast or cancer cells in vitro. Results also carry over to in vivo. C57BL/6 mice exposed to whole-body irradiation with 0.2 Gy dose of 56Fe and injected subcutaneously with LLC tumor cells showed a significant augmentation in tumor growth and growth rate in the irradiated group. Additionally, nude mice exposed to whole-body 56Fe radiation and injected intravenously with A549 cancer cells 3 h post-irradiation demonstrated a significant enhancement in lung colonization capacity when compared with the sham-irradiated control mice injected.
These results together suggest cell and tissue-level responses to 56Fe irradiation may act to overcome major cancer progression-level bottlenecks including those related to angiogenesis, cell proliferation and invasion. This is of significant concern for cancer risk estimations pertinent to NASA as achieving these cancer hallmark processes can make the difference between a radiation-induced cancer cell progressing to a clinically detectable cancer in astronauts or not. In conclusion, we demonstrate a strong radiation quality dependence for space radiation carcinogenesis risk manifested through influences on intercellular interactions in the progression phase of carcinogenesis. Fig. 1.Heatmaps of selected differentially regulated major angiogenesis genes after proton and 56Fe ion radiation in HMVECs and NHDF. Cells were treated with either 0, 0.5, 1 or 2 Gy of proton radiation or 0, 0.2, 0.4 or 1 Gy of 56Fe ion dose. Among the major regulated genes were VEGF, HIF-1A and IL-6; they were down-regulated by proton radiation and up-regulated by iron radiation. Fig. 2.Immunofluorescence images of lungs of C57BL/6 mice treated with 0, 0.2 or 1 Gy of 56Fe ion dose and stained 6 h later. Pro-angiogenic factors VEGF and MMP9 were increased in mice that received the 56Fe ion treatment.
PMCID: PMC3941549
5.  Autoantibodies against Muscarinic Receptors in Breast Cancer: Their Role in Tumor Angiogenesis 
PLoS ONE  2013;8(2):e57572.
The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10−8 M) and carbachol (10−9 M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.
PMCID: PMC3583830  PMID: 23460876
6.  Fibroblast growth factor regulation of neovascularization 
Current opinion in hematology  2008;15(3):215-220.
Purpose of review
Fibroblast growth factors (FGFs) are potent angiogenic inducers; however, their precise roles in angiogenesis have not been well understood. In this review, we will focus on specific roles played by FGFs in neovascularization.
Recent findings
Whereas FGFs promote a strong angiogenic response, it has been suggested that FGF-induced angiogenesis requires activation of the vascular endothelial growth factor (VEGF) system. Recent findings have endorsed this view: indirect contribution of FGF signaling to vascular development. A study using embryoid bodies demonstrated a non-immediate role played by FGFR1 in vasculogenesis since VEGF supplementation was sufficient to promote vascular development in Fgfr1-/- embryoid bodies. Moreover, another line of evidence indicated that myocardial FGF signaling is essential for mouse coronary development. The key role of FGF signaling is Hedgehog activation, which induces VEGF expression and formation of the coronary vasculature.
In addition to VEGF interaction, FGFs can control neovascularization by influencing other growth factors and chemokines such as PDGF, HGF and MCP-1, contributing to development of mature vessels and collateral arteries.
Although FGFs are potent angiogenic factors, they may indirectly control neovascularization in concert with other growth factors. Thus, the unique role played by FGFs might be organization of various angiogenic pathways and coordination of cell-cell interactions in this process.
PMCID: PMC2745288  PMID: 18391788
fibroblast growth factor; vascular endothelial growth factor; platelet-derived growth factor; angiogenesis; arteriogenesis
7.  Basic Fibroblast Growth Factor Contributes to a Shift in the Angioregulatory Activity of Retinal Glial (Müller) Cells 
PLoS ONE  2013;8(7):e68773.
Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK−1/−2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under physiological conditions or transient hypoxia seem to provide an anti-angiogenic environment, but long-lasting hypoxia causes the release of bFGF, which might significantly co-stimulate neovascularization in the retina.
PMCID: PMC3701643  PMID: 23861940
8.  Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9–mediated progenitor cell mobilization 
Mast cells accumulate in tissues undergoing angiogenesis during tumor growth, wound healing, and tissue repair. Mast cells can secrete angiogenic factors such as vascular endothelial growth factor (VEGF). Ionizing irradiation has also been shown to have angiogenic potential in malignant and nonmalignant diseases. We observed that low-dose irradiation fosters mast cell–dependent vascular regeneration in a limb ischemia model. Irradiation promoted VEGF production by mast cells in a matrix metalloproteinase-9 (MMP-9)–dependent manner. Irradiation, through MMP-9 up-regulated by VEGF in stromal and endothelial cells, induced the release of Kit-ligand (KitL). Irradiation-induced VEGF promoted migration of mast cells from the bone marrow to the ischemic site. Irradiation-mediated release of KitL and VEGF was impaired in MMP-9–deficient mice, resulting in a reduced number of tissue mast cells and delayed vessel formation in the ischemic limb. Irradiation-induced vasculogenesis was abrogated in mice deficient in mast cells (steel mutant, Sl/Sld mice) and in mice in which the VEGF pathway was blocked. Irradiation did not induce progenitor mobilization in Sl/Sld mice. We conclude that increased recruitment and activation of mast cells following irradiation alters the ischemic microenvironment and promotes vascular regeneration in an ischemia model. These data show a novel mechanism of neovascularization and suggest that low-dose irradiation may be used for therapeutic angiogenesis to augment vasculogenesis in ischemic tissues.
PMCID: PMC2212942  PMID: 16157686
9.  Activation of Multiple Signaling Pathways Is Critical for Fibroblast Growth Factor 2- and Vascular Endothelial Growth Factor-Stimulated Ovine Fetoplacental Endothelial Cell Proliferation1 
Biology of reproduction  2007;78(1):143-150.
Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by NG-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.
PMCID: PMC2441762  PMID: 17901071
AKT1; endothelial cell proliferation; FGF2; growth factors; kinases; MAPK3/1; nitric oxide; pregnancy; vascular endothelial growth factor
10.  Human Apurinic/Apyrimidinic Endonuclease siRNA Inhibits the Angiogenesis Induced by X-Ray Irradiation in Lung Cancer Cells 
Objective: Radiotherapy is an important and effective treatment method for non-small cell lung cancer (NSCLC). Nonetheless, radiotherapy can alter the expression of proangiogenic molecules and induce angiogenesis. Human apurinic/apyrimidinic endonuclease (APE1) is a multifunctional protein, which has DNA repair and redox function. Our previous studies indicated APE1 is also a crucial angiogenic regulator. Thus, we investigated the effect of APE1 on radiation-induced angiogenesis in lung cancer and its underlying mechanism.
Methods: Tumor specimens of 136 patients with NSCLC were obtained from 2003 to 2008. The APE1 and vascular endothelial growth factor (VEGF) expression, as well as microvessel density (MVD) were observed with immunohistochemistry in tumor samples. Human lung adenocarcinoma A549 cells were treated with Ad5/F35-APE1 siRNA and/or irradiation, and then the cells were used for APE1 analysis by Western blot and VEGF analysis by RT-PCR and ELISA. To elucidate the underline mechanism of APE1 on VEGF expression, HIF-1α protein level was determined by Western blot, and the DNA binding activity of HIF-1α was detected by EMSA. Transwell migration assay and capillary-like structure assay were used to observe the migration and capillary-like structure formation ability of human umbilical veins endothelial cells (HUVECs) that were co-cultured with Ad5/F35-APE1 siRNA and (or) irradiation treated A549 cells culture medium.
Results: The high expression rates of APE1 and VEGF in NSCLC were 77.94% and 66.18%, respectively. The expressions of APE1 was significantly correlated with VEGF and MVD (r=0.369, r=0.387). APE1 and VEGF high expression were significantly associated with reduced disease free survival (DFS) time. The high expressions of APE1 and VEGF on A549 cells were concurrently induced by X-ray irradiation in a dose-dependent manner. Silencing of APE1 by Ad5/F35-APE1 siRNA significantly decreased DNA binding activity of HIF-1α and suppressed the expression of VEGF in A549 cells, moreover, significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by irradiated A549 cells.
Conclusion: Our results indicate that APE1 may play a crucial role in angiogenesis induced by irradiation. Administration of Ad5/F35-APE1 siRNA during radiotherapy could be a potent adjuvant therapeutic approach to enhance the radiotherapy response, effectively eliminate metastasis and improve the efficacy of radiotherapy for NSCLC.
PMCID: PMC3675501  PMID: 23781133
Human apurinic/apyrimidinic endonuclease; angiogenesis; irradiation; lung cancer.
11.  Tpl2 Inhibitors Thwart Endothelial Cell Function in Angiogenesis and Peritoneal Dissemination12 
Neoplasia (New York, N.Y.)  2013;15(9):1036-1048.
Angiogenesis is critical in the development of cancer, which involves several angiogenic factors in its peritoneal dissemination. The role of protein tumor progression locus 2 (Tpl2) in angiogenic factor-related endothelial cell angiogenesis is still unclear. To understand the precise mechanism(s) of Tpl2 inhibition in endothelial cells, this study investigated the role of Tpl2 in mediating angiogenic signals using in vitro, in vivo, and ex vivo models. Results showed that inhibition of Tpl2 inhibitor significantly reduced peritoneal dissemination in a mouse model by positron emission tomography/computed tomography imaging. Simultaneously, inhibiting Tpl2 blocked angiogenesis in tumor nodules and prevented angiogenic factor-induced proliferating cell nuclear antigen (PCNA) in endothelial cells. Vascular endothelial growth factor (VEGF) or chemokine (C-X-C motif) ligand 1 (CXCL1) increased Tpl2 kinase activity and phosphorylation in a dose- and time-dependent manner. Furthermore, Tpl2 inhibition or ablation by siRNA prevented the angiogenic signal-induced tube formation in Matrigel plug assay or aortic ring assay. Inhibiting Tpl2 also prevented the angiogenic factor-induced chemotactic motility and migration of endothelial cells. Tpl2 inhibition by CXCL1 or epidermal growth factor in endothelial cells was associated with inactivation of CCAAT/enhancer binding protein β, nuclear factor κ light-chain enhancer of activated B cells, and activating protein 1 and suppression of VEGF expression. Thus, Tpl2 inhibitors thwart Tpl2-regulated VEGF by inactivating transcription factors involved in angiogenic factor-triggered endothelial cell angiogenesis. These results suggest that the therapeutic inhibition of Tpl2 may extend beyond cancer and include the treatment of other diseases involving pathologic angiogenesis.
PMCID: PMC3769883  PMID: 24027429
12.  Endothelial Membrane Remodeling Is Obligate for Anti-Angiogenic Radiosensitization during Tumor Radiosurgery 
PLoS ONE  2010;5(8):e12310.
While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either “normalize” dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.
Methodology/Principal Findings
Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C16-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase+/+ mice or asmase−/− littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase−/− mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.
These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.
PMCID: PMC2924400  PMID: 20808818
13.  Carbon Ion Radiation Inhibits Glioma and Endothelial Cell Migration Induced by Secreted VEGF 
PLoS ONE  2014;9(6):e98448.
This study evaluated the effects of carbon ion and X-ray radiation and the tumor microenvironment on the migration of glioma and endothelial cells, a key process in tumorigenesis and angiogenesis during cancer progression. C6 glioma and human microvascular endothelial cells were treated with conditioned medium from cultures of glioma cells irradiated at a range of doses and the migration of both cell types, tube formation by endothelial cells, as well as the expression and secretion of migration-related proteins were evaluated. Exposure to X-ray radiation-conditioned medium induced dose-dependent increases in cell migration and tube formation, which were accompanied by an upregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2 and -9 expression. However, glioma cells treated with conditioned medium of cells irradiated at a carbon ion dose of 4.0 Gy showed a marked decrease in migratory potential and VEGF secretion relative to non-irradiated cells. The application of recombinant VEGF165 stimulated migration in glioma and endothelial cells, which was associated with increased FAK phosphorylation at Tyr861, suggesting that the suppression of cell migration by carbon ion radiation could be via VEGF-activated FAK signaling. Taken together, these findings indicate that carbon ion may be superior to X-ray radiation for inhibiting tumorigenesis and angiogenesis through modulation of VEGF level in the glioma microenvironment.
PMCID: PMC4043910  PMID: 24893038
14.  PI3-Kinase/p38 Kinase-Dependent E2F1 Activation Is Critical for Pin1 Induction in Tamoxifen-Resistant Breast Cancer Cells 
Molecules and Cells  2011;32(1):107-111.
Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. We have shown that Pin1, a peptidyl prolyl isomerase, is consistently overexpressed in TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) and plays a key role in the enhanced angiogenic potential of TAMR-MCF-7 cells. In the present study, we focused on signaling pathways for Pin1 up-regulation in TAMR-MCF-7 cells. Relative to MCF-7 cells, Pin1 gene transcription and E2 transcription factor1 (E2F1) expression were enhanced in TAMR-MCF-7 cells. E2F1 siRNA significantly reduced both the protein expression and the promoter transcriptional activity of Pin1. Activities of phosphatidylinositol 3-kinase (PI3K), extracellular signalregulated kinase (ERK) and p38 kinase were all higher in TAMR-MCF-7 cells than in control MCF-7 cells and the enhanced Pin1 and E2F1 expression in TAMR-MCF-7 cells was reversed by inhibition of PI3K or p38 kinase. Moreover, the higher production of vascular endothelial growth factor (VEGF) in TAMR-MCF-7 cells was significantly diminished by suppression of PI3K or p38 kinase. These results suggest that Pin1 overexpression and subsequent VEGF production in TAMR-MCF-7 cells are mediated through PI3-kinase or p38 kinase-dependent E2F1 activation.
PMCID: PMC3887657  PMID: 21573702
E2F1; p38 kinase; PI3-kinase; Pin1; tamoxifen-resistant breast cancer; VEGF
15.  Coupling In vitro and In vivo Paradigm Reveals a Dose Dependent Inhibition of Angiogenesis Followed by Initiation of Autophagy by C6-Ceramide 
The activity of N-hexanoyl-D-erythro-sphingosine, a C6-ceramide against angiogenesis was tested in vitro and in vivo. The effect of ceramide in inhibiting MCF-7 cancer cells was also determined. The aim of this study was to potentiate the effect of ceramide as anti-angiogenic compound that can regulate tumor induced angiogenesis.
C6-ceramide inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) tube formation in a dose-dependent manner within 24 hours. Ceramide at concentrations between 12.5 and 25 μM inhibited the viability of MCF-7 cells and reduced VEGF-induced cell migration in 24 hours. At 50 μM, ceramide induced MCF-7 cell death via autophagy as demonstrated by accumulation of MDC in ceramide-treated MCF-7 vacuoles. The expression of VEGF was reduced and the levels of cathepsin D in MCF-7 increased. In vivo, 50 μM ceramide caused a 40% reduction of new vessel formation in the CAM assay within 24 hours. Zebrafish exposed to 100 - 400 μM ceramide had a distinct disruption of blood vessel development at 48 hours post-fertilization. Ceramide-exposed embryos also had primary motoneurons exhibiting abnormal axonal trajectories and ectopic branching. Ceramide induced cell-death was not detected in the zebrafish assay. Collectively, these data indicate that ceramide is a potent anti-angiogenic compound and that the mechanism underlying its anti-angiogenic capabilities does not rely upon the induction of apoptosis.
PMCID: PMC3107471  PMID: 21647331
Ceramide; angiogenesis; zebrafish; MCF-7 cell-line; VEGF; apoptosis; autophagy
16.  Paeonol Oxime Inhibits bFGF-Induced Angiogenesis and Reduces VEGF Levels in Fibrosarcoma Cells 
PLoS ONE  2010;5(8):e12358.
We previously reported the anti-angiogenic activity of paeonol isolated from Moutan Cortex. In the present study, we investigated the negative effect of paeonol oxime (PO, a paeonol derivative) on basic fibroblast growth factor (bFGF)-mediated angiogenesis in human umbilical vein endothelial cells (HUVECs) (including tumor angiogenesis) and pro-survival activity in HT-1080 fibrosarcoma cell line.
Methodology/Principal Findings
We showed that PO (IC50  = 17.3 µg/ml) significantly inhibited bFGF-induced cell proliferation, which was achieved with higher concentrations of paeonol (IC50 over 200 µg). The treatment with PO blocked bFGF-stimulated migration and in vitro capillary differentiation (tube formation) in a dose-dependent manner. Furthermore, PO was able to disrupt neovascularization in vivo. Interestingly, PO (25 µg/ml) decreased the cell viability of HT-1080 fibrosarcoma cells but not that of HUVECs. The treatment with PO at 12.5 µg/ml reduced the levels of phosphorylated AKT and VEGF expression (intracellular and extracelluar) in HT-1080 cells. Consistently, immunefluorescence imaging analysis revealed that PO treatment attenuated AKT phosphorylation in HT-1080 cells.
Taken together, these results suggest that PO inhibits bFGF-induced angiogenesis in HUVECs and decreased the levels of PI3K, phospho-AKT and VEGF in HT-1080 cells.
PMCID: PMC2925949  PMID: 20808805
17.  Radiotherapy Suppresses Angiogenesis in Mice through TGF-βRI/ALK5-Dependent Inhibition of Endothelial Cell Sprouting 
PLoS ONE  2010;5(6):e11084.
Radiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions.
Principal Findings
Here we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-β type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation.
These results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.
PMCID: PMC2884035  PMID: 20552031
18.  Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer 
Strahlentherapie Und Onkologie  2014;190(12):1154-1162.
The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells.
Materials and methods
Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase–polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay.
Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells.
Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.
PMCID: PMC4240909  PMID: 24989178
Radiation; Vascular endothelial growth factor C; Lung cancer cells; Endothelial cells; PI3K/Akt/mTOR signaling pathway; Strahlung; Vaskulärer endothelialer Wachstumsfaktor C; Lungenkrebszellen; Endothelzellen; PI3K/Akt/mTOR-Signalweg
19.  Expression of the pro-angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 by human breast carcinomas is responsive to nutrient deprivation and endoplasmic reticulum stress 
Molecular Cancer  2004;3:4.
The expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-κB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-κB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines.
We found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153).
These results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.
PMCID: PMC343294  PMID: 14738568
Angiogenesis; glucose; glutamine; breast cancer; glucose-regulated protein 78 (GRP78); growth arrest and DNA damage inducible gene 153 (GADD153); tunicamycin; brefeldin A; thapsigargin; ionomycin; hypoxia
20.  VEGF and Pleiotrophin Modulate the Immune Profile of Breast Cancer 
Cancers  2010;2(2):970-988.
Angiogenesis, the sprouting of the existing vascular network to form new vessels, is required for the growth of solid tumors. For this reason, the primary stimulant of angiogenesis, vascular endothelial growth factor-A (VEGF), is an attractive target for tumor therapy. In fact, there are currently numerous anti-VEGF therapies in clinical development for the treatment of various cancers, including breast cancer. VEGF signals through two primary VEGF receptors, VEGFR1 and VEGFR2. VEGFR2 is the primary angiogenic receptor, and VEGFR1 has been implicated in macrophage chemotaxis and tumor cell survival and invasion. It has only been appreciated recently that the VEGFRs are expressed not only on endothelial cells and tumor cells but also on many host immune cells. Therefore, to better understand the effects of anti-VEGF therapy it is important to consider the effects of VEGF on all cells in the tumor microenvironment, including immune cells. Bevacizumab (Avastin®, Genetech), which binds VEGF and inhibits interaction with VEGFR1 and VEGFR2, was approved for the treatment of metastatic HER2/NEU-negative breast cancer in 2008, however, the majority of human mammary tumors are either innately resistant or will acquire resistance to anti-VEGF therapy. This suggests that these tumors activate alternate angiogenesis pathways. Pleiotrophin (PTN) is an important angiogenic cytokine in breast cancer and is expressed at high levels in approximately 60% of human breast tumors. PTN functions as an angiogenic factor and promotes remodeling of the tumor microenvironment as well as epithelial-mesenchymal transition (EMT). In addition, PTN can have profound effects on macrophage phenotype. The present review focuses on the functions of VEGF and PTN on immune cell infiltration and function in breast cancer. Furthermore, we will discuss how anti-VEGF therapy modulates the immune cell profile.
PMCID: PMC3835113  PMID: 24281102
VEGF; pleiotrophin; macrophage; anti-VEGF; angiogenesis
21.  Suppression of Choroidal Neovascularization Through Inhibition of APE1/Ref-1 Redox Activity 
The redox function of APE1/Ref-1 is a key regulator in pathological angiogenesis, such as retinal neovascularization and tumor growth. In this study, we examined whether inhibition of APE1/Ref-1 redox function by a small molecule inhibitor E3330 suppresses experimental choroidal neovascularization (CNV) in vitro and in vivo.
Primate choroid endothelial cells (CECs) received treatment of 0 to 100 μM E3330 alone or cotreatment of E3330 and 500 μg/mL anti-VEGF antibody bevacizumab. Choroid endothelial cell angiogenic function was examined by cell proliferation, migration, and tube formation assays. The effects of E3330 on NF-κB and STAT3 signaling pathways were determined by reporter gene assay, Western blot, and ELISA. Laser-induced CNV mouse model was used to test the effects of E3330 in vivo. Potential toxicity of E3330 was evaluated by TUNEL assay.
The E3330 of 25 to 100 μM dose-dependently suppressed CEC proliferation, migration, and tube formation, in the absence of noticeable cell toxicity. Lower doses of E3330 (10–20 μM) reduced the transcriptional activity of NF-κB and STAT3 without affecting protein phosphorylation of both molecules. At the same time, E3330 downregulated MCP-1 production in CECs. The antiangiogenic effect of E3330 was comparable and additive to bevacizumab. The E3330 effectively attenuated the progression of laser-induced CNV in mice after a single intravitreal injection.
The APE1/Ref-1 redox function regulates multiple transcription factors and inflammatory molecules, and is essential for CEC angiogenesis. Specific inhibition of APE1/Ref-1 redox function with E3330 may represent a promising novel treatment for wet AMD.
Selective inhibition of APE1/Ref-1 redox function has significant anti-angiogenic effect on choroidal neovascularization both in vitro and in vivo.
PMCID: PMC4106251  PMID: 24970265
APE1/Ref-1; redox function; transcription factors; choroidal neovascularization; antiangiogenesis
22.  Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen. 
Molecular Medicine  1995;1(2):182-193.
BACKGROUND: New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells. MATERIALS AND METHODS: Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production. RESULTS: Following 6-24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE. CONCLUSIONS: From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.
PMCID: PMC2229943  PMID: 8529097
23.  Glioblastoma-derived Leptin Induces Tube Formation and Growth of Endothelial Cells: Comparison with VEGF Effects 
BMC Cancer  2011;11:303.
Leptin is a pleiotropic hormone whose mitogenic and angiogenic activity has been implicated in the development and progression of several malignancies, including brain tumors. In human brain cancer, especially in glioblastoma multiforme (GBM), leptin and its receptor (ObR) are overexpressed relative to normal tissue. Until present, the potential of intratumoral leptin to exert proangiogenic effects on endothelial cells has not been addressed. Using in vitro models, we investigated if GBM can express leptin, if leptin can affect angiogenic and mitogenic potential of endothelial cells, and if its action can be inhibited with specific ObR antagonists. Leptin effects were compared with that induced by the best-characterized angiogenic regulator, VEGF.
We found that GBM cell lines LN18 and LN229 express leptin mRNA and LN18 cells secrete detectable amounts of leptin protein. Both lines also expressed and secreted VEGF. The conditioned medium (CM) of LN18 and LN 229 cultures as well as 200 ng/mL pure leptin or 50 ng/mL pure VEGF stimulated proliferation of human umbilical vein endothelial cells (HUVEC) at 24 h of treatment. Mitogenic effects of CM were ~2-fold greater than that of pure growth factors. Furthermore, CM treatment of HUVEC for 24 h increased tube formation by ~5.5-fold, while leptin increased tube formation by ~ 80% and VEGF by ~60% at 8 h. The mitogenic and angiogenic effects of both CM were blocked by Aca 1, a peptide ObR antagonist, and by SU1498, which inhibits the VEGF receptor. The best anti-angiogenic and cytostatic effects of Aca1 were obtained with 10 nM and 25 nM, respectively, while for SU1498, the best growth and angiogenic inhibition was observed at 5 μM. The combination of 5 μM SU1498 and Aca1 at 25 nM (growth inhibition) or at 10 nM (reduction of tube formation) produced superior effects compared with single agent treatments.
Our data provide the first evidence that LN18 and LN 229 human GBM cells express leptin mRNA and might produce biologically active leptin, which can stimulate tube formation and enhance proliferation of endothelial cells. Furthermore, we demonstrate for the first time that a peptide ObR antagonist inhibits proangiogenic and growth effects of leptin on endothelial cells, and that the pharmacological potential of this compound might be combined with drugs targeting the VEGF pathway.
PMCID: PMC3146945  PMID: 21771332
24.  Therapeutics targeting angiogenesis: Genetics and epigenetics, extracellular miRNAs and signaling networks (Review) 
Angiogenesis is a process of neovascular formation from pre-existing blood vessels, which consists of sequential steps for vascular destabilization, angiogenic sprouting, lumen formation and vascular stabilization. Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietin, Notch, transforming growth factor-β (TGF-β), Hedgehog and WNT signaling cascades orchestrate angiogenesis through the direct or indirect regulation of quiescence, migration and the proliferation of endothelial cells. Small-molecule compounds and human/humanized monoclonal antibodies interrupting VEGF signaling have been developed as anti-angiogenic therapeutics for cancer and neovascular age-related macular degeneration (AMD). Gene or protein therapy delivering VEGF, FGF2 or FGF4, as well as cell therapy using endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been developed as pro-angiogenic therapeutics for ischemic heart disease and peripheral vascular disease. Anti-angiogenic therapy for cancer and neovascular AMD is more successful than pro-angiogenic therapy for cardiovascular diseases, as VEGF-signal interruption is technically feasible compared with vascular re-construction. Common and rare genetic variants are detected using array-based technology and personal genome sequencing, respectively. Drug and dosage should be determined based on personal genotypes of VEGF and other genes involved in angiogenesis. As epigenetic alterations give rise to human diseases, polymer-based hydrogel film may be utilized for the delivery of drugs targeting epigenetic processes and angiogenesis as treatment modalities for cardiovascular diseases. Circulating microRNAs (miRNAs) in exosomes and microvesicles are applied as functional biomarkers for diagnostics and prognostics, while synthetic miRNAs in polymer-based nanoparticles are applicable for therapeutics. A more profound understanding of the spatio-temporal interactions of regulatory signaling cascades and advances in personal genotyping and miRNA profiling are required for the optimization of targeted therapy.
PMCID: PMC3812243  PMID: 23863927
bevacizumab; sunitinib; sorafenib; dovitinib; ponatinib; AZD4547; breast cancer; lung cancer; colorectal cancer; gastric cancer; field cancerization; poly(lactic-co-glycolic acid); chitosan; vascular medicine
25.  Low Doses of Ionizing Radiation Promote Tumor Growth and Metastasis by Enhancing Angiogenesis 
PLoS ONE  2010;5(6):e11222.
Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.
PMCID: PMC2888592  PMID: 20574535

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