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1.  Studies of Vascular Endothelial Growth Factor in Asthma and Chronic Obstructive Pulmonary Disease 
Vascular endothelial growth factor (VEGF) is a potent stimulator of vascular angiogenesis, permeability, and remodeling that also plays important roles in wound healing and tissue cytoprotection. To begin to define the roles of VEGF in diseases like asthma and COPD, we characterized the effects of lung-targeted transgenic VEGF165 and defined the innate immune pathways that regulate VEGF tissue responses. The former studies demonstrated that VEGF plays an important role in Th2 inflammation because, in addition to stimulating angiogenesis and edema, VEGF induced eosinophilic inflammation, mucus metaplasia, subepithelial fibrosis, myocyte hyperplasia, dendritic cell activation, and airways hyperresponsiveness via IL-13–dependent and -independent mechanisms. VEGF was also produced at sites of aeroallergen-induced Th2 inflammation, and VEGF receptor blockade ameliorated adaptive Th2 inflammation and Th2 cytokine elaboration. The latter studies demonstrated that activation of the RIG-like helicase (RLH) innate immune pathway using viral pathogen–associated molecular patterns such as Poly(I:C) or viruses ameliorated VEGF-induced tissue responses. In accord with these findings, Poly(I:C)-induced RLH activation also abrogated aeroallergen-induced Th2 inflammation. When viewed in combination, these studies suggest that VEGF excess can contribute to the pathogenesis of Th2 inflammatory disorders such as asthma and that abrogation of VEGF signaling via RLH activation can contribute to the pathogenesis of viral disorders such as virus-induced COPD exacerbations. They also suggest that RLH activation may be a useful therapeutic strategy in asthma and related disorders.
PMCID: PMC3359071  PMID: 22052929
asthma; chronic obstructive pulmonary disease; virus; RIG-like helicase; mitochondrial antiviral signaling molecule
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.  Targeting Neuropilin-1 to Inhibit VEGF Signaling in Cancer: Comparison of Therapeutic Approaches 
PLoS Computational Biology  2006;2(12):e180.
Angiogenesis (neovascularization) plays a crucial role in a variety of physiological and pathological conditions including cancer, cardiovascular disease, and wound healing. Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Multiple VEGF receptors are expressed on endothelial cells, including signaling receptor tyrosine kinases (VEGFR1 and VEGFR2) and the nonsignaling co-receptor Neuropilin-1. Neuropilin-1 binds only the isoform of VEGF responsible for pathological angiogenesis (VEGF165), and is thus a potential target for inhibiting VEGF signaling. Using the first molecularly detailed computational model of VEGF and its receptors, we have shown previously that the VEGFR–Neuropilin interactions explain the observed differential effects of VEGF isoforms on VEGF signaling in vitro, and demonstrated potent VEGF inhibition by an antibody to Neuropilin-1 that does not block ligand binding but blocks subsequent receptor coupling. In the present study, we extend that computational model to simulation of in vivo VEGF transport and binding, and predict the in vivo efficacy of several Neuropilin-targeted therapies in inhibiting VEGF signaling: (a) blocking Neuropilin-1 expression; (b) blocking VEGF binding to Neuropilin-1; (c) blocking Neuropilin–VEGFR coupling. The model predicts that blockade of Neuropilin–VEGFR coupling is significantly more effective than other approaches in decreasing VEGF–VEGFR2 signaling. In addition, tumor types with different receptor expression levels respond differently to each of these treatments. In designing human therapeutics, the mechanism of attacking the target plays a significant role in the outcome: of the strategies tested here, drugs with similar properties to the Neuropilin-1 antibody are predicted to be most effective. The tumor type and the microenvironment of the target tissue are also significant in determining therapeutic efficacy of each of the treatments studied.
Neuropilin is a co-receptor for some of the isoforms of the vascular endothelial growth factor (VEGF) family. The presence of Neuropilin on endothelial or other cells increases binding of these isoforms to their signaling receptor VEGFR2, thus increasing pro-angiogenesis signaling and stimulating vascular growth. Neuropilin is thus a suitable target for anti-angiogenesis therapy, which holds promise for the treatment of vasculature-dependent diseases such as cancer and diabetic retinopathy. In this study, Mac Gabhann and Popel perform computational simulations of VEGF transport in breast cancer, using a previously validated model of VEGF–VEGF receptor interactions, as well as geometrical information on the tumor itself—tumor cells, vasculature, and extracellular matrix. Three different molecular therapies targeting Neuropilin are tested in silico, and the simulations predict that one of these therapies will be effective at reducing VEGFR2 signaling in certain types (or subtypes) of tumors, while the others will not. Thus, we demonstrate that identification of a target molecule is not sufficient; different therapeutic strategies targeting the same molecule may result in different outcomes.
PMCID: PMC1761657  PMID: 17196035
4.  Endogenous, or therapeutically induced, type I interferon responses differentially modulate Th1/Th17-mediated autoimmunity in the CNS 
Immunology and Cell Biology  2012;90(5):505-509.
Different viruses trigger pattern recognition receptor systems, such as Toll-like receptors or cytosolic RIG-I like helicases (RLH), and thus induce early type I interferon (IFN-I) responses. Such responses may confer protection until adaptive immunity is activated to an extent that the pathogen can be eradicated. Interestingly, the same innate immune mechanisms that are relevant for early pathogen defense have a role in ameliorating experimental autoimmune encephalomyelitis (EAE), a rodent model of human multiple sclerosis. We and others found that mice devoid of a component of the IFN-I receptor (Ifnar1−/−) showed significantly enhanced autoimmune disease of the central nervous system (CNS). A detailed analysis revealed that in wild-type mice IFN-I triggering of myeloid cells was instrumental in reducing brain damage. A more recent study indicated that similar to Ifnar1−/− mice, RLH-signaling-deficient mice showed enhanced autoimmune disease of the CNS as well. Moreover, when peripherally treated with synthetic RLH ligands wild-type animals with EAE disease showed reduced clinical scores. Under such conditions, IFN-I receptor triggering of dendritic cells had a crucial role. The therapeutic effect of treatment with RLH ligands was associated with negative regulation of Th1 and Th17 T-cell responses within the CNS. These experiments are consistent with the hypothesis that spatiotemporal conditions of, and cell types involved in, disease-ameliorating IFN-I responses differ significantly, depending on whether they were endogenously induced in the context of EAE pathogenesis within the CNS or upon therapeutic RLH triggering in the periphery. It is attractive to speculate that RLH triggering represents a new strategy to treat multiple sclerosis by stimulating endogenous immunoregulatory IFN-I responses.
PMCID: PMC3365287  PMID: 22430251
experimental autoimmune encephalomyelitis; interferon-beta; new therapy approach
5.  The role of AMP-activated protein kinase in the functional effects of vascular endothelial growth factor-A and -B in human aortic endothelial cells 
Vascular Cell  2011;3:9.
Vascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis.
Human aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK.
Incubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca2+/calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.
These data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.
PMCID: PMC3094250  PMID: 21507243
6.  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
7.  Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability 
The formation of unstable, leaky neovessels underlies the pathogenesis of many chronic inflammatory diseases. Granzyme B (GZMB) is an immune-derived serine protease that accumulates in the extracellular matrix (ECM) during chronic inflammation and is capable of cleaving fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM through its interaction with FN. As GZMB levels are elevated in chronic inflammatory diseases that are associated with increased vascular permeability, the role of GZMB in the regulation of VEGF bioavailability and vascular permeability were assessed.
Methods and Results
GZMB was added to either VEGF-bound to FN or VEGF-bound to endothelial cell (EC)-derived ECM. Supernatants containing released VEGF were assessed to determine VEGF activity by treating EC and evaluating VEGF receptor-2 (VEGFR2) phosphorylation. GZMB released VEGF from both FN and from EC-derived matrix, while GZMB inhibition prevented FN cleavage and VEGF release. GZMB-mediated VEGF release resulted in significant phosphorylation of VEGFR2. The role of GZMB-mediated VEGF release in altering vascular permeability was also assessed in vivo using a Miles/Evan’s Blue permeability assay. GZMB induced a significant VEGF-dependent increase in vascular permeability in vivo that was reduced in the presence of an anti-VEGF neutralizing antibody. Inflammatory-mediated vascular leakage was also assessed in GZMB-KO mice using a delayed-type hypersensitivity model. GZMB-KO mice exhibited reduced microvascular leakage compared to C57\B6 controls.
GZMB increases vascular permeability in part through the proteolytic release of ECM-sequestered VEGF leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.
PMCID: PMC4074428  PMID: 24791744 CAMSID: cams4292
Fibronectin; Granzyme B; Inflammation; VEGF; Vascular permeability
8.  AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1) 
Cytokine  2013;64(1):337-342.
The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine- glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3′UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.
PMCID: PMC3770750  PMID: 23769804
AUF1; hnRNP D; VEGF; soluble VEGFR-1; sFlt-1; AUF1-RGG peptide; AU-rich elements; AURE; inflammation
9.  TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA 
PLoS ONE  2009;4(5):e5674.
In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.
Principal Findings
Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.
Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.
PMCID: PMC2682567  PMID: 19479062
10.  Computational Model of Vascular Endothelial Growth Factor Spatial Distribution in Muscle and Pro-Angiogenic Cell Therapy 
PLoS Computational Biology  2006;2(9):e127.
Members of the vascular endothelial growth factor (VEGF) family of proteins are critical regulators of angiogenesis. VEGF concentration gradients are important for activation and chemotactic guidance of capillary sprouting, but measurement of these gradients in vivo is not currently possible. We have constructed a biophysically and molecularly detailed computational model to study microenvironmental transport of two isoforms of VEGF in rat extensor digitorum longus skeletal muscle under in vivo conditions. Using parameters based on experimental measurements, the model includes: VEGF secretion from muscle fibers; binding to the extracellular matrix; binding to and activation of endothelial cell surface VEGF receptors; and internalization. For 2-D cross sections of tissue, we analyzed predicted VEGF distributions, gradients, and receptor binding. Significant VEGF gradients (up to 12% change in VEGF concentration over 10 μm) were predicted in resting skeletal muscle with uniform VEGF secretion, due to non-uniform capillary distribution. These relative VEGF gradients were not sensitive to extracellular matrix composition, or to the overall VEGF expression level, but were dependent on VEGF receptor density and affinity, and internalization rate parameters. VEGF upregulation in a subset of fibers increased VEGF gradients, simulating transplantation of pro-angiogenic myoblasts, a possible therapy for ischemic diseases. The number and relative position of overexpressing fibers determined the VEGF gradients and distribution of VEGF receptor activation. With total VEGF expression level in the tissue unchanged, concentrating overexpression into a small number of adjacent fibers can increase the number of capillaries activated. The VEGF concentration gradients predicted for resting muscle (average 3% VEGF/10 μm) is sufficient for cellular sensing; the tip cell of a vessel sprout is approximately 50 μm long. The VEGF gradients also result in heterogeneity in the activation of blood vessel VEGF receptors. This first model of VEGF tissue transport and heterogeneity provides a platform for the design and evaluation of therapeutic approaches.
It is not currently possible to experimentally quantify the gradients of protein concentration in the extracellular space in vivo. However, the concentration gradients of vascular endothelial growth factor (VEGF) are essential for both initiation and directed guidance of new blood vessels. The authors develop a computational model of VEGF transport in tissue in vivo (skeletal muscle, though the method is applicable to other tissues and other proteins) with realistic geometry and including biophysical interactions of VEGF, its receptors, and the extracellular matrix. Using this model, the authors predict for the first time the distribution of VEGF concentration and VEGF receptor activation throughout the tissue. VEGF concentration gradients are significant, up to 12% change in VEGF concentration over 10 μm in resting muscle. Transplanting VEGF-overexpressing myocytes (for therapeutic induction of blood vessel growth) increases the gradients significantly. Endothelial cells in sprouting vessels are approximately 50 μm long, and therefore the predicted gradients across the cell are high and sufficient for chemotactic guidance of the new vessels. The VEGF concentration gradients also result in significant heterogeneity in the activation of VEGF receptors on blood vessels throughout the tissue, a possible reason for the sporadic nature of sprout initiation.
PMCID: PMC1570371  PMID: 17002494
11.  Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies 
BMC Systems Biology  2011;5:193.
Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer.
Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10-5 cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF121 comprises at least 25% of the VEGF secreted by tumor cells.
This study explores the optimal drug characteristics required for an anti-VEGF agent to have a therapeutic effect and the tumor-specific properties that influence the response to therapy. Our model provides a framework for investigating the use of VEGF-neutralizing drugs for personalized medicine treatment strategies.
PMCID: PMC3229549  PMID: 22104283
12.  Transforming Growth Factor-β1 Downregulates Vascular Endothelial Growth Factor-D Expression in Human Lung Fibroblasts via the Jun NH2-Terminal Kinase Signaling Pathway 
Molecular Medicine  2014;20(1):120-134.
Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.
PMCID: PMC3960396  PMID: 24515257
13.  Intrauterine Pulmonary Hypertension Impairs Angiogenesis In Vitro 
Rationale: Mechanisms that impair angiogenesis in neonatal persistent pulmonary hypertension (PPHN) are poorly understood.
Objectives: To determine if PPHN alters fetal pulmonary artery endothelial cell (PAEC) phenotype and impairs growth and angiogenesis in vitro, and if altered vascular endothelial growth factor–nitric oxide (VEGF–NO) signaling contributes to this abnormal phenotype.
Methods: Proximal PAECs were harvested from fetal sheep that had undergone partial ligation of the ductus arteriosus in utero (PPHN) and age-matched control animals. Growth and tube formation ± VEGF and NO stimulation and inhibition were studied in normal and PPHN PAECs. Western blot analysis was performed for VEGF, VEGF receptor-2 (VEGF-R2), and endothelial NO synthase (eNOS) protein content. NO production with VEGF administration was measured in normal and PPHN PAECs.
Measurements and Main Results: PPHN PAECs demonstrate decreased growth and tube formation in vitro. VEGF and eNOS protein expression were decreased in PPHN PAECs, whereas VEGF-R2 protein expression was not different. VEGF and NO increased PPHN PAEC growth and tube formation to values achieved in normal PAECs. VEGF inhibition decreased growth and tube formation in normal and PPHN PAECs. NOS inhibition decreased growth in normal and PPHN PAECs, but tube formation was only reduced in normal PAECs. NO reversed the inhibitory effects of VEGF-R2 inhibition on tube formation in normal and PPHN PAECs. VEGF increased NO production in normal and PPHN PAECs.
Conclusions: PPHN in utero causes sustained impairment of PAEC phenotype in vitro, with reduced PAEC growth and tube formation and down-regulation of VEGF and eNOS protein. VEGF and NO enhanced growth and tube formation of PPHN PAECs.
PMCID: PMC2176095  PMID: 17823355
persistent pulmonary hypertension of the newborn; angiogenesis; vascular endothelial growth factor; nitric oxide; endothelial cells
14.  Lung vascular endothelial growth factor expression induces local myeloid dendritic cell activation 
Clinical immunology (Orlando, Fla.)  2009;132(3):371-384.
We previously demonstrated that vascular endothelial growth factor (VEGF) expression in the murine lung increases local CD11c+ MHCII+ DC number and activation. In this study, employing a multicolor flow cytometry, we report increases in both myeloid (mDC) and plasmacytoid (pDC) DC in the lungs of VEGF transgenic (tg) compared to WT mice. Lung pDC from VEGF tg mice exhibited higher levels of activation with increased expression of MHCII and costimulatory molecules. As VEGF tg mice display an asthma-like phenotype and lung mDC play a critical role in asthmatic setting, studies were undertaken to further characterize murine lung mDC. Evaluations of sorted mDC from VEGF tg lungs demonstrated a selective upregulation of cathepsin K, MMP-8, -9, -12, and -14, and chemokine receptors as compared to those obtained from WT control mice. They also had increased VEGFR2 but downregulated VEGFR1 expression. Analysis of chemokine and regulatory cytokine expression in these cells showed an upregulation of macrophage chemotactic protein-3 (MCP-3), thymus-expressed chemokine (TECK), secondary lymphoid organ chemokine (SLC), macrophage-derived chemokine (MDC), IL-1β, IL-6, IL-12 and IL-13. The antigen (Ag) OVA-FITC uptake by lung DC and the migration of Ag-loaded DC to local lymph nodes were significantly increased in VEGF tg mice compared to WT mice. Thus, VEGF may predispose the lung to inflammation and/or repair by activating local DC. It regulates lung mDC expression of innate immunity effector molecules. The data presented here demonstrate how lung VEGF expression functionally affects local mDC for the transition from the innate response to a Th2-type inflammatory response.
PMCID: PMC2780370  PMID: 19553159
Rodent; lung; inflammation; dendritic cells; cell activation
15.  The early responses of VEGF and its receptors during acute lung injury: implication of VEGF in alveolar epithelial cell survival 
Critical Care  2006;10(5):R130.
The function of the vascular endothelial growth factor (VEGF) system in acute lung injury (ALI) is controversial. We hypothesized that the role of VEGF in ALI may depend upon the stages of pathogenesis of ALI.
To determine the responses of VEGF and its receptors during the early onset of ALI, C57BL6 mice were subjected to intestinal ischemia or sham operation for 30 minutes followed by intestinal ischemia-reperfusion (IIR) for four hours under low tidal volume ventilation with 100% oxygen. The severity of lung injury, expression of VEGF and its receptors were assessed. To further determine the role of VEGF and its type I receptor in lung epithelial cell survival, human lung epithelial A549 cells were treated with small interference RNA (siRNA) to selectively silence related genes.
IIR-induced ALI featured interstitial inflammation, enhancement of pulmonary vascular permeability, increase of total cells and neutrophils in the bronchoalveolar lavage (BAL), and alveolar epithelial cell death. In the BAL, VEGF was significantly increased in both sham and IIR groups, while the VEGF and VEGF receptor (VEGFR)-1 in the lung tissues were significantly reduced in these two groups. The increase of VEGF in the BAL was correlated with the total protein concentration and cell count. Significant negative correlations were observed between the number of VEGF or VEGFR-1 positive cells, and epithelial cells undergoing cell death. When human lung epithelial A549 cells were pre-treated with 50 nM of siRNA either against VEGF or VEGFR-1 for 24 hours, reduced VEGF and VEGFR-1 levels were associated with reduced cell viability.
These results suggest that VEGF may have dual roles in ALI: early release of VEGF may increase pulmonary vascular permeability; reduced expression of VEGF and VEGFR-1 in lung tissue may contribute to the death of alveolar epithelial cells.
PMCID: PMC1751039  PMID: 16968555
16.  Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity 
PLoS Pathogens  2012;8(7):e1002747.
Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.
Author Summary
In response to pathogens, such as viruses and bacteria, infected cells defend themselves by generating a set of cytokines called type I interferon (IFN). Since Type I IFN (namely IFN alpha and beta) are potent antiviral agents, understanding the cellular mechanisms by which infected cells produce type I IFN is required to identify novel cellular targets for future antiviral therapies. Notably, a protein called Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) was demonstrated to be an essential mediator of this antiviral response. However, how TRAF3 reacts in response to a viral infection is still not totally understood. We now demonstrate that, through its capacity to interact with other proteins (namely Sec16A and p115) that normally control protein secretion, TRAF3 resides close to the nucleus in uninfected cells, in a region called the ER-to-Golgi Intermediate Compartment (ERGIC). Following viral infection, the ERGIC reorganizes into small punctate structures allowing TRAF3 to associate with Mitochondrial AntiViral Signaling (MAVS), an essential adaptor of the anti-viral type I IFN response. Thus, our study reveals an unpredicted role of the protein secretion system for the proper localization of TRAF3 and the antiviral response.
PMCID: PMC3390413  PMID: 22792062
17.  Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16 
Oncotarget  2015;6(12):10222-10238.
Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.
PMCID: PMC4496351  PMID: 25912305
VEGF; transgenic mice; pulmonary tumorigenesis; magnetic resonance imaging (MRI); miRNA therapy
18.  Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding 
eLife  2014;3:e03720.
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1VEGF−). Nrp1VEGF− mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.
eLife digest
Blood flows through blood vessels to carry oxygen and nutrients towards, and waste away from, the cells of the body. New blood vessels are formed not only during development but also throughout life as part of normal tissue growth and repair. However, blood vessels may also form as a consequence of diseases, such as cancer. For example, tumors often stimulate the growth of new blood vessels to ensure a good supply of blood carrying nutrients and oxygen. As such, some anti-cancer therapies try to stop blood vessels from developing in an attempt to slow down or prevent tumor growth.
New blood vessels often form by branching off from existing vessels. One molecule that stimulates this branching process is called vascular endothelial growth factor (or VEGF for short). Three ‘receptor’ proteins found on the outside of cells can bind to the VEGF molecule and then trigger a response inside the cell that guides the development of new blood vessels. VEGF and its receptor proteins—including one called NRP1—are being investigated as a possible target for drugs that could treat cancer and other diseases affecting blood vessels. However, the exact mechanisms that control the formation of new blood vessels are not fully understood, which makes it difficult to develop these treatments.
Now Gelfand et al. have created mice whose NRP1 receptors cannot bind VEGF. These mice unexpectedly survive to adulthood and develop normal blood vessels. This outcome is in contrast to mice that lack NRP1, which normally die as embryos and have severe defects with their nerves and blood vessels. Gelfand et al. instead found that mice that only lack NRP1 in the cells of their blood vessels had less of another receptor protein called VEGFR2 on the surface of these cells. This result suggests that NRP1 controls blood vessel development, not by binding to VEGF but by affecting how much of the VEGFR2 receptor is available to interact with VEGF.
These findings challenge the long-held view of how NRP1 functions and lead Gelfand et al. to suggest a new mechanism: NRP1 interacts with VEGFR2, rather than with VEGF, to control the formation of new blood vessels. Future work will aim to uncover how these interactions regulate the normal development of blood vessels, and if other molecules that bind to NRP1 are involved in this process. Furthermore, these findings may help to guide the on-going efforts to develop drugs that target NRP1 into treatments that are effective against diseases that involve problems with blood vessels—including diabetes, immune disorders, and cancer.
PMCID: PMC4197402  PMID: 25244320
Neuropilin-1; developmental angiogenesis; VEGFR2; VEGF; mouse genetics; mouse
19.  Specific VEGF sequestering to biomaterials: Influence of serum stability 
Acta biomaterialia  2013;9(11):8823-8831.
Vascular endothelial growth factor (VEGF) was originally discovered as a tumor-derived factor that is able to induce endothelial cell behavior associated with angiogenesis. It has been implicated during wound healing for the induction of endothelial cell proliferation, tube formation and blood vessel remodeling. However, previous investigations into the biological effect of VEGF concluded that a particular range of growth factor concentrations are required for healthy vasculature to form, motivating recent studies to regulate VEGF activity via molecular sequestering to biomaterials. Numerous VEGF sequestering strategies have been developed, and they have typically relied on extracellular matrix mimicking moieties that are not specific for VEGF and can affect many growth factors simultaneously. We describe here a strategy for efficient, specific VEGF sequestering with poly(ethylene glycol) (PEG) microspheres, using peptides designed to mimic VEGF receptor type 2 (VEGFR2). By immobilizing two distinct peptides with different serum stabilities, we examined the effect of serum on the specific interaction between peptide-containing PEG microspheres and VEGF. We addressed the hypothesis that VEGF sequestering in serum-containing solutions would be influenced by the serum stability of the VEGF-binding peptide. We further hypothesized that soluble VEGF could be sequestered in serum-containing cell culture media, resulting in decreased VEGF-dependent proliferation of human umbilical vein endothelial cells. We show that soluble VEGF concentration can be effectively regulated in serum-containing environments via specific molecular sequestering, which suggests potential clinical applications.
PMCID: PMC4149317  PMID: 23816648
Angiogenesis; VEGF; Sequestration; Poly(ethylene glycol); Extracellular matrix
20.  Peroxiredoxin 1 Stimulates Endothelial Cell Expression of VEGF via TLR4 Dependent Activation of HIF-1α 
PLoS ONE  2012;7(11):e50394.
Chronic inflammation leads to the formation of a pro-tumorigenic microenvironment that can promote tumor development, growth and differentiation through augmentation of tumor angiogenesis. Prostate cancer (CaP) risk and prognosis are adversely correlated with a number of inflammatory and angiogenic mediators, including Toll-like receptors (TLRs), NF-κB and vascular endothelial growth factor (VEGF). Peroxiredoxin 1 (Prx1) was recently identified as an endogenous ligand for TLR4 that is secreted from CaP cells and promotes inflammation. Inhibition of Prx1 by CaP cells resulted in reduced expression of VEGF, diminished tumor vasculature and retarded tumor growth. The mechanism by which Prx1 regulates VEGF expression in normoxic conditions was investigated in the current study. Our results show that incubation of mouse vascular endothelial cells with recombinant Prx1 caused increases in VEGF expression that was dependent upon TLR4 and required hypoxia inducible factor-1 (HIF-1) interaction with the VEGF promoter. The induction of VEGF was also dependent upon NF-κB; however, NF-κB interaction with the VEGF promoter was not required for Prx1 induction of VEGF suggesting that NF-κB was acting indirectly to induce VEGF expression. The results presented here show that Prx1 stimulation increased NF-κB interaction with the HIF-1α promoter, leading to enhanced promoter activity and increases in HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. Prx1 induced HIF-1 also promoted NF-κB activity, suggesting the presence of a positive feedback loop that has the potential to perpetuate Prx1 induction of angiogenesis. Strikingly, inhibition of Prx1 expression in CaP was accompanied with reduced expression of HIF-1α. The combined findings of the current study and our previous study suggest that Prx1 interaction with TLR4 promotes CaP growth potentially through chronic activation of tumor angiogenesis.
PMCID: PMC3503895  PMID: 23185615
21.  M27 Expressed by Cytomegalovirus Counteracts Effective Type I Interferon Induction of Myeloid Cells but Not of Plasmacytoid Dendritic Cells 
Journal of Virology  2014;88(23):13638-13650.
In healthy individuals, the functional immune system effectively confines human cytomegalovirus (CMV) replication, while viral immune evasion and persistence preclude sterile immunity. Mouse CMV (MCMV) is a well-established model to study the delicate CMV-host balance. Effective control of MCMV infection depends on the induction of protective type I interferon (IFN-I) responses. Nevertheless, it is unclear whether in professional antigen-presenting cell subsets MCMV-encoded evasins inhibit the induction of IFN-I responses. Upon MCMV treatment, enhanced expression of MCMV immediate-early and early proteins was detected in bone marrow cultures of macrophages and myeloid dendritic cells compared with plasmacytoid dendritic cell cultures, whereas plasmacytoid dendritic cells mounted more vigorous IFN-I responses. Experiments with Toll-like receptor (TLR)- and/or RIG-I like helicase (RLH)-deficient cell subsets revealed that upon MCMV treatment of myeloid cells, IFN-I responses were triggered independently of TLR and RLH signaling, whereas in plasmacytoid dendritic cells, IFN-I induction was strictly TLR dependent. Macrophages and myeloid dendritic cells treated with either UV-inactivated MCMV or live MCMV that lacked the STAT2 antagonist M27 mounted significantly higher IFN-I responses than cells treated with live wild-type MCMV. In contrast, plasmacytoid dendritic cells responded similarly to UV-inactivated and live MCMV. These experiments illustrated that M27 not only inhibited IFN-I-mediated receptor signaling, but also evaded the induction of IFN responses in myeloid dendritic cells. Furthermore, we found that additional MCMV-encoded evasins were needed to efficiently shut off IFN-I responses of macrophages, but not of myeloid dendritic cells, thus further elucidating the subtle adjustment of the host-pathogen balance.
IMPORTANCE MCMV may induce IFN-I responses in fibroblasts and epithelial cells, as well as in antigen-presenting cell subsets. We focused on the analysis of IFN-I responses of antigen-presenting cell subsets, including plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages, which are all triggered by MCMV to mount IFN-I responses. Interestingly, myeloid dendritic cells and macrophages, but not plasmacytoid dendritic cells, are readily MCMV infected and support viral gene expression. As expected from previous studies, plasmacytoid dendritic cells sense MCMV Toll-like receptor 9 (TLR9) dependently, whereas in myeloid cells, IFN-I induction is entirely TLR and RLH independent. MCMV-encoded M27 does not impair the IFN-I induction of plasmacytoid dendritic cells, while in myeloid dendritic cells, it reduces IFN-I responses. In macrophages, M27 plus other, not yet identified evasins profoundly inhibit the induction of IFN-I responses. Collectively, these results illustrate that MCMV has evolved diverse mechanisms to differentially modulate IFN-I responses in single immune cell subsets.
PMCID: PMC4248974  PMID: 25231302
22.  Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2 
Angiogenesis and lymphangiogenesis mediated by vascular endothelial growth factors (VEGFs) are main features of chronic inflammation and tumors. Secreted phospholipases A2 (sPLA2s) are overexpressed in inflammatory lung diseases and cancer and they activate inflammatory cells by enzymatic and receptor-mediated mechanisms. We investigated the effect of sPLA2s on the production of VEGFs from human macrophages purified from the lung tissue of patients undergoing thoracic surgery. Primary macrophages express VEGF-A, VEGF-B, VEGF-C, and VEGF-D at both mRNA and protein level. Two human sPLA2s (group IIA and group X) induced the expression and release of VEGF-A and VEGF-C from macrophages. Enzymatically-inactive sPLA2s were as effective as the active enzymes in inducing VEGF production. Me-Indoxam and RO092906A, two compounds that block receptor-mediated effects of sPLA2s, inhibited group X-induced release of VEGF-A. Inhibition of the MAPK p38 by SB203580 also reduced sPLA2-induced release of VEGF-A. Supernatants of group X-activated macrophages induced an angiogenic response in chorioallantoic membranes that was inhibited by Me-Indoxam. Stimulation of macrophages with group X sPLA2 in the presence of adenosine analogs induced a synergistic increase of VEGF-A release and inhibited TNF-α production through a cooperation between A2A and A3 receptors. These results demonstrate that sPLA2s induce production of VEGF-A and VEGF-C in human macrophages by a receptor-mediated mechanism independent from sPLA2 catalytic activity. Thus, sPLA2s may play an important role in inflammatory and/or neoplastic angiogenesis and lymphangiogenesis.
PMCID: PMC3073479  PMID: 20357262
23.  Uridine Composition of the Poly-U/UC Tract of HCV RNA Defines Non-Self Recognition by RIG-I 
PLoS Pathogens  2012;8(8):e1002839.
Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5′-triphosphate (5′-ppp) on the viral RNA in association with a 3′ poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine “core” of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5′-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5′-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5′-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.
Author Summary
Pathogen recognition receptors (PRRs) are critical components of the innate immune response to viral pathogens, and function in the host to recognize pathogen-associated molecular patterns (PAMPs) in viral proteins or nucleic acids. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic PRR that senses viral RNA inside an infected cell. RIG-I recognizes hepatitis C virus (HCV) RNA as non-self through the presence of both a 5′-triphosphate (5′-ppp) and a 3′ poly-U/UC tract within the viral RNA. Here we examined the RNA structure-function attributes that define the HCV poly-U/UC tract as non-self to RIG-I, including nucleotide composition. We found that the 34 nucleotide poly-uridine “core” (U-core) within the HCV poly-U/UC tract RNA was required for non-self recognition by RIG-I, and interspersed ribocytosine nucleotides were also important to induce optimal RIG-I signaling. RIG-I/RNA binding studies revealed that RIG-I formed weaker interactions with HCV RNAs lacking poly-U sequences, and RNA interaction with multiple domains of RIG-I was required to activate signaling. Finally, RIG-I recognition of the U-core within the poly-U/UC tract activated anti-HCV responses in vitro and hepatic innate immune responses in vivo. Our studies identify long poly-uridine sequences with interspersed ribocytosines as an HCV PAMP motif that drives optimal RIG-I signaling.
PMCID: PMC3410852  PMID: 22912574
24.  Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia☆ 
Neurobiology of Disease  2014;71:245-259.
Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform.
We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event – leading to the preferential expression of VEGF-A165b over VEGF165a – prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a.
After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain.
We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.
Graphical abstract
•The different vegf-a splice variants, VEGF-A165a and VEGF-A165b have pro- and anti-nociceptive actions respectively.•Pro-nociceptive actions of VEGF-A165a are dependent on TRPV1.•Alternative pre-mRNA splicing underpins peripheral sensitization by VEGF-A isoforms in normal and neuropathic animals.
PMCID: PMC4194316  PMID: 25151644
VEGF-A, vascular endothelial growth factor-A; SRPK1, serine arginine protein kinase 1; SRSF1, serine arginine splice factor 1; VEGFR2, vascular endothelial growth factor receptor 2; IB4, isolectin B4; TRPV1, transient receptor potential vanilloid 1; CV, conduction velocity; PSNI, partial saphenous nerve ligation injury; DRG, dorsal root ganglia; Vascular endothelial growth factor A; Alternative mRNA splicing; Neuropathy; Nociceptors
25.  Endothelial NADPH oxidase 4 mediates vascular endothelial growth factor receptor 2–induced intravitreal neovascularization in a rat model of retinopathy of prematurity 
Molecular Vision  2014;20:231-241.
NADPH oxidase–generated reactive oxygen species (ROS) are implicated in angiogenesis. Isoforms of NADPH oxidase NOX1, NOX2, and NOX4 are reported to be expressed in endothelial cells (ECs). Of these, NOX1 and NOX2 have been reported to contribute to intravitreal neovascularization (IVNV) in oxygen-induced retinopathy (OIR) models. In this study, we tested the hypothesis that the isoform NOX4 in ECs contributed to vascular endothelial growth factor (VEGF)–induced angiogenesis and IVNV.
Isoforms of NADPH oxidase mRNA were measured in several types of cultured vascular ECs: human retinal microvascular ECs (hRMVECs), choroidal ECs (CECs), and human umbilical vascular ECs (HUVECs) using real-time PCR. Newborn rat pups and dams were placed into an OIR model that cycled oxygen concentration between 50% and 10% every 24 h for 14 days, and then were placed in room air (RA) for an additional 4 days (rat OIR model). NOX4 expression in retinal lysates from the RA–raised pups at postnatal day 0 (P0), P14, and P18 was determined with western blots. STAT3 activation was determined as the ratio of phosphorylated STAT3 to total STAT3 with western blot analysis of retinal lysates from pups raised in RA or from the rat OIR model at P18. Semiquantitative assessment of the density of NOX4 colabeling with lectin-stained retinal ECs was determined by immunolabeling of retinal cryosections from P18 pups in OIR or in RA. In hRMVECs transfected with NOX4 siRNA and treated with VEGF or control, 1) ROS generation was measured using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester fluorescence assay and 2) phosphorylated VEGF receptor 2 and STAT3, and total VEGFR2 and STAT3 were measured in western blot analyses. VEGF-stimulated hRMVEC proliferation was measured following transfection with NOX4 siRNA or STAT3 siRNA, or respective controls.
NOX4 was the most prevalent isoform of NADPH oxidase in vascular ECs. NOX4 expression in retinal lysates was significantly decreased during development in RA. Compared to RA, the expression of retinal NOX4 increased at P18. At p18 OIR, semiquantitative assessment of the density of lectin and NOX4 colabeling in retinal vascular ECs was greater in retinal cryosections and activated STAT3 was greater in retinal lysates when compared to the RA-raised pups. In cultured hRMVECs, knockdown of NOX4 by siRNA transfection inhibited VEGF-induced ROS generation. VEGF induced a physical interaction of phosphorylated-VEGFR2 and NOX4. Knockdown of NOX4: 1) reduced VEGFR2 activation but did not abolish it and 2) abolished STAT3 activation in response to VEGF. Knockdown of either NOX4 or STAT3 inhibited VEGF-induced EC proliferation.
Our data suggest that in a model representative of human retinopathy of prematurity, NOX4 was increased at a time point when IVNV developed. VEGF-activated NOX4 led to an interaction between VEGF-activated VEGFR2 and NOX4 that mediated EC proliferation via activation of STAT3. Altogether, our results suggest that NOX4 may regulate VEGFR2-mediated IVNV through activated STAT3.
PMCID: PMC3945806  PMID: 24623966

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