BACKGROUND

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor and a regulator of physiological and pathological angiogenesis. Four different proteins are produced by alternative splicing of a unique transcript generated from a single-copy gene. Knowledge of the chromosomal location of the VEGF gene would help in determining a linkage to any known human congenital syndrome and/or to known chromosomal rearrangements in tumors.

RESULTS

A human chromosome mapping panel was used to assign the VEGF gene to human chromosomes by polymerase chain reaction using VEGF-specific oligonucleotide primers. Amplified DNA fragments were fractionated on a 1% agarose gel. A single band of the expected size was obtained only from the DNA of those hybrid cell lines that contained the human chromosome 6. Three YAC clones containing the VEGF gene were obtained by screening the ICI Diagnostics library. In situ hybridization was then used to locate the VEGF gene in the 6p21.3 region.

CONCLUSIONS

The location of the VEGF gene in the 6p21.3 region is a potential starting point for a linkage study. In addition, the isolation of YAC clones containing the VEGF gene will contribute to the construction of the physical map of this chromosomal region.

The development of a vascular supply is essential not only for organ development and differentiation during embryogenesis but also for wound healing and reproductive functions in the adult Folkman, 1995). Angiogenesis is also implicated in the pathogenesis of a variety of disorders: proliferative retinopathies, age-related macular degeneration, tumors, rheumatoid arthritis, and psoriasis (Folkman, 1995; Garner, 1994). Several potential regulators of angiogenesis have been identified, including fibroblast growth factor-a (aFGF), bFGF, transforming growth factor-alpha (TGF-alpha), TGF-beta, hepatocyte growth factor/scatter factor (HGF/SF), tumor necrosis factor-alpha (TNF-alpha), angiogenin, and interleukin-8 (IL-8) (Folkman and Shing, 1992; Risau, 1997). More recently, the angiopoietins, the ligands of the Tie-2 receptor (Suri et al., 1996; Maisonpierre et al., 1997), have been identified. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific mitogen. The finding that VEGF was potent and specific for vascular endothelial cells and, unlike bFGF, freely diffusible, led to the hypothesis that this molecule plays a unique role in the regulation of physiological and pathological angiogenesis (Ferrara and Henzel, 1989: Leung et al., 1989). Over the last few years, several additional members of the VEGF gene family have been identified, including placenta growth factor (PIGF) (Maglione et al., 1991,1993), VEGF-B (Olofsson et al., 1996), VEGF-C (Joukov et al., 1996; Lee et al., 1996), and VEGF-D (Orlandini et al., 1996. Achen et al., 1998). There is compelling evidence that VEGF plays an essential role in the development and differentiation of the cardiovascular system (Ferrara and Davis-Smyth, 1997).

Angiogenesis and inflammation are central processes through which the tumor microenvironment influences tumor growth. We have demonstrated recently that peroxisome proliferator-activated receptor (PPAR)alpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of thrombospondin (TSP)-1 and prevents tumor growth. Hence, we speculated that pharmacologic activation of PPARalpha would promote tumor growth. Surprisingly, the PPARalpha agonist fenofibrate potently suppressed primary tumor growth in mice. This effect was not mediated by cancer-cell-autonomous antiproliferative mechanisms but by the inhibition of angiogenesis and inflammation in the host tissue. Although PPARalpha-deficient tumors were still susceptible to fenofibrate, absence of PPARalpha in the host animal abrogated the potent antitumor effect of fenofibrate. In addition, fenofibrate suppressed endothelial cell proliferation and VEGF production, increased TSP-1 and endostatin, and inhibited corneal neovascularization. Thus, both genetic abrogation of PPARalpha as well as its activation by ligands cause tumor suppression via overlapping antiangiogenic pathways. These findings reveal the potential utility of the well tolerated PPARalpha agonists beyond their use as lipid-lowering drugs in anticancer therapy. Our results provide a mechanistic rationale for evaluating the clinical benefits of PPARalpha agonists in cancer treatment, alone and in combination with other therapies.

For solid tumors and metastatic lesions, tumor vascularity is a critical factor in assessing response to therapy. Here we report the first example, to our knowledge, of (64)Cu-labeled vascular endothelial growth factor 121 (VEGF(121)) for PET of VEGF receptor (VEGFR) expression in vivo.

METHODS

VEGF(121) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and then labeled with (64)Cu for small-animal PET of mice bearing different sized U87MG human glioblastoma xenografts. Blocking experiments and ex vivo histopathology were performed to confirm the in vivo results.

RESULTS

There were 4.3 +/- 0.2 DOTA molecules per VEGF(121), and the VEGFR2 binding affinity of DOTA-VEGF(121) was comparable to VEGF(121). (64)Cu labeling of DOTA-VEGF(121) was achieved in 90 +/- 10 min and the radiolabeling yield was 87.4% +/- 3.2%. The specific activity of (64)Cu-DOTA-VEGF(121) was 3.2 +/- 0.1 GBq/mg with a radiochemical purity of >98%. Small-animal PET revealed rapid, specific, and prominent uptake of (64)Cu-DOTA-VEGF(121) in small U87MG tumors (high VEGFR2 expression) but significantly lower and sporadic uptake in large U87MG tumors (low VEGFR2 expression). No appreciable renal clearance of (64)Cu-DOTA-VEGF(121) was observed, although the kidney uptake was relatively high likely due to VEGFR1 expression. Blocking experiments, immunofluorescence staining, and western blot confirmed the VEGFR specificity of (64)Cu-DOTA-VEGF(121).

CONCLUSIONS

Successful demonstration of the ability of (64)Cu-DOTA-VEGF(121) to visualize VEGFR expression in vivo may allow for clinical translation of this radiopharmaceutical for imaging tumor angiogenesis and guiding antiangiogenic treatment, especially patient selection and treatment monitoring of VEGFR-targeted cancer therapy.

Exercise and muscle contractions create a powerful stimulus for structural remodeling of the vasculature. An increase in flow velocity through a vessel increases shear stress, a major stimulus for enlargement of conduit vessels. This leads to an endothelial-dependent, nitric oxide-dependent enlargement of the vessel. Increased flow within muscle, in the absence of contractions, leads to an enhanced capillarity by intussusceptive angiogenesis, a process of capillary splitting by intraluminal longitudinal divide. In contrast, sprouting angiogenesis requires extensive endothelial cell proliferation, with degradation of the extracellular matrix to permit migration and tube formation. This occurs during muscle adaptations to chronic contractions and/or muscle overload. The angiogenic growth factor VEGF appears to be an important element in angiogenesis. Recent advances in research have identified hemodynamic and mechanical stimuli that upregulate angiogenic processes, demonstrated a complexity of potent growth factors and interactions with their corresponding receptors, detected an interaction of cellular signaling events, and identified important tissue reorganization processes that must be coordinated to effect vascular remodeling. It is likely that much of this information is applicable to the vascular remodeling that occurs in response to exercise and/or muscle contractions.

Aberrant genetic alternations in human gliomas, such as amplification of epidermal growth factor receptor, mutation and/or deletion of tumor suppressor gene PTEN, and mutations of PIK3CA, contribute to constitutive activation of the phosphatidylinositol 3-kinase (PI3K) pathway. We investigated the potential antitumor activity of NVP-BEZ235, which is a novel dual PI3K/mammalian target of rapamycin (mTOR) inhibitor in gliomas. The compound suppressed glioma cell proliferation with IC(50) values in the low nanomolar range by specifically inhibiting the activity of target proteins including Akt, S6K1, S6, and 4EBP1 in the PI3K/Akt/mTOR signaling pathway. NVP-BEZ235 treatment of glioma cell lines led to G(1) cell cycle arrest and induced autophagy. Furthermore, expression of the vascular endothelial growth factor (VEGF), which is an important angiogenic modulator in glioma cells, was significantly decreased, suggesting that NVP-BEZ235 may also exert an antiangiogenic effect. Preclinical testing of the therapeutic efficacy of NVP-BEZ235 showed that it significantly prolonged the survival of tumor-bearing animals without causing any obvious toxicity. Tumor extracts harvested from animals after treatment showed that the compound inhibited the activity of target proteins in the PI3K/Akt/mTOR cascade. Immunohistochemical analyses also showed a significant reduction in staining for VEGF von Willebrand factor (factor VIII) in NVP-BEZ235-treated tumor sections compared with controls, further confirming that NVP-BEZ235 has an antiangiogenic effect in vivo. We conclude from these findings that NVP-BEZ235 antagonizes PI3K and mTOR signaling and induces cell cycle arrest, down-regulation of VEGF, and autophagy. These results warrant further development of NVP-BEZ235 for clinical trials for human gliomas or other advanced cancers with altered PI3K/Akt/mTOR signaling.

The hypoxia-inducible transcription factor-1 (HIF-1) is central to a number of pathological processes through the induction of specific genes such as vascular endothelial growth factor (VEGF). Even though HIF-1 is highly regulated by cellular oxygen levels, other elements of the inflammatory and tumor microenvironment were shown to influence its activity under normal oxygen concentration. Among others, recent studies indicated that transforming growth factor (TGF) beta increases the expression of the regulatory HIF-1alpha subunit, and induces HIF-1 DNA binding activity. Here, we demonstrate that TGFbeta acts on HIF-1alpha accumulation and activity by increasing HIF-1alpha protein stability. In particular, we demonstrate that TGFbeta markedly and specifically decreases both mRNA and protein levels of a HIF-1alpha-associated prolyl hydroxylase (PHD), PHD2, through the Smad signaling pathway. As a consequence, the degradation of HIF-1alpha was inhibited as determined by impaired degradation of a reporter protein containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. Moreover, inhibition of the TGFbeta1 converting enzyme, furin, resulted in increased PHD2 expression, and decreased basal HIF-1alpha and VEGF levels, suggesting that endogenous production of bioactive TGFbeta1 efficiently regulates HIF-1-targeted genes. This was reinforced by results from HIF-1alpha knock-out or HIF-1alpha-inhibited cells that show impairment in VEGF production in response to TGFbeta. This study reveals a novel mechanism by which a growth factor controls HIF-1 stability, and thereby drives the expression of specific genes, through the regulation of PHD2 levels.

OBJECTIVE

Stress has long been believed to influence carcinogenesis, but little is known about physiological mechanisms that may underlie these effects. We have recently observed lower levels of vascular endothelial growth factor (VEGF) in ovarian cancer patients with greater social support, whereas higher VEGF was found in patients with greater distress. The goal of this study was to examine possible mechanisms underlying these relationships.

METHODS

The effects of stress-related mediators including norepinephrine (NE), epinephrine, isoproterenol (a nonspecific beta-adrenergic agonist), and cortisol on the production of VEGF by the ovarian cell lines SKOV3 and EG were investigated.

RESULTS

NE and isoproterenol significantly enhanced VEGF production by SKOV3 cells, and all three of the adrenergic agonists enhanced VEGF production by EG cells. These effects were blocked by the beta antagonist propranolol, supporting a role for beta-adrenergic receptors in these effects. Reverse transcriptase-PCR studies indicated constitutive expression of beta-1 and beta-2 adrenergic receptors on both cell lines. Effects of cortisol on VEGF production varied according to the specific cell line and dose, with stimulating effects on SKOV3 at pharmacologic doses (1000 nM) and on EG at physiological stress level doses (10 nM), and inhibitory effects on EG at pharmacologic doses. Although priming with cortisol blunted NE-induced VEGF production from both cell lines at 3 h, significant increases in VEGF were still seen. Priming with cortisol enhanced isoproterenol-induced VEGF production from SKOV3.

CONCLUSIONS

These findings provide the first experimental evidence of a pathway by which biobehavioral stress mediators could directly contribute to the progression of ovarian tumors.

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague-Dawley rats (n=9). Following implantation, the local release profiles were determined by measuring the activity of (125)I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, microCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p=0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p=0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.

OBJECTIVE

Vascular endothelial growth factor (VEGF)-C/VEGF-receptor 3 (VEGF-R3) signal plays a significant role in lymphangiogenesis and tumor metastasis based on its effects on lymphatic vessels. However, little is known about the effect of inhibiting VEGF-R3 on lymphangiogenesis and lymph node metastases using a small-molecule kinase inhibitor.

METHODS

We evaluated the effect of E7080, a potent inhibitor of both VEGF-R2 and VEGF-R3 kinase, and bevacizumab on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of human breast cancer using MDA-MB-231 cells that express excessive amounts of VEGF-C. Lymphangiogenesis was determined by lymphatic vessel density (LVD) and angiogenesis by microvessel density (MVD).

RESULTS

In contrast to MDA-MB-435 cells, which expressed a similar amount of VEGF to MDA-MB-231 cells with an undetectable amount of VEGF-C, only MDA-MB-231 exhibited lymphangiogenesis in the primary tumor. E7080 but not bevacizumab significantly decreased LVD within the MDA-MB-231 tumor. E7080 and bevacizumab decreased MVD in both the MDA-MB-231 and MDA-MB-435 models. E7080 significantly suppressed regional lymph nodes and distant lung metastases of MDA-MB-231, whereas bevacizumab significantly inhibited only lung metastases. E7080 also decreased both MVD and LVD within the metastatic nodules at lymph nodes after resection of the primary tumor.

CONCLUSIONS

Inhibition of VEGF-R3 kinase with E7080 effectively decreased LVD within MDA-MB-231 tumors, which express VEGF-C. Simultaneous inhibition of both VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy to control regional lymph node and distant lung metastases.

The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

BACKGROUND

Bevacizumab (Avastin; rhuMab VEGF), a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), significantly prolongs survival when added to intravenous 5-fluorouracil-based chemotherapy in first-line metastatic colorectal cancer (CRC) treatment. Because antiangiogenic agents might inhibit wound healing, we assessed postoperative wound healing complications in two randomized trials of 5 mg/kg bevacizumab in CRC treatment.

METHODS

We assessed the wound healing complications in patients who: (1) underwent cancer surgery 28-60 days before study treatment and (2) underwent major surgery during study treatment. Cases were reviewed for wound healing complications occurring < or = 60 days after surgery.

RESULTS

With cancer surgery 28-60 days before study treatment, wound healing complications occurred in 3/230 (1.3%) bevacizumab-treated patients and 1/194 (0.5%) control patients. With major surgery during study treatment, 10/75 bevacizumab-treated patients (13%) and 1/29 control patients (3.4%) had wound healing complications. Bevacizumab-treated patients experienced complications with surgery < or = 30 and 31-60 days after the last dose.

CONCLUSIONS

Bevacizumab administered in combination with 5-fluorouracil/leucovorin-based chemotherapy 28-60 days after primary cancer surgery caused no increased risk of wound healing complications compared with chemotherapy alone. While wound healing complications were increased in patients who had major surgery during bevacizumab therapy, the majority of bevacizumab-treated patients experienced no complications.

BALB-neuT mice expressing an activated rat c-erbB-2/neu transgene under the mouse mammary tumor virus long terminal repeat show enhanced hematopoiesis with hyperproduction of myeloid-derived suppressor cells (MDSC) because of vascular endothelial growth factor (VEGF) secreted by the tumor. Here, we show that both tumor and stromal cells express matrix metalloproteinase-9 (MMP-9), thereby increasing the levels of pro-MMP-9 in the sera of tumor-bearing mice. Treatment with amino-biphosphonates impaired tumor growth, significantly decreased MMP-9 expression and the number of macrophages in tumor stroma, and reduced MDSC expansion both in bone marrow and peripheral blood by dropping serum pro-MMP-9 and VEGF. We dissected the role of tumor-derived MMP-9 from that secreted by stromal leukocytes by transplanting bone marrow from MMP-9 knockout mice into BALB-neuT mice. Although bone marrow progenitor-derived MMP-9 had a major role in driving MDSC expansion, amino-biphosphonate treatment of bone marrow chimeras further reduced both myelopoiesis and the supportive tumor stroma, thus enhancing tumor necrosis. Moreover, by reducing MDSC, amino-biphosphonates overcome the tumor-induced immune suppression and improved the generation and maintenance of antitumor immune response induced by immunization against the p185/HER-2. Our data reveal that suppression of MMP-9 activity breaks the vicious loop linking tumor growth and myeloid cell expansion, thus reducing immunosuppression. Amino-biphosphonates disclose a specific MMP-9 inhibitory activity that may broaden their application above their current usage.

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.

Ischemia is a known stimulus for vascular growth. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are believed to contribute to new blood vessel growth, but the mechanism for this contribution is unknown. To elucidate how BM cells are able to form new blood vessels, a novel murine model of soft tissue ischemia was developed in lethally irradiated mice with BM reconstituted from either tie2/lacZ or ROSA/green fluorescent protein (GFP) mice (n = 24). BM-derived EPCs were recruited to ischemic tissue within 72 hours, and the extent of recruitment was directly proportional to the degree of tissue ischemia. At 7 days, there were persistently elevated levels of vascular endothelial growth factor (VEGF) (2.5-fold) and circulating VEGF receptor-2/CD11(-) (flk-1(+)/CD11(-)) cells (18-fold) which correlated with increased numbers of BM-derived EPCs within ischemic tissue. The cells were initially located extravascularly as proliferative clusters. By day 14, these clusters coalesced into vascular cords, which became functional vessels by day 21. In vitro examination of human EPCs from healthy volunteers (n = 10) confirmed that EPC proliferation, adhesion, and chemotaxis were all significantly stimulated in hypoxic conditions. We conclude that BM-derived cells produce new blood vessels via localized recruitment, proliferation, and differentiation of circulating cells in a sequence of events markedly different from existing paradigms of angiogenesis.

Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfer restores neovascularization in a mouse model of diabetes.

Sprouting angiogenesis is a dynamic process in which endothelial cells collectively migrate, shape new lumenized tubes, make new connections, and remodel the nascent network into a hierarchically branched and functionally perfused vascular bed. Endothelial cells in the nascent sprout adopt two distinct cellular phenotypes--known as tip and stalk cells--with specialized functions and gene expression patterns. VEGF and Notch signaling engage in an intricate cross talk to balance tip and stalk cell formation and to regulate directed tip cell migration and stalk cell proliferation. In this article, we summarize the current knowledge and implications of the tip/stalk cell concepts and the quantitative and dynamic integration of VEGF and Notch signaling in tip and stalk cell selection.

The role of angiogenesis in atherosclerosis and other cardiovascular diseases has emerged as a major unresolved issue. Angiogenesis has attracted interest from opposite perspectives. Angiogenic cytokine therapy has been widely regarded as an attractive approach both for treating ischemic heart disease and for enhancing arterioprotective functions of the endothelium; conversely, a variety of studies suggest that neovascularization contributes to the growth of atherosclerotic lesions and is a key factor in plaque destabilization leading to rupture. Here, we critically review the evidence supporting a role for angiogenesis and angiogenic factors in atherosclerosis and neointima formation, emphasizing the problems raised by some of the landmark studies and the suitability of animal models of atherosclerosis and neointimal thickening for investigating the role of angiogenesis. Because many of the relevant studies have focused on the role of vascular endothelial growth factor (VEGF), we consider this work in the wider context of VEGF biology and in light of recent experience from clinical trials of VEGF and other angiogenic cytokines for ischemic heart disease. Also discussed are recent findings suggesting that, although angiogenesis may contribute to neointimal growth, it is not required for the initiation of intimal thickening. Our assessment of the evidence leads us to conclude that, although microvessels are a feature of advanced human atherosclerotic plaques, it remains unclear whether angiogenesis either plays a central role in the development of atherosclerosis or is responsible for plaque instability. Furthermore, current evidence from clinical trials of both proangiogenic and antiangiogenic therapies does not suggest that inhibition of angiogenesis is likely to be a viable therapeutic strategy for cardiovascular disease.

VEGF can be secreted in multiple isoforms with variable affinity for extracellular proteins and different abilities to induce vascular morphogenesis, but the molecular mechanisms behind these effects remain unclear. Here, we show molecular distinctions between signaling initiated from soluble versus matrix-bound VEGF, which mediates a sustained level of VEGFR2 internalization and clustering. Exposure of endothelial cells to matrix-bound VEGF elicits prolonged activation of VEGFR2 with differential phosphorylation of Y1214, and extended activation kinetics of p38. These events require association of VEGFR2 with beta1 integrins. Matrix-bound VEGF also promotes reciprocal responses on beta1 integrin by inducing its association with focal adhesions; a response that is absent upon exposure to soluble VEGF. Inactivation of beta1 integrin blocks the prolonged phosphorylation of Y1214 and consequent activation of p38. Combined, these results indicate that when in the context of extracellular matrix, activation of VEGFR2 is distinct from that of soluble VEGF in terms of recruitment of receptor partners, phosphorylation kinetics, and activation of downstream effectors.

The low rate of approval of novel anti-cancer agents underscores the need for better preclinical models of therapeutic response as neither xenografts nor early-generation genetically engineered mouse models (GEMMs) reliably predict human clinical outcomes. Whereas recent, sporadic GEMMs emulate many aspects of their human disease counterpart more closely, their ability to predict clinical therapeutic responses has never been tested systematically. We evaluated the utility of two state-of-the-art, mutant Kras-driven GEMMs--one of non-small-cell lung carcinoma and another of pancreatic adenocarcinoma--by assessing responses to existing standard-of-care chemotherapeutics, and subsequently in combination with EGFR and VEGF inhibitors. Standard clinical endpoints were modeled to evaluate efficacy, including overall survival and progression-free survival using noninvasive imaging modalities. Comparisons with corresponding clinical trials indicate that these GEMMs model human responses well, and lay the foundation for the use of validated GEMMs in predicting outcome and interrogating mechanisms of therapeutic response and resistance.

There is growing evidence for a connection between inflammation and tumor development, and the nuclear factor kappa B (NF-kappaB), a proinflammatory transcription factor, is hypothesized to promote tumorigenesis. Although the genetic evidence for the hypothesis has been lacking, recent papers have lent credence to this hypothesis. It has been reported that constitutive NF-kappaB activation in inflammatory bowel diseases (IBDs) increases risk of colorectal cancer (CRC) in the patients with the number of years of active disease. NF-kappaB activation might induce cellular transformation, mediate cellular proliferation, prevent the elimination of pre-neoplastic and fully malignant cells by up-regulating the anti-apoptosis proteins. Furthermore, NF-kappaB may contribute to the progression of CRC by regulating the expression of diverse target genes that are involved in cell proliferation (Cyclin D1), angiogenesis (VEGF, IL-8, COX2), and metastasis (MMP9). These findings implicate NF-kappaB inhibition as an important therapeutic target in CRC. However, due to lack of knowledge about the specific roles of different NF-kappaB subunits in different stage of carcinogenesis, and compounds to block specific subunits of NF-kappaB family, it will be a long time before the coming of targeting NF-kappaB in CRC therapy.

Electroconvulsive seizure therapy (ECS) is a clinically proven treatment for depression and is often effective even in patients resistant to chemical antidepressants. However, the molecular mechanisms underlying the therapeutic efficacy of ECS are not fully understood. One theory that has gained attention is that ECS and other antidepressants increase the expression of select neurotrophic factors that could reverse or block the atrophy and cell loss resulting from stress and depression. To further address this topic, we examined the expression of other neurotrophic-growth factors and related signaling pathways in the hippocampus in response to ECS using a custom growth factor microarray chip. We report the regulation of several genes that are involved in growth factor and angiogenic-endothelial signaling, including neuritin, stem cell factor, vascular endothelial growth factor (VEGF), VGF (nonacronymic), cyclooxygenase-2, and tissue inhibitor of matrix metalloproteinase-1. Some of these, as well as other growth factors identified, including VEGF, basic fibroblast growth factor, and brain-derived neurotrophic factor, have roles in mediating neurogenesis and cell proliferation in the adult brain. We also examined gene expression in the choroid plexus and found several growth factors that are enriched in this vascular tissue as well as regulated by ECS. These data suggest that an amplification of growth factor signaling combined with angiogenic mechanisms could have an important role in the molecular action of ECS. This study demonstrates the applicability of custom-focused microarray technology in addressing hypothesis-driven questions regarding the action of antidepressants.

BACKGROUND

Vascular endothelial growth factor (VEGF) plays an important role in many diseases of the posterior pole that are characterized by macular edema and/or intraocular neovascularization. Recently anti-VEGF agents such as ranibizumab and pegaptanib sodium have been shown to be beneficial in the treatment of choroidal neovascularization (CNV) secondary to age-related macular degeneration (ARMD). However in most parts of the world, both pegaptanib sodium and ranibizumab are not readily available. Bevacizumab, a humanized recombinant monoclonal IgG antibody that binds and inhibits all VEGF isoforms, has been proposed as an alternative treatment option.

METHODS

A total of 1,265 consecutive patients were injected with bevacizumab for diseases such as proliferative diabetic retinopathy, diabetic macular edema, retinal vein occlusions, and CNV of several etiologies including ARMD at eight Latin American institutions from 1 September 2005 to 31 January 2006. Of these 1,265, 92 were excluded because they were injected once and lost to follow-up. The remaining 1,173 patients constitute the subjects of this retrospective, multicenter, open label, uncontrolled interventional case series that reports the cumulative systemic and ocular adverse events following intravitreal bevacizumab during 12 months of follow-up. Patients were examined at baseline and then monthly. If the patients were unable to attend the 12-month visit, a telephone interview was conducted to assess for possible systemic complications.

RESULTS

A total of 4,303 intravitreal injections of bevacizumab on 1,310 eyes was reported. All 1,173 patients were accounted for at the 12-month visit. Systemic adverse events were reported in 18 (1.5%) patients. These included seven (0.59%) cases of an acute elevation of systemic blood pressure, six (0.5%) cerebrovascular accidents, five (0.4%) myocardial infarctions, two (0.17%) iliac artery aneurysms, two (0.17%) toe amputations and five (0.4%) deaths. Ocular complications included seven (0.16%) bacterial endophthalmitis, seven (0.16%) tractional retinal detachments, four (0.09%) uveitis, and a case (0.02%) each of rhegmatogenous retinal detachment and vitreous hemorrhage.

CONCLUSIONS

Despite the limited follow-up, repeated intravitreal injections of either 1.25 mg or 2.5 mg of bevacizumab appears to be safe and well tolerated during the 1st year.