MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important roles in various cellular functions and tumor development. Recent studies have indicated that miR-21 is one of the important miRNAs associated with tumor growth and metastasis, but the role and molecular mechanism of miR-21 in regulating tumor angiogenesis remain to be elucidated. In this study, miR-21 was overexpressed by transfecting pre-miR-21 into human prostate cancer cells and tumor angiogenesis was assayed using chicken chorioallantoic membrane (CAM). We found that overexpression of miR-21 in DU145 cells increased the expression of HIF-1α and VEGF, and induced tumor angiogenesis. AKT and extracellular regulated kinases (ERK) 1/2 are activated by miR-21. Inhibition of miR-21 by the antigomir blocked this process. Overexpression of the miR-21 target, PTEN, also inhibited tumor angiogenesis by partially inactivating AKT and ERK and decreasing the expression of HIF-1 and VEGF. The AKT and ERK inhibitors, LY294002 and U0126, suppressed HIF-1α and VEGF expression and angiogenesis. Moreover, inhibition of HIF-1α expression alone abolished miR-21-inducing tumor angiogenesis, indicating that HIF-1α is required for miR-21-upregulated angiogenesis. Therefore, we demonstrate that miR-21 induces tumor angiogenesis through targeting PTEN, leading to activate AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression; HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.

BACKGROUND

Many studies have shown that angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors. Recently, several angiogenic factors have been identified. Vascular endothelial growth factor (VEGF) is thought to be one such angiogenic factor and is also thought to be a selective mitogen for endothelial cells. We investigated the correlation between the expression of VEGF and the progression of gastric carcinoma.

METHODS

One hundred twenty-nine specimens resected from patients with gastric carcinoma were investigated by staining with a polyclonal antibody against VEGF. Correlations between the expression of VEGF, microvessel density, and various clincopathologic factors were studied.

RESULTS

Microvessel density, determined by immunostaining for Factor VIII related antigen, was significantly higher in VEGF-positive tumors than in VEGF-negative tumors. VEGF positivity was correlated with vessel involvement, lymph node metastasis, and liver metastasis. Moreover, patients with VEGF-positive tumors had a significantly poorer prognosis than those with VEGF-negative tumors. Multivariate analysis indicated that the expression of VEGF is an independent prognostic factor in patients with gastric cancer. According to the mode of recurrence, the frequency of hepatic metastases was significantly increased among patients with VEGF-positive tumors.

CONCLUSIONS

The expression of VEGF may be a good prognostic indicator for patients with gastric carcinoma and may also be useful as a predictor of the mode of recurrence in patients with gastric carcinoma.

The growth of solid tumors in vivo beyond 1-2 mm in diameter requires induction and maintenance of an angiogenic response. This can occur through the release of various angiogenic growth factors from tumor cells. One such factor is vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a secreted and specific mitogen for vascular endothelial cells. We show that one of the most commonly encountered genetic changes detected in human cancer, i.e., expression of mutant ras oncogenes, is associated with marked up-regulation of VEGF/VPF in transformed epithelial cells. Thus, elevation of the levels of both VEGF/VPF mRNA and secreted functional protein were detected in human and rodent tumor cell lines expressing mutant K-ras or H-ras oncogenes, respectively. Genetic disruption of the mutant K-ras allele in human colon carcinoma cells was associated with a reduction in VEGF/VPF activity. Furthermore, pharmacological disruption of mutant RAS protein function in H-ras transformed rat intestinal epithelial cells by treatment with L-739,749 (a protein farnesyltransferase inhibitor) caused a significant suppression of VEGF/VPF. The results suggest that dominantly acting ras oncogenes may contribute to the growth of solid tumors in vivo not only by a direct effect on tumor cell proliferation but also indirectly, i.e., by facilitating tumor angiogenesis. Hence, pharmacologically targeting mutant ras oncogenes could conceivably suppress solid tumor growth in vivo, in part, by inhibiting tumor-induced angiogenesis.

Blood-retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, but the molecular changes that cause this pathology are unclear. Occludin is a transmembrane component of interendothelial tight junctions that may regulate permeability at the BRB. In this study, we examined the effects of vascular endothelial growth factor (VEGF) and diabetes on vascular occludin content and barrier function. Sprague-Dawley rats were made diabetic by intravenous streptozotocin injection, and age-matched animals served as controls. After 3 months, BRB permeability was quantified by intravenous injection of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), Mr 66 kDa, and 10-kDa rhodamine-dextran (R-D), followed by digital image analysis of retinal sections. Retinal fluorescence intensity for FITC-BSA increased 62% (P < or = 0.05), but R-D fluorescence did not change significantly. Occludin localization at interendothelial junctions was confirmed by immunofluorescence, and relative protein content was determined by immunoblotting of retinal homogenates. Retinal occludin content decreased approximately 35% (P < or = 0.03) in the diabetic versus the control animals, whereas the glucose transporter GLUT1 content was unchanged in rat retinas. Additionally, treatment of bovine retinal endothelial cells in culture with 0.12 nmol/l or 12 nmol/l VEGF for 6 h reduced occludin content 46 and 54%, respectively. These data show that diabetes selectively reduces retinal occludin protein expression and increases BRB permeability. Our findings suggest that the elevated VEGF in the vitreous of patients with diabetic retinopathy increases vascular permeability by downregulating occludin content. Decreased tight junction protein expression may be an important means by which diabetes causes increased vascular permeability and contributes to macular edema.

Angiogenesis, or formation of new blood vessels from pre-existing ones, is essential for normal development and wound healing/reproductive functions in adults. Abnormal regulation of angiogenesis has been implicated in the pathogenesis of several disorders, including cancer. Vascular endothelial growth factor (VEGF)-A is a pivotal stimulator of angiogenesis because its binding to VEGF receptors has been shown to promote endothelial cell migration and proliferation, two key features required for the development of new blood vessels. In addition, VEGF-A increases vascular permeability, which may also contribute to angiogenesis and tumor growth. Recognition of the central role of VEGF-A in angiogenesis has led to the hypothesis that its inhibition may represent a novel and effective approach to the treatment of cancer and other conditions characterized by pathologic angiogenesis. Several lines of evidence support this idea, and early clinical experience with the humanized anti-VEGF-A monoclonal antibody bevacizumab (Avastin, rhuMAb-VEGF; Genentech, South San Francisco, CA) has been encouraging. Clinical efficacy of antiangiogenic therapy with bevacizumab is being evaluated in several phase 3 trials in various types of cancer, as well as in patients with age-related macular degeneration.

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription-PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF >> 1 ng/10(6) cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin-Sepharose-enriched supernatant mainly detected the isoform VEGFVEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis.

The central role of vascular endothelial growth factor (VEGF) in angiogenesis in health and disease makes it attractive both as a therapeutic target for anti-angiogenic drugs and as a pro-angiogenic cytokine for the treatment of ischaemic heart disease. While VEGF binds to two receptor protein tyrosine kinases, VEGFR1 (Flt-1) and VEGFR2 (KDR), most biological functions of VEGF are mediated via VEGFR2, and the role of VEGFR1 is currently unknown. Neuropilin-1, a non-tyrosine kinase transmembrane molecule, may function as a co-receptor for VEGFR2. Considerable progress has recently been made towards delineating the signal transduction pathways distal to activation of VEGFR2. Activation of the mitogen-activated protein kinase, protein kinase C and Akt pathways are all strongly implicated in mediating diverse cellular biological functions of VEGF, including cell survival, proliferation, the generation of nitric oxide and prostacyclin and angiogenesis. Upregulation of metalloproteinases, activation of focal adhesion kinase and interactions between VEGF receptors and integrins are strongly implicated in VEGF-induced endothelial cell migration. Recent findings suggest important roles for the vasodilators nitric oxide and prostacyclin, in linking post-receptor signaling networks to downstream biological effects and in mediating some in vivo endothelial functions of VEGF.

Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.

The overexpression in tumor cells of (proto)-oncogenic receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or ErbB2/neu (also known as HER-2) is generally thought to contribute to the development of solid tumors primarily through their effects on promoting uncontrolled cell proliferation. However, agents that antagonize the function of the protein products encoded by these (proto)-oncogenes are known to behave in vivo in a cytotoxic-like manner. This implies that such oncogenes may regulate critical cell survival functions, including angiogenesis. The latter could occur as a consequence of regulation of relevant growth factors by such oncogenes. We therefore sought to determine whether EGFR or ErbB2/neu may contribute to tumor angiogenesis by examining their effects on the expression of vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF), one of the most important of all known inducers of tumor angiogenesis. We found that in vitro treatment of EGFR-positive A431 human epidermoid carcinoma cells, which are known to be heavily dependent on VEGF/VPF in vivo as an angiogenesis growth factor, with the C225 anti-EGFR neutralizing antibody caused a dose-dependent inhibition of VEGF protein expression. Prominent suppression of VEGF/VPF expression in vivo, as well as a significant reduction in tumor blood vessel counts, were also observed in established A431 tumors shortly after injection of the antibody as few as four times into nude mice. Transformation of NIH 3T3 fibroblasts with mutant ErbB2/neu, another EGFR-like oncogenic tyrosine kinase, resulted in a significant induction of VEGF/VPF, and the magnitude of this effect was further elevated by hypoxia. Moreover, treatment of ErbB2/neu-positive SKBR-3 human breast cancer cells in vitro with a specific neutralizing anti-ErbB2/neu monoclonal antibody (4D5) resulted in a dose-dependent reduction of VEGF/VPF protein expression. Taken together, the results suggest that oncogenic properties of EGFR and ErbB2/neu may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors such as VEGF/VPF. These genetic changes may cooperate with epigenetic/environmental effects such as hypoxia to maximally stimulate VEGF/VPF expression. Therapeutic disruption of EGFR or ErbB2/neu protein function in vivo may therefore result in partial suppression of angiogenesis, a feature that could enhance the therapeutic index of such agents in vivo and endow them with anti-tumor effects, the magnitude of which may be out of proportion with their observed cytostatic effects in monolayer tissue culture.

OBJECTIVE

Dysfunctional tumor vessels can be a significant barrier to effective cancer therapy. However, increasing evidence suggests that vascular endothelial growth factor (VEGF) inhibition can effect transient "normalization" of the tumor vasculature, thereby improving tumor perfusion and, consequently, delivery of systemic chemotherapy. We sought to examine temporal changes in tumor vascular function in response to the anti-VEGF antibody, bevacizumab.

METHODS

Established orthotopic neuroblastoma xenografts treated with bevacizumab were evaluated at serial time points for treatment-associated changes in intratumoral vascular physiology, penetration of systemically administered chemotherapy, and efficacy of combination therapy.

RESULTS

After a single bevacizumab dose, a progressive decrease in tumor microvessel density to <30% of control was observed within 7 days. Assessment of the tumor microenvironment revealed a rapid, sustained decrease in both tumor vessel permeability and tumor interstitial fluid pressure, whereas intratumoral perfusion, as assessed by contrast-enhanced ultrasonography, was improved, although this latter change abated by 1 week. Intratumoral drug delivery mirrored these changes; penetration of chemotherapy was improved by as much as 81% when given 1 to 3 days after bevacizumab, compared with when both drugs were given concomitantly, or 7 days apart. Finally, administering topotecan to tumor-bearing mice 3 days after bevacizumab resulted in greater tumor growth inhibition (36% of control size) than with monotherapy (88% bevacizumab, 54% topotecan) or concomitant administration of the two drugs (44%).

CONCLUSIONS

Bevacizumab-mediated VEGF blockade effects alterations in tumor vessel physiology that allow improved delivery and efficacy of chemotherapy, although careful consideration of drug scheduling is required to optimize antitumor activity.

Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells, and it promotes angiogenesis in vivo. Here we report that VEGF(165) has neurotrophic actions on cultured adult mouse superior cervical ganglia (SCG) and dorsal root ganglia (DRG), measured as axonal outgrowth. Maximal effect was observed at 10-50 ng/ml for SCG and 100 ng/ml for DRG. VEGF-induced axonal outgrowth was inhibited by the mitogen-activated protein kinase kinase inhibitor PD 98059 but not by the protein kinase inhibitor K252a. VEGF also increased survival of both neurons and satellite cells and the number of proliferating Schwann cells. Immunocytochemistry and immunoblotting revealed that VEGF was expressed in virtually all nerve cells in the SCG but only in a population of small-diameter (<35 micrometers) neurons representing approximately 30% of the neurons in DRG. Immunostaining showed that the VEGF receptor fetal liver kinase receptor (flk-1) was found on nerve cell bodies in DRG and to a lesser extent on neurons in SCG. Growth cones of regenerating axons from both types of ganglia exhibited flk-1 immunoreactivity, as did Schwann cells. We conclude that VEGF has both neurotrophic and mitogenic activity on cells in the peripheral nervous system.

Glioblastoma (GBM) is a highly malignant, rapidly progressive astrocytoma that is distinguished pathologically from lower grade tumors by necrosis and microvascular hyperplasia. Necrotic foci are typically surrounded by "pseudopalisading" cells-a configuration that is relatively unique to malignant gliomas and has long been recognized as an ominous prognostic feature. Precise mechanisms that relate morphology to biologic behavior have not been described. Recent investigations have demonstrated that pseudopalisades are severely hypoxic, overexpress hypoxia-inducible factor (HIF-1), and secrete proangiogenic factors such as VEGF and IL-8. Thus, the microvascular hyperplasia in GBM that provides a new vasculature and promotes peripheral tumor expansion is tightly linked with the emergence of pseudopalisades. Both pathologic observations and experimental evidence have indicated that the development of hypoxia and necrosis within astrocytomas could arise secondary to vaso-occlusion and intravascular thrombosis. This emerging model suggests that pseudopalisades represent a wave of tumor cells actively migrating away from central hypoxia that arises after a vascular insult. Experimental glioma models have shown that endothelial apoptosis, perhaps resulting from angiopoetin-2, initiates vascular pathology, whereas observations in human tumors have clearly demonstrated that intravascular thrombosis develops with high frequency in the transition to GBM. Tissue factor, the main cellular initiator of thrombosis, is dramatically upregulated in response to PTEN loss and hypoxia in human GBM and could promote a prothrombotic environment that precipitates these events. A prothrombotic environment also activates the family of protease activated receptors (PARs) on tumor cells, which are G-protein-coupled and enhance invasive and proangiogenic properties. Vaso-occlusive and prothrombotic mechanisms in GBM could readily explain the presence of pseudopalisading necrosis in tissue sections, the rapid peripheral expansion on neuroimaging, and the dramatic shift to an accelerated rate of clinical progression resulting from hypoxia-induced angiogenesis.

Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.

Since the NF-kappaB/relA transcription factor is constitutively activated in human prostate cancer cells, we determined whether blocking NF-kappaB/relA activity in human prostate cancer cells affected their angiogenesis, growth, and metastasis in an orthotopic nude mouse model. Highly metastatic PC-3M human prostate cancer cells were transfected with a mutated IkappaBalpha (IkappaBalphaM), which blocks NF-kappaB activity. Parental (PC-3M), control vector-transfected (PC-3M-Neo), and IkappaBalphaM-transfected (PC-3M-IkappaBalphaM) cells were injected into the prostate gland of nude mice. PC-3M and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastasis, whereas PC-3M-IkappaBalphaM cells produced slow growing tumors with low metastatic potential. NF-kappaB signaling blockade significantly inhibited in vitro and in vivo expression of three major proangiogenic molecules, VEGF, IL-8, and MMP-9, and hence decreased neoplastic angiogenesis. Inhibition of NF-kappaB activity in PC-3M cells also resulted in the downregulation of MMP-9 mRNA and collagenase activity, resulting in decreased invasion through Matrigel. Collectively, these data suggest that blockade of NF-kappaB activity in PC-3M cells inhibits angiogenesis, invasion, and metastasis.

Recent studies in experimental animals have shown that combining antiangiogenic therapy with radiation can enhance tumor response. Whether this enhancement is mainly attributable to angiogenesis inhibition, endothelial cell radiosensitivity, tumor cell apoptosis, or a decrease in the number of hypoxic cells (improved oxygenation) is not known. We designed this study to discern the role of tumor oxygenation. We chose an anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody (mAb) which has a known target, human VEGF. We also measured interstitial fluid pressure (IFP) to test the hypothesis that the decreased vascular permeability induced by the anti-VEGF mAb can lower IFP. The effect of anti-VEGF mAb on vascular density, partial oxygen tension (pO2), and apoptosis was also measured. Athymic NCr/Sed nu/nu mice bearing 6-mm xenograft of the human glioblastoma multiforme (U87), or colon adenocarcinoma (LS174T) were treated with anti-VEGF mAb injected i.p. on alternate days for a total of six injections at a dosage of 100 microg/injection/mouse. For combined anti-VEGF and radiation, single radiation doses were given under normal blood flow (20 and 30 Gy) or clamped hypoxic conditions (30 and 40 Gy) 24 h after the sixth injection of mAb. The inhibition of the growth of U87 and LS174T tumors by the anti-VEGF mAb was associated with a significant reduction in tumor vascular density and a relatively small increase in the number of apoptotic cells. Compared with size-matched controls, IFP decreased by 74% in LS174T, and 73% in U87 in mice treated with anti-VEGF mAb. After antibody treatment PO2 increased significantly in U87, but did not change in LS174T tumors. Combined treatment induced in U87 tumors a tumor-growth delay (TGD) which was greater than additive; in LS174T except for the 40-Gy hypoxic group, the effect was only additive. In both U87 and LS174T the TGD induced by the antibody was independent of oxygen levels in the tumor at the time of radiation. The fact that the increase in TGD occurred under both normoxic and hypoxic conditions suggests that anti-VEGF mAb treatment can compensate for the resistance to radiation induced by hypoxia.

Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91(phox-/-) mice, suggesting that ROS derived from gp91(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival.

OBJECTIVE

Standard cytotoxic chemotherapy has limited efficacy in metastatic neuroendocrine tumor patients. Neuroendocrine tumors express vascular endothelial growth factor (VEGF) and its receptor (VEGFR). Sunitinib malate, an oral tyrosine kinase inhibitor, has activity against VEGFRs as well as platelet-derived growth factor receptors, stem-cell factor receptor, glial cell line-derived neurotrophic factor, and FMS-like tyrosine kinase-3. We evaluated the efficacy of sunitinib in a two-cohort, phase II study of advanced carcinoid and pancreatic neuroendocrine tumor patients.

METHODS

Patients were treated with repeated 6-week cycles of oral sunitinib (50 mg/d for 4 weeks, followed by 2 weeks off treatment). Patients were observed for response, survival, and adverse events. Patient-reported outcomes were assessed.

RESULTS

Among 109 enrolled patients, 107 received sunitinib (carcinoid, n = 41; pancreatic endocrine tumor, n = 66). Overall objective response rate (ORR) in pancreatic endocrine tumor patients was 16.7% (11 of 66 patients), and 68% (45 of 66 patients) had stable disease (SD). Among carcinoid patients, ORR was 2.4% (one of 41 patients), and 83% (34 of 41 patients) had SD. Median time to tumor progression was 7.7 months in pancreatic neuroendocrine tumor patients and 10.2 months in carcinoid patients. One-year survival rate was 81.1% in pancreatic neuroendocrine tumor patients and 83.4% in carcinoid patients. No significant differences from baseline in patient-reported quality of life or fatigue were observed during treatment.

CONCLUSIONS

Sunitinib has antitumor activity in pancreatic neuroendocrine tumors; its activity against carcinoid tumors could not be definitively determined in this nonrandomized study. Randomized trials of sunitinib in patients with neuroendocrine tumors are warranted.

Studies of the last decades have revealed the importance of angiogenesis for normal growth and for the pathogenesis of numerous diseases. Much less studied is lymphangiogenesis, the growth of lymphatic vessels, which drain extravasated fluid, proteins, and cells and transport them back to the venous circulation. Nonetheless, insufficient lymphangiogenesis causes incapacitating lymphedema, while lymphatic growth around tumors may facilitate metastatic spread of malignant cells that ultimately kill the patient. The recent discovery of the key lymphangiogenic factors VEGF-C and VEGF-D and their receptor VEGFR-3 has allowed novel insights into how the lymphatic vessels and blood vessels coordinately grow and affect human disease. In addition, these studies have opened novel diagnostic and therapeutic avenues for the treatment of lymphedema and metastasis. This overview highlights the recent insights and developments in the field of lymphatic vascular research.

We investigated the interaction between angiogenic and osteogenic factors in bone formation and bone healing with ex vivo gene therapy using muscle-derived stem cells genetically engineered to express human bone morphogenetic protein-4 (BMP4), VEGF, or VEGF-specific antagonist (soluble Flt1). Our results show that although VEGF alone did not improve bone regeneration, it acted synergistically with BMP4 to increase recruitment of mesenchymal stem cells, to enhance cell survival, and to augment cartilage formation in the early stages of endochondral bone formation. These early effects, coupled with accelerated cartilage resorption, eventually led to a significant enhancement of bone formation and bone healing. The beneficial effect of VEGF on bone healing elicited by BMP4 depends critically on the ratio of VEGF to BMP4, with an improper ratio leading to detrimental effects on bone healing. Finally, we show that soluble Flt1 inhibits bone formation elicited by BMP4. Thus, VEGF plays an important role in bone formation elicited by BMP4, and it can significantly enhance BMP4-elicited bone formation and regeneration through multiple mechanisms. This study has important implications for the formulation of new strategies to improve bone healing through increasing mesenchymal stem cell recruitment and survival, in combination with muscle-derived stem cell-based gene therapy.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.

PURPOSE To evaluate the safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics, and preliminary anticancer activity of ramucirumab (IMC-1121B), a fully human immunoglobulin G(1) monoclonal antibody targeting the vascular endothelial growth factor receptor (VEGFR)-2. PATIENTS AND METHODS Patients with advanced solid malignancies were treated once weekly with escalating doses of ramucirumab. Blood was sampled for PK studies throughout treatment. The effects of ramucirumab on circulating vascular endothelial growth factor-A (VEGF-A), soluble VEGFR-1 and VEGFR-2, tumor perfusion, and vascularity using dynamic contrast-enhanced magnetic resonance imaging were assessed. Results Thirty-seven patients were treated with 2 to 16 mg/kg of ramucirumab. After one patient each developed dose-limiting hypertension and deep venous thrombosis at 16 mg/kg, the next lower dose (13 mg/kg) was considered the MTD. Nausea, vomiting, headache, fatigue, and proteinuria were also noted. Four (15%) of 27 patients with measurable disease had a partial response (PR), and 11 (30%) of 37 patients had either a PR or stable disease lasting at least 6 months. PKs were characterized by dose-dependent elimination and nonlinear exposure consistent with saturable clearance. Mean trough concentrations exceeded biologically relevant target levels throughout treatment at all dose levels. Serum VEGF-A increased 1.5 to 3.5 times above pretreatment values and remained in this range throughout treatment at all dose levels. Tumor perfusion and vascularity decreased in 69% of evaluable patients. CONCLUSION Objective antitumor activity and antiangiogenic effects were observed over a wide range of dose levels, suggesting that ramucirumab may have a favorable therapeutic index in treating malignancies amenable to VEGFR-2 inhibition.

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.