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Extracellular matrix (ECM) proteins secreted by blood-brain barrier (BBB) endothelial cells (ECs) are implicated in cell trafficking. We discovered that the expression of ECM epidermal growth factor-like protein 7 (EGFL7) is increased in the CNS vasculature of patients with multiple sclerosis (MS), and in mice with experimental autoimmune encephalomyelitis (EAE). Perivascular CD4 T lymphocytes colocalize with ECM-bound EGFL7 in MS lesions. Human and mouse activated T cells upregulate EGFL7 ligand αvβ3 integrin and can adhere to EGFL7 through integrin αvβ3. EGFL7-knockout (KO) mice show earlier onset of EAE and increased brain and spinal cord parenchymal infiltration of T lymphocytes. Importantly, EC-restricted EGFL7-KO is associated with a similar EAE worsening. Finally, treatment with recombinant EGFL7 improves EAE, reduces MCAM expression, and tightens the BBB in mouse. Our data demonstrate that EGFL7 can limit CNS immune infiltration and may represent a novel therapeutic avenue in MS.

Lessons Learned: These negative phase II results for parsatuzumab highlight the challenges of developing an agent intended to enhance the efficacy of vascular endothelial growth factor inhibition without the benefit of validated pharmacodynamic biomarkers or strong predictive biomarker hypotheses.Any further clinical development of anti-EGFL7 is likely to require new mechanistic insights and biomarker development for antiangiogenic agents., Background: EGFL7 (epidermal growth factor-like domain 7) is a tumor-enriched vascular extracellular matrix protein that supports endothelial cell survival. This phase II trial evaluated the efficacy of parsatuzumab (also known as MEGF0444A), a humanized anti-EGFL7 IgG 1 monoclonal antibody, in combination with modified FOLFOX6 (mFOLFOX6) (folinic acid, 5-fluorouracil, and oxaliplatin) bevacizumab in patients with previously untreated metastatic colorectal cancer (mCRC)., Methods: One-hundred twenty-seven patients were randomly assigned to parsatuzumab, 400 mg, or placebo, in combination with mFOLFOX6 plus bevacizumab, 5 mg/kg. Treatment cycles were repeated every 2 weeks until disease progression or unacceptable toxicity for a maximum of 24 months, with the exception of oxaliplatin, which was administered for up to 8 cycles., Results: The progression-free survival (PFS) hazard ratio was 1.17 (95% confidence interval [CI], 0.71-1.93; p  = .548). The median PFS was 12 months for the experimental arm versus 11.9 months for the control arm. The hazard ratio for overall survival was 0.97 (95% CI, 0.46-2.1; p  = .943). The overall response rate was 59% in the parsatuzumab arm and 64% in the placebo arm. The adverse event profile was similar in both arms., Conclusions: There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first-line mCRC. The Oncologist 2017;22:375-e30., (© AlphaMed Press; the data published online to support this summary is the property of the authors.)

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Endothelial cell growth factor (ECGF) was recently identified as the first autoantigen known to be a target of T cell and B cell responses in ~20% of patients with antibiotic-refractory Lyme arthritis. The goal of the current study was to look for a pathologic correlate between ECGF autoantibody responses and histologic findings in synovial tissue., Methods: Synovial tissue was examined from 14 patients with antibiotic-refractory Lyme arthritis and 6 patients with other forms of chronic inflammatory arthritis, primarily rheumatoid arthritis. The tissue sections were subjected to chemical and immunostaining, and IgG antibody responses to ECGF were determined by enzyme-linked immunosorbent assay (ELISA). Each finding was ranked for statistical analysis., Results: In each disease, synovial tissue showed synovial hypertrophy, vascular proliferation, immune cell infiltrates, and fibrosis. However, among the 14 patients with antibiotic-refractory arthritis, 8 (57%) had obliterative microvascular lesions in the tissue, compared with none of the 6 patients with other forms of chronic inflammatory arthritis (P = 0.04). Among the patients with Lyme arthritis, 5 (36%) had autoantibody responses to ECGF, and all 5 had obliterative lesions, as compared with only 3 of 9 patients who lacked ECGF antibody responses (P = 0.009). Moreover, the magnitude of ECGF antibody responses correlated directly with the extent of obliterative lesions (P = 0.02) and with greater vascularity in the tissue (P = 0.05)., Conclusion: The correlations of ECGF autoantibody reactivity with obliterative microvascular lesions imply that these autoantibodies may be involved in the obliterative process, suggesting that anti-ECGF antibodies have specific pathologic consequences in the synovial tissue of patients with antibiotic-refractory Lyme arthritis., (Copyright © 2014 by the American College of Rheumatology.)

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.

Objective: Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role., Methods: Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera., Results: Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis., Conclusion: T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness., (Copyright © 2013 by the American College of Rheumatology.)

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.

Downregulating the leukocyte adhesion molecules expressed by endothelial cells that line tumor blood vessels can limit the entry of immune effector cells into the tumor mass, thereby contributing to tumoral immune escape. Egfl7 (also known as VE-statin) is a secreted protein specifically expressed by endothelial cells in normal tissues and by cancer cells in various human tumors. High levels of Egfl7 correlate with higher tumor grade and poorer prognosis. Here we show that expression of Egfl7 in breast and lung carcinoma cells accelerates tumor growth and metastasis in immunocompetent mice but not in immunodeficient mice. Tumors expressing Egfl7 were infiltrated relatively poorly by immune cells and were characterized by reduced levels of immunostimulatory cytokines [IFN-γ, interleukin-12 (IL-12)] and fewer endothelial adhesion molecules [intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)]. In vitro studies revealed that Egfl7 inhibited the expression of leukocyte adhesion molecules by endothelial cells, preventing lymphocyte adhesion. In contrast, Egfl7 did not exert any effects on immune cell activation. Human breast cancer lesions expressing high levels of Egfl7 also expressed less ICAM-1 and VCAM-1 in their blood vessels, also indicating an inverse correlation between expression levels of Egfl7 and IFN-γ. Thus, Egfl7 expression in tumors promotes tumor progression by reducing the expression of endothelial molecules that mediate immune cell infiltration. Our findings highlight a novel mechanism through which tumors escape immune control.

Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10(-6)M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.

Although stem cell transplantation (SCT) is being used for hematopoietic reconstitution following high-dose chemotherapy for malignancy, it involves certain serious transplant-related complications such as graft-versus-host disease (GVHD). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) plays important roles in regulating cell death, immune response, and inflammation. However, the role of soluble TRAIL (sTRAIL) after SCT is poorly understood. In this study, 42 patients underwent SCT; 22 patients received allogeneic SCT, while the remaining 20 received autologous SCT. In these patients, levels of sTRAIL, cytokines, and soluble factors were measured by enzyme-linked immunosorbent assay (ELISA). In addition, a basic study of the generation of endothelial cell-derived microparticle (EDMP) by TNF-alpha and soluble Fas ligand (sFasL) was conducted. sFasL and EDMP exhibited significant elevation in the early phase (2-3 weeks) after SCT. In addition, the elevation of IL-6, TNF-alpha, and sIL-2R after allogeneic SCT was observed. EDMP also exhibited changes similar to sFasL. The patients with high sTRAIL exhibited significant decrease of sFasL and EDMP as compared with those without high sTRAIL. TNF-alpha and sFasL induced an increase in procoagulant and apoptotic markers in endothelial cells, and EDMP shedding was observed. Furthermore, sTRAIL inhibited the EDMP elevation caused by TNF-alpha and sFasL. The apoptotic markers such as sFasL and sTRAIL exhibited particular changes after SCT. Our results suggest that sTRAIL generation after allogeneic SCT relates to the prevention of GVHD.

Background: Behçet's syndrome is a chronic systemic immuno-inflammatory disorder affecting multiple organs with generalized vasculitis of arteries and veins. Although the aetiology is still unknown, endothelial dysfunction is one of the most prominent features in Behçet's syndrome. The skin pathergy reaction (SPR) is a non-specific hyperreactive lesion formation that is one of the major features and diagnostic criteria of the disease. It develops after 24-48 h at the site of the needle-prick, especially in the exacerbation period, and it is very similar to the erythematous papules or pustules that appear spontaneously in patients with Behçet's syndrome. Therefore, an investigation into the formation of the SPR lesion may contribute to the pathophysiology of skin lesions of this unique disorder., Objective: To evaluate the immunological features of SPR formation by assessing the immunohistochemical staining of cell adhesion molecules and endothelial growth factor markers such as E-selectin, P-selectin and endoglin (CD 105)., Methods: Patients with Behçet's syndrome showing positive (n = 15) or negative (n = 10) SPR and 15 age- and sex-matched hospital-based healthy control subjects from a similar ethnic background were included in this study. Patients were divided into active and inactive stage by clinical findings and acute-phase reactant parameters including erythrocyte sedimentation rate (ESR) and neutrophil count. Punch biopsy specimens were obtained both from the lesion site on the forearms at 48 h and from normal skin approximately 5 cm adjacent to the SPR site. A biopsy was also obtained from the test application site in Behçet's syndrome patients with negative SPR and healthy volunteers. Biopsy specimens were then evaluated by immunohistochemical staining., Results: Immunohistochemical examination demonstrated a mixed inflammatory cell infiltrate around the vessels and skin appendages that extended somewhat into the deep dermis. A positive segmental staining of E-selectin and P-selectin was noted in the endothelial cells of biopsies obtained from the patients with positive SPR. A positive segmental staining of CD 105 in the endothelial cells was also observed in the same group of patients. However, the immunostaining of the same markers was found to be negative in the biopsies obtained from normal skin of SPR-positive patients, SPR-negative patients and healthy control subjects. Both acute-phase reactant levels were significantly higher in the active stage than in inactive patients or healthy controls., Conclusion: Interaction of cellular adhesion molecules together with endothelial proliferation may play an important role in the formation of SPR lesions in patients with Behçet's syndrome. The involvement of the vascular endothelium in a large number of diseases including Behçet's syndrome supports the importance of vascular-specific adhesion molecules for their aetiopathogenesis.

Single-chain antibodies (scFv) have an enormous potential for clinical application. However, rapid blood clearance and difficulties in large-scale production of active scFvs have limited the practical use of these antibody fragments. Recently, an anti-vascular endothelial growth factor (VEGF) scFv (scFv V65) was selected in our laboratory from a human antibody phage-display library. This antibody was able to reduce tumor growth in mice by approximately 50%. Here, we employ a gene therapy strategy for sustained in vivo expression of scFv V65 and its derivatives. scFv fusion proteins containing parts of the constant IgG1 region were generated (minibody and scFv V65-Fc) to increase the serum half-life of the scFv V65. Systemic administration of recombinant adenovirus encoding scFv V65 resulted in substantial tumor inhibition. This effect could be improved by multiple virus injections. We found that the efficacy of different scFv V65 formats was dependent on the mode of administration: whereas scFv V65-Fc was the most efficient when expressed locally, scFv V65 was superior when delivered systemically. Our results show that therapeutic levels of scFv V65 can be obtained by systemic injection of recombinant adenoviruses. Therefore, therapeutic gene delivery of scFv is a feasible strategy that overcomes several limitations of conventional antibody therapy.

Nonsteroidal anti-inflammatory drugs are known to suppress the occurrence and progression of malignancies such as colorectal cancers. However, the precise mechanism of these actions remains unknown. We have evaluated the role of an inducible cyclo-oxygenase (COX-2) in tumor-associated angiogenesis and tumor growth, and identified the downstream molecules involved using a ddy mouse model of sponge angiogenesis, which mimics tumor angiogenesis and is COX-2 and vascular endothelial growth factor (VEGF) dependent. In this model, VEGF expression was down-regulated by selective COX-2 inhibition with NS-398. To find out the involvement of COX-2/VEGF pathway in tumor-associated angiogenesis, we estimated angiogenesis occurring around implanted Millipore chambers containing sarcoma-180 (S-180) cells or Lewis lung carcinoma cells. Daily oral administration of NS-398 or of aspirin, a nonselective COX inhibitor, suppressed angiogenesis seen around the Millipore chambers. S-180 cells implanted in ddy mice formed substantial tumors with extensive angiogenesis markedly suppressed by aspirin and COX-2 inhibitors NS-398 and JTE522, but not by mofezolac, an inhibitor of constitutive COX-1. Tumor-associated angiogenesis was also significantly suppressed by a neutralizing antibody against VEGF. S-180 tumor growth in the subcutaneous tissues was also suppressed by aspirin, COX-2 selective inhibitors, and the VEGF antibody, but not by the COX-1 inhibitor. These results demonstrate that the inhibition of the COX-2/VEGF-dependent pathway was effective in tumor-associated angiogenesis, tumor growth, and tumor metastasis.

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study, we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs), evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti-vascular endothelial growth factor (anti-VEGF) and/or anti-interleukin 6 (anti-IL-6) antibodies neutralized this inhibitory effect, confirming that VEGF and IL-6, at least in part, mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo, immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor alpha (TNF-alpha) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs, and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6), DCs only (SI = 9.3), or MM lysate-pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM.

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Objective: To review tumor angiogenesis, identify potential targets for anti-angiogenic cancer therapy, and highlight certain anti-angiogenic agents in clinical trials., Data Source: Research articles, abstracts, review articles, and book chapters., Conclusion: Tumor angiogenesis is a complex, multistep process that provides a target for antineoplastic therapy whereby tumor neovasculature is interrupted at various steps in the angiogenic process. Clinical trials are investigating the application and efficacy of anti-angiogenic agents., Implications for Nursing Practice: Oncology nurses must continually increase their knowledge with the onset of newer, targeted agents. This will provide a background for educating and caring for the patient who is receiving anti-angiogenic therapy.

The hypothesis that tumor growth is angiogenesis dependent has been documented by a considerable body of direct and indirect experimental data and has generated intense basic and pharmaceutical-related interest. In contrast, the study of endothelial cell tumors has been modest by comparison. Hemangioma is the most common tumor of any kind seen in infancy and also, perhaps, the least understood. We compared a mouse hemangioma-derived cell line (EOMA) and primary human endothelial cells (HUVEC) for their proliferative behavior and molecular alterations. EOMA cells intrinsically expressed vascular endothelial growth factor (VEGF), which acts in an autocrine manner, resulting in an increase in CD1 expression and cell proliferation, both of which were inhibited by anti-VEGF neutralizing antibodies. Such an autocrine loop is supported by constitutive VEGF receptor (Flk-1) tyrosine phosphorylation, Flk-1 and Flt-1 nuclear localization, and mitogen-activated protein kinase activation. beta-catenin was also found to exhibit significant nuclear localization and constitutively associate with Flk-1 and Flt-1 in EOMA cells but much less so in HUVEC, and immunoprecipitated Flk-1 was able to phosphorylate purified beta-catenin in an immune complex kinase assay. EOMA cells were also noted to express reduced levels of N-cadherin and gamma-catenin compared with HUVEC. Interestingly, sequestration of endogenous VEGF in EOMA cultures resulted in a dramatic decrease in nuclear beta-catenin and a reduction in CD1 levels, whereas addition of exogenous VEGF elicited increased nuclear beta-catenin localization and increased CD1 levels in HUVEC. The possible contributions of VEGF signaling pathways, cell junction component expression levels, and phosphorylation states to endothelial cell transformation and proliferation are discussed.

This study tested the hypothesis that combination of antiangiogenic therapy and tumor immunotherapy of cancer is synergistic. To inhibit angiogenesis, mice were immunized with dendritic cells (DCs) transfected with mRNA that encode products that are preferentially expressed during neoangiogenesis: vascular endothelial growth factor receptor-2 (VEGFR-2) and Tie2 expressed in proliferating endothelial cells, and vascular endothelial growth factor (VEGF) expressed in the angiogenic stroma as well as the tumor cells used in this study. Immunization of mice against VEGF or VEGFR-2 stimulated cytotoxic T lymphocyte (CTL) responses and led to partial inhibition of angiogenesis. Antiangiogenic immunity was not associated with morbidity or mortality except for a transient impact on fertility seen in mice immunized against VEGFR-2, but not VEGF. Tumor growth was significantly inhibited in mice immunized against VEGF, VEGFR-2, and Tie2, either before tumor challenge or in the setting of pre-existing disease in murine B16/F10.9 melanoma and MBT-2 bladder tumor models. Coimmunization of mice against VEGFR-2 or Tie2 and total tumor RNA exhibited a synergistic antitumor effect. Synergism was also observed when mice were coimmunized with various combinations of defined tumor-expressed antigens, telomerase reverse transcriptase (TERT) or TRP-2, and VEGF or VEGFR-2. This study shows that coimmunizing mice against angiogenesis-associated and tumor-expressed antigens can deliver 2 compatible and synergistic cancer treatment modalities via a common treatment, namely immunization.

Background: Mutations in the tumor-suppressor gene VHL cause oversecretion of vascular endothelial growth factor by clear-cell renal carcinomas. We conducted a clinical trial to evaluate bevacizumab, a neutralizing antibody against vascular endothelial growth factor, in patients with metastatic renal-cell carcinoma., Methods: A randomized, double-blind, phase 2 trial was conducted comparing placebo with bevacizumab at doses of 3 and 10 mg per kilogram of body weight, given every two weeks; the time to progression of disease and the response rate were primary end points. Crossover from placebo to antibody treatment was allowed, and survival was a secondary end point., Results: Minimal toxic effects were seen, with hypertension and asymptomatic proteinuria predominating. The trial was stopped after the interim analysis met the criteria for early stopping. With 116 patients randomly assigned to treatment groups (40 to placebo, 37 to low-dose antibody, and 39 to high-dose antibody), there was a significant prolongation of the time to progression of disease in the high-dose--antibody group as compared with the placebo group (hazard ratio, 2.55; P<0.001). There was a small difference, of borderline significance, between the time to progression of disease in the low-dose--antibody group and that in the placebo group (hazard ratio, 1.26; P=0.053). The probability of being progression-free for patients given high-dose antibody, low-dose--antibody, and placebo was 64 percent, 39 percent, and 20 percent, respectively, at four months and 30 percent, 14 percent, and 5 percent at eight months. At the last analysis, there were no significant differences in overall survival between groups (P>0.20 for all comparisons)., Conclusions: Bevacizumab can significantly prolong the time to progression of disease in patients with metastatic renal-cell cancer., (Copyright 2003 Massachusetts Medical Society)

Cancer cachexia is a poorly understood syndrome of anorexia, weight loss, and muscle wasting that negatively impacts quality of life and survival in cancer patients. Research has clearly implicated pro-inflammatory cytokines in the biology of cancer cachexia. More recent research implicates products of arachidonic acid and suggests that cachexia may be a chronic inflammatory condition rather than a nutritional aberration. To date, nutritional support to slow weight loss has focused primarily on increasing calorie intake. Alternatively, many foods contain factors that can modulate the synthesis or activity of pro-inflammatory mediators, especially the synthesis of prostaglandin E2 from arachidonic acid. These factors and foods are sometimes called nutraceuticals, and research is needed to evaluate their efficacy in combating cancer cachexia.

Utilizing in utero aortopulmonary vascular graft placement, we developed a lamb model of congenital heart disease and increased pulmonary blood flow. We showed previously that these lambs have increased pulmonary vessel number at 4 wk of age. To determine whether this was associated with alterations in VEGF signaling, we investigated vascular changes in expression of VEGF and its receptors, Flt-1 and KDR/Flk-1, in the lungs of shunted and age-matched control lambs during the first 8 wk of life. Western blot analysis demonstrated that VEGF, Flt-1, and KDR/Flk-1 expression was higher in shunted lambs. VEGF and Flt-1 expression was increased at 4 and 8 wk of age (P <0.05). However, KDR/Flk-1 expression was higher in shunted lambs only at 1 and 4 wk of age (P <0.05). Immunohistochemical analysis demonstrated that, in control and shunted lambs, VEGF localized to the smooth muscle layer of vessels and airways and to the pulmonary epithelium while increased VEGF expression was localized to the smooth muscle layer of thickened media in remodeled vessels in shunted lambs. VEGF receptors were localized exclusively in the endothelium of pulmonary vessels. Flt-1 was increased in the endothelium of small pulmonary arteries in shunted animals at 4 and 8 wk of age, whereas KDR/Flk-1 was increased in small pulmonary arteries at 1 and 4 wk of age. Our data suggest that increased pulmonary blood flow upregulates expression of VEGF and its receptors, and this may be important in development of the vascular remodeling in shunted lambs.

Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an in vivo study to investigate the relation between VEGF-blocking therapy, tumoral blood vessels, and intratumoral uptake of anticancer drugs. Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.

Endometriosis is a disease in which the lining of the uterus (endometrium), shed at the time of menstruation, becomes established at sites such as the peritoneum and ovaries. These explants develop a rich blood supply that enables them to survive and grow. We hypothesized that inhibitors of angiogenesis would prevent this growth by disrupting sensitive vessels supplying endometriotic lesions. Vessels sensitive to angiogenic antagonism have few associations with pericyte cells. The vessels supplying human endometriotic lesions were immunohistochemically characterized and found to be predominantly pericyte free. A model in which human endometrium is implanted into nude mice was used to test the effects of two antagonists of the angiogenic growth factor, vascular endothelial cell growth factor A. Soluble truncated receptor (flt-1; P = 0.002) and an affinity-purified antibody to human vascular endothelial cell growth factor A (P = 0.03) significantly inhibited the growth of nude mouse explants. Pericyte-free vessels were shown to supply endometrial lesions in nude mice and were disrupted in lesions taken from soluble flt-1-treated mice. In summary, antiangiogenic agents inhibited the growth of explants in an in vivo model of endometriosis by disrupting the vascular supply, and this effect is likely to apply to the human disease. These findings suggest that antiangiogenic agents may provide a novel therapeutic approach for the treatment of endometriosis.

The tumor microenvironment plays a critical role in determining the fate of tumor cells. We have previously reported that adhesion of human myeloma and leukemia cell lines to the extracellular matrix protein, fibronectin, confers a multidrug-resistant phenotype. Mechanisms associated with this cell adhesion-mediated drug resistance are drug-type specific. In the present study, we examined the influence of bone marrow stromal cells (BMSCs) on myeloma cell response to the topoisomerase II inhibitor, mitoxantrone. Apoptosis was inhibited by more than 50% when cells were adhered to BMSCs as compared to myeloma cells maintained in suspension. To investigate the mechanisms contributing to the resistance of myeloma cells in contact with BMSCs, we examined the protective effects of BMSCs under four separate conditions: (1) direct cell contact; (2) BMSCs conditioned medium; (3) medium conditioned by coculturing myeloma cells in direct contact with BMSCs; and (4) medium conditioned by coculturing myeloma cells and BMSCs without direct physical contact. Conditioned medium from BMSCs alone was not sufficient to protect myeloma cells from drug-induced apoptosis; however, soluble factors produced during the myeloma-BMSCs interaction decreased the sensitivity of myeloma cells to mitoxantrone, suggesting a dynamic interaction between myeloma cells and BMSCs. We also found that myeloma cells in direct contact with BMSCs underwent growth arrest, whereas soluble factors produced by myeloma cells-BMSCs coincubation stimulated the proliferation of myeloma cells. These data show that both cell-cell adhesion of BMSCs with myeloma cells and soluble factors induced by this cell-cell interaction are involved in the protection of myeloma cells from mitoxantrone-induced apoptosis; however, the mechanisms contributing to the drug resistance are different.

Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis induced by electrical stimulation in skeletal muscle. Less is known about the role of arachidonic acid metabolites in the control of growth of blood vessels in vivo. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE) on the angiogenesis induced by electrical stimulation in skeletal muscle. The tibialis anterior and extensor digitorum longus muscles of rats were stimulated for 7 days. Electrical stimulation significantly increased the 20-HETE formation and angiogenesis in the muscles, which was blocked by chronic treatment with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or 1-aminobenzotriazole (ABT). Chronic treatment with either HET0016 or ABT did not block the increases in VEGF protein expression in both muscles. To analyze the role of VEGF on 20-HETE formation, additional rats were treated with VEGF-neutralizing antibody (VEGF Ab). VEGF Ab blocked the increases of 20-HETE formation induced by stimulation. These results place 20-HETE in the downstream signaling pathway for angiogenesis and show that both VEGF and 20-HETE are involved in the angiogenesis induced by electrical stimulation in skeletal muscle.

Acute eosinophilic pneumonia (AEP) is associated with the presence of diffuse pulmonary infiltrates on the chest radiograph and an increased number of eosinophils and an elevation of interleukin (IL)-5 levels in bronchoalveolar lavage (BAL) fluid. Vascular endothelial growth factor (VEGF) is a constitutively expressed protein encoded by messenger ribonucleic acid in human eosinophils and is released following stimulation with IL-5. However, the roles of IL-5 and VEGF in the pathogenesis or activity of this disease have not been clarified. The authors investigated the cells and the levels of these two factors in BAL fluid in five AEP patients and five normal controls before and after corticosteroid treatment. The absolute number of eosinophils-mL(-1), IL-5 and VEGF levels in patients before treatment were higher than in controls (53.8 versus 0.3 x 10(4) x mL(-1), 490.1 versus 5.2 pg x mL(-1) and 643.0 versus 133.9 pg x mL(-1), respectively). IL-5 and VEGF rapidly decreased to the control level in parallel with clinical improvement. The relationship between eosinophilia and IL-5 and VEGF levels was strongly significant. Elevated interleukin-5 in the lung may initiate the recruitment of eosinophils and enhance the release of mediators, such as vascular endothelial growth factor from eosinophils, which, in turn, increases the permeability of blood vessels.

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria. The role of vascular endothelial growth factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These studies suggest that neutralization of physiologic levels of VEGF, a key endothelial survival factor, may lead to proteinuria. In the present study, we evaluated the potential of anti-VEGF neutralizing antibodies and sFlt-1 in the induction of proteinuria. Our studies demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular endothelial cell detachment and hypertrophy, in association with down-regulation of nephrin, a key epithelial protein in the glomerular filtration apparatus. These studies suggest that down-regulation or neutralization of circulating VEGF may play an important role in the induction of proteinuria in various kidney diseases, some forms of cancer therapy and also in women with preeclampsia.

Bone marrow stromal cells (MSCs) are pluripotent cells capable of differentiation into several tissue types. This present study was performed to determine their functional neoangiogenic potential in vivo. Whole bone marrow was harvested from C57Bl/6 mice, and the adherent cellular fraction was culture expanded for 14 doublings. These MSCs were resuspended in Matrigel and their angiogenic effect assessed in isogenic recipients. At 2 weeks postimplantation, the mean vascular density in Matrigel plugs containing 2 x 10(6) MSCs/ml was 41+/-5.0 blood vessels (BVs)/mm(2) compared to 0.5+/-0.7 for empty Matrigel (P<0.001). In comparison, Matrigel plugs containing either recombinant murine VEGF 165 at 50 ng/ml or bovine bFGF at 1000 ng/ml generated 21+/-5 and 11+/-2.0 BV/mm(2), respectively. Arteriogenesis was observed only in the MSC-containing implants. With the use of LacZ retroviral labeling of ex vivo expanded MSCs, we show that approximately 10% of LacZ(+)MSCs differentiated into CD31(+) and VEGF(+) endothelial cells. More than 99% of the neoangiogenic phenomena arose from recruitment of host-derived LacZ(null) vascular structures. Neutralizing anti-VEGF antibodies inhibited the MSC-initiated angiogenic response in vivo by 85% (P<0.001). In conclusion, MSCs have the ability to effectively recruit and participate in angiogenesis and arteriogenesis de novo and VEGF plays a central role in the observed host-derived angiogenic response. We propose that ex vivo expanded autologous MSCs may serve as cell therapy to promote therapeutic angiogenesis.

A number of studies have provided indirect evidence that angiogenesis is involved in tumour growth and metastasis formation of renal cell carcinoma (RCC). Nude mice bearing xenografts of human Caki-I RCC were treated i.p. for 3 weeks with a murine monoclonal antibody against vascular endothelial growth factor, VEGF (VEGF-ab). Tumour growth and mRNA expression of human and murine VEGF and murine VE-Cadherin in the tumours were measured. After 3 weeks of therapy, the tumour volume in the control nude mice was 548 +/- 98 mm3 compared to the tumours in the nude mice treated with VEGF-ab (122 +/- 24 mm3, p < 0.01). Treatment with VEGF-ab significantly reduced mRNA expression of murine VEGF-120 and murine VE-Cadherin (p < 0.05 and p < 0.01, respectively). The mRNA expression of human VEGF (hVEGF165, hVEGF189) and murine VEGF (mVEGF164) was unchanged due to antibody treatment. The mean percentage of apoptotic cells in tumours harvested from antibody-treated animals was significantly lower than in tumours from the control-treated animals (p < 0.02). These findings demonstrate for the first time that VEGF-ab significantly inhibit the growth of Caki-1 RCC in vivo.

It has been suggested that endometrial angiogenesis in response to the sex steroids oestrogen and progesterone is mediated at a local level via compounds such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), acting through their respective tyrosine kinase receptors. The aim of the present study was to use SUGEN tyrosine kinase receptor angiogenic inhibitor compounds SU5416, SU5402, SU11652 and SU11685, to determine whether VEGF, FGF or PDGF play a role in mediating endometrial endothelial cell proliferation after administration of oestrogen and progesterone. Endometrial endothelial cell proliferation was induced in adult ovariectomized mice by either oestrogen alone for 24 h (E1), or a regimen using oestrogen alone, then progesterone with low dose oestrogen, followed by progesterone with high-dose oestrogen (PE) over a total of 7 days. Each angiogenesis inhibitor compound was injected daily for 4 days (100 mg kg(-1) day(-1), s.c.) before endometrial tissue collection at either the E1 or PE stage. This study also evaluated the effect of VEGF antiserum (0.2 ml, i.p.) on endothelial cell proliferation at the E1 stage. All four angiogenic inhibitor compounds significantly reduced endothelial cell proliferation activity at the E1 and PE stages. The greatest reduction in the endothelial cell proliferative index was at the E1 stage in the group treated with the VEGF receptor inhibitor SU5416 (2.5 +/- 0.7% versus 27.9 +/- 1.1%, P < 0.001), with a reduction of similar magnitude in the group treated with anti-VEGF antibody. At the PE stage, all four inhibitors significantly reduced endothelial cell proliferation to a similar extent, indicating that VEGF, FGF and PDGF are all involved. These results demonstrate that endometrial angiogenesis after acute oestrogen treatment is primarily mediated by VEGF, but that under the influence of combined oestrogen and progesterone, FGF and PDGF are also probably involved.

Context: Vascular endothelial growth factor (VEGF), an angiogenic and chemotactic peptide, is abundantly expressed in normal lung tissue, especially in alveolar and bronchial epithelium, glandular cells of the bronchi, and activated alveolar macrophages., Objective: To investigate the role of VEGF in progressively impaired lung function as the major complication and cause of death in septic patients., Materials and Methods: We evaluated pulmonary VEGF expression in lung autopsy material from septic patients who had been cared for by intensive care medicine using enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical methods., Results: Compared with expression in nonseptic control individuals (n = 10), pulmonary VEGF expression as determined by ELISA was significantly (P <.001) decreased in septic patients (n = 8). As monitored by RT-PCR, mRNA for the 2 splice variants, VEGF(121) and VEGF(165), and for VEGFR-2/KDR were expressed in both groups, the yields being lower in the sepsis group. Samples from septic patients lacked or showed only sparse immunoreaction on bronchial and alveolar epithelium, whereas this reaction was strong in all control samples. However, alveolar macrophages were similarly immunopositive in both groups., Conclusions: The precise underlying mechanisms for the distinctly different expression of pulmonary VEGF in septic patients and nonseptic control individuals are not clear at present. Particularly the role of VEGF in the development of sepsis-induced lung injury and acute respiratory distress syndrome in mechanically ventilated patients suffering from severe sepsis remains to be clarified.

Background: Hepatoblastoma is the most common primary hepatic malignancy of childhood, frequently presenting as advanced disease. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and survival factor critical to growth and angiogenesis in many human cancers. Inhibition of VEGF effectively suppresses tumorigenesis in multiple experimental models. The authors hypothesized that anti-VEGF antibody would alter vascular architecture and impede tumor growth in experimental hepatoblastoma., Methods: The Institutional Animal Care and Use Committee of Columbia University approved all protocols. Xenografts were established in athymic mice by intrarenal injection of cultured human hepatoblastoma cells. Anti-VEGF antibody (100 microg/dose) or vehicle was administered intraperitoneally 2 times per week for 5 weeks. At week 6, 10 control/treated mice were killed and remaining animals maintained without treatment until week 8. Tumor weights were compared by Kruskal-Wallis analysis, and vascular alterations ascertained by fluorescein angiography and specific immunostaining., Results: Anti-VEGF antibody significantly inhibited tumor growth at 6 weeks (1.85 g +/- 0.60 control, 0.05 +/- 0.03 antibody, P <.0003). In comparison with controls, treated xenografts showed decreased vascularity and dilated surviving vessels with prominent vascular smooth muscle elements., Conclusions: Specific anti-VEGF therapy inhibits neoangiogenesis and significantly suppresses tumor growth in experimental hepatoblastoma. Surviving vasculature displays dilation and increased vascular smooth muscle. Anti-VEGF agents may represent new therapeutic alternatives for children with advanced disease., (Copyright 2003, Elsevier Science (USA). All rights reserved.)

Delivery of DNA mixed with a degradable matrix carrier was supposed to improve transgene expression. Using a rabbit hind-limb ischemia model, we tested the angiogenic potency of plasmid encoding human vascular endothelial growth factor (pSG5-VEGF165) entrapped in fibrin sealant. Animals were injected intramuscularly with 500 microg of pSG5-VEGF165 or control plasmid, dissolved in saline (PBS) or fibrin glue. After 14 days, presence of delivered constructs and expression of transgene was confirmed in injected muscles of all animals. There were no significant differences in the levels of human VEGF mRNA and protein between VEGF-PBS and VEGF-fibrin groups (Mann-Whitney test). Accordingly, pSG5-VEGF165 regardless of the way of delivery, induced similar increases in capillary density within treated muscles (ANOVA). Control plasmid did not show any effects. In conclusion, injection of pSG5-VEGF165 into ischemic adductor muscle leads to synthesis of human VEGF and increases the number of capillaries. Fibrin carrier does not influence its angiogenic potential.

Vascular endothelial growth factor (VEGF)-C is a member of the VEGF family. VEGF-C is involved in developmental lymphangiogenesis and may be important in pathological lymphangiogenesis, lymphatic invasion and metastasis in carcinoma. We describe the development of an indirect enzyme-linked immunosorbent (ELISA) assay for the quantification of VEGF-C in plasma. Capture of VEGF-C was achieved using goat anti-human VEGF-C antibody, followed by detection with rabbit anti-human VEGF-C antibody. The sensitivity of the assay was amplified using the biotin-avidin and enhanced chemiluminescence (ECL) systems. The assay was highly sensitive and reproducible with a detection range of 0.4-100 U/ml and the intra- and inter-assay variations were less than 8%. Substitutional tests demonstrated that the assay was specific for VEGF-C with no cross-reaction with VEGF-A or VEGF-D. Practical application of the assay was evaluated in 41 colorectal cancer patients and 31 controls. Median plasma levels of VEGF-C were 35.0 U/ml (range: 17.4-75.9 U/ml) in colorectal cancer patients in contrast to 11.5 U/ml (range: 5.4-21.5 U/ml) in controls (p<0.001). Moreover, VEGF-C levels tended to be elevated in patients with advanced disease compared to early disease, but this was not statistically significant owing to a relatively small number of patients in each group. Immunoprecipitation and immunoblotting confirmed detection of VEGF-C in plasma and revealed that two forms of VEGF-C were present in the plasma corresponding to approximately 40 and approximately 80 kDa. The measurement of plasma VEGF-C offers opportunities to explore clinical applications in the management of malignancy, in particular in the prediction of lymphatic spread and in other lymphangiogenesis-related diseases.

The ability to induce endothelial cell proliferation is a common feature of human pathogenic Bartonella species. Recent data have indicated that bartonellae can provoke angioproliferation by at least two independent mechanisms: directly, by triggering proliferation and inhibiting apoptosis of endothelial cells; and indirectly, by stimulating a paracrine angiogenic loop of vascular endothelial growth factor production by infected macrophages. A NF-kappaB-mediated acute inflammatory reaction of the Bartonella-infected endothelium appears to be critical for the recruitment of monocytes/macrophages and the initiation and maintenance of a paracrine angiogenic loop. Given that bartonellae effectively adhere to and invade endothelial cells, their ability to trigger angioproliferation might represent a dedicated pathogenic strategy for expanding the bacterial host cell habitat.

Objectives: Recurrent hypoxemia has been proposed as an important pathophysiological mechanism underlying sudden infant death syndrome (SIDS). However, conflicting results emerged when xanthines were used as markers for hypoxia. The vascular endothelial growth factor (VEGF) gene is highly sensitive to changes in tissue partial oxygen tension, and changes in genomic and protein expression occur even after changes in oxygenation within the physiologic range., Methods: For determining whether hypoxia precedes SIDS, VEGF levels were measured using an enzyme-linked immunosorbent assay in the cerebrospinal fluid (CSF) of 51 SIDS infants and in 33 additional control infants who died of an identifiable cause. In addition, 6 rats that had a chronically implanted catheter in the lateral ventricle were exposed to a short hypoxic challenge, and VEGF concentrations were measured in CSF at various time points for 24 hours. Another set of 6 rats were killed with a pentobarbital overdose, and VEGF CSF levels were obtained at different time points after death., Results: Mean VEGF concentrations in CSF were 308.2 +/- 299.1 pg/dL in the SIDS group and 85.1 +/- 82.9 pg/dL in those who died of known causes. Mean postmortem delay averaged 22 hours for both groups. In rat experiments, hypoxic exposures induced time-dependent increases in VEGF, peaking at 12 hours and returning to baseline at 24 hours. Postmortem duration in the animals was associated with gradual increases in VEGF that reached significance only at 36 hours., Conclusions: We conclude that VEGF CSF concentrations are significantly higher in infants who die of SIDS. We postulate that hypoxia is a frequent event that precedes the sudden and unexpected death of these infants.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine which plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF-A 121, 145, 165, 189, 206). VEGF is essential for tumor angiogenesis and several studies have correlated elevated VEGF levels with tumor stage, metastases and progression. Antibody phage display was employed to isolate two scFv antibody fragments, D8 and F10, with specificity for the VEGF165 isoform. It was shown by ELISA and competitive immunohistochemistry that each clone bound to VEGF165 but not VEGF121. Immunohistochemistry with D8 and F10 on colorectal carcinoma and adenoma sections revealed positive staining similar to that shown by a polyclonal VEGF antibody. The scFv antibody fragments, D8 and F10, will be useful in specifically detecting circulating VEGF165 in cancer patients as most studies to date have quantified the total level of circulating VEGF (121 and 165). These reagents will allow further elucidation of the role of VEGF in tumor angiogenesis.

Background: Idiopathic membranous nephropathy (IMN), a principal disease of glomerular capillaries, was investigated for some aspects of glomerular capillary injury and repair (angiogenesis)., Methods: Fifteen cases of IMN were studied immunohistochemically for expression of the endothelial cell antigen platelet endothelial cell adhesion molecule-1 (PECAM-1[CD31]) and the angiogenesis-stimulating factors vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). An equal number of normal control kidneys of fetal and mature origin were tested for the same antigens., Results: Normal tissues expressed PECAM-1 in both glomerular and interstitial endothelial cells, whereas VEGF and TP were expressed in the tubular epithelium. IMN was characterized by complete or partial loss of PECAM-1 expression from glomerular capillaries and a parallel gain/expression of this antigen by the tubular epithelium. In addition, VEGF and TP expression was lost or considerably reduced from tubular cells of IMN., Conclusion: We hypothesize that PECAM-1 expression by tubular epithelial cells represents uptake of CD31(+) cell-surface fragments released by glomerular endothelial cells after glomerular damage. The damage is confounded by the failure of angiogenic mechanisms to promote glomerular angiogenesis (repair) because both VEGF and TP stimulation by the tubular epithelium is eliminated. It is suggested that immunohistochemical detection of VEGF or TP in the tubular epithelium may be useful in understanding the pathogenesis of IMN.

The field of angiogenesis research has seen an explosion of knowledge within the last 10 years. More than 3500 angiogenesis-related papers are presently being published per year compared to the less than 200 annual papers published in the early 1990s. Paralleling the progress in the field of basic angiogenesis research, translational research has led to the identification of more than 100 angiomanipulatory compounds. Presently, more than 40 substances are in various phases of clinical trials. The prospect of these exciting developments is presently dampened by the negative outcome of some advanced clinical trials. Thus, following euphoria and disillusion, the field is presently experiencing that translational clinical research requires endurance to eventually accomplish the successful implementation of angiomanipulatory therapies in the clinical setting. The present article provides an overview of the field of angiogenesis research and summarizes ongoing efforts aimed at developing angiomanipulatory therapies.

The realization that the growth and spread of tumors are dependent on angiogenesis has created new avenues of research designed to help us to better understand cancer biology and to facilitate the development of new therapeutic strategies. However, the process of angiogenesis consists of multiple sequential and interdependent steps with a myriad of positive and negative regulators of angiogenesis being involved. The survival of tumors and thus their metastases are dependent on the balance of endogenous angiogenic and antiangiogenic factors such that the outcome favors increased angiogenesis. Several growth factors have been identified that regulate angiogenesis in colon cancer; the most important of these is vascular endothelial growth factor. In addition, specific integrins such as alphavbeta3 and alpha5beta1 mediate endothelial cell survival and have been shown to be overexpressed on the endothelium of colon cancer. These angiogenic mediators thus serve as targets for therapy of metastatic colon cancer and have shown promise in preclinical trials.

We evaluated the effectiveness of vascular endothelial growth factor (VEGF) blockade alone and in combination with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU, nimustine), a cytotoxic agent commonly used in the treatment of malignant gliomas, to eradicate tumors of human glioblastoma cell lines implanted in SCID (severe combined immunodeficiency) mice. ACNU, but not cisplatin and etoposide, elevated VEGF expression in a glioma cell line in vitro. VEGF antibody alone inhibited glioma growth in vivo as a result of angiogenesis inhibition. The combination with ACNU resulted in an additive effect for inhibition of glioma growth. ACNU also induced VEGF up-regulation in glioma tissues, which was decreased with VEGF antibody treatment. One of the mechanisms of the additive effect of the VEGF antibody and ACNU combination is the blockade of VEGF up-regulation induced by ACNU. As such, the combination of antiangiogenic therapy with conventional therapy is promising for glioma treatment in the future.

The authors previously showed that C34+ progenitor cells are mobilized into the peripheral blood in tumor bearers. The current study used a murine Lewis lung carcinoma (LLC) model to examine if the CD34+ cells can be chemoattracted to a tumor excision site as the first step to inducing differentiation of the attracted CD34+ cells into immune stimulatory dendritic cells (DC). Upon tumor excision, gelatin sponges were implanted into the surgical site and infused with phosphate-buffered saline or vascular endothelial cell growth factor (VEGF). The implants were removed after 4, 7, 14, and 21 days and analyzed for their cellular content. The incorporation of VEGF into implants increased the accumulation of CD34+ cells early after implantation. By day 21, the CD34+ cell content declined. However, the numbers of DCs became increased in the VEGF-containing sponges and this persisted throughout the 3-week duration of the study. The VEGF-containing implants contained a lower percentage of CD11b+ myeloid cells for the first 2 weeks after implantation as compared with the control implants. By 21 days, myeloid cell numbers declined in the control and VEGF implants. Since endothelial cell precursors have a common precursor with myeloid progenitor cells, the implants were also examined for their endothelial cell content. With increasing time after implantation, the number of endothelial cells and formation of vessel-like structures became greater in the VEGF-containing implant as compared with the control implants. These results show the feasibility of using VEGF-containing sponges to attract DC precursors and to increase the number of DCs at the tumor excision site.