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1.
Transplant Proc ; 38(10): 3266-7, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17175244

ABSTRACT

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. The exact molecular and cellular mechanisms contributing to obliterative lesion formation are unknown. Pathological characteristics of OB are epithelial damage, peribronchial inflammation, and increasing obliteration of bronchioli. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that exerts proinflammatory effects by increasing endothelial permeability and inducing expression of endothelial adhesion molecules. We investigated the role of VEGF in the development of OB in rat tracheal allografts and the role of VEGF receptors (VEGFR)-1 and -2 in the development of OB in mouse tracheal allografts. In nontreated allografts, with increasing loss of epithelium and airway occlusion, VEGF messenger RNA (mRNA) and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene led to a decrease in epithelial necrosis but increased luminal occlusion by >50% compared with AdLacZ-treated rat tracheal allografts. When compared with the control immunoglobulin (Ig)G group, simultaneous treatment with antibodies against VEGFR-1 and -2 significantly lowered the degree of luminal occlusion of mouse tracheal allografts.


Subject(s)
Trachea/transplantation , Transplantation, Homologous/physiology , Transplantation, Isogeneic/physiology , Vascular Endothelial Growth Factor A/physiology , Adenoviridae/genetics , Animals , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats , Rats, Inbred WF , Transplantation, Heterotopic , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-1/physiology , Vascular Endothelial Growth Factor Receptor-2/physiology , beta-Galactosidase/genetics
2.
Br J Cancer ; 94(11): 1710-7, 2006 Jun 05.
Article in English | MEDLINE | ID: mdl-16685275

ABSTRACT

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.


Subject(s)
Adenocarcinoma/physiopathology , Cell Movement/physiology , Colonic Neoplasms/physiopathology , Vascular Endothelial Growth Factor Receptor-1/physiology , src-Family Kinases/genetics , src-Family Kinases/metabolism , Adenocarcinoma/enzymology , Cell Line, Tumor , Colonic Neoplasms/enzymology , Enzyme Activation , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Plasmids , RNA, Small Interfering/genetics
3.
Kidney Int ; 69(10): 1741-8, 2006 May.
Article in English | MEDLINE | ID: mdl-16572116

ABSTRACT

Polycystic kidney disease (PKD) is associated with mutations in PKD1 and PKD2 and vascular abnormalities. The links between the epithelial and vascular defects, however, are poorly understood. Vascular endothelial growth factor (VEGF) has been shown to be critical for normal kidney development. In animal models, blockade of VEGF in the perinatal period can lead to abnormal glomerular development, impaired nephrogenesis, proteinuria, and renal failure. We hypothesized that brief blockade of VEGF signaling during early postnatal kidney development can lead to renal cyst development. On days 2 and 4 of life, CD-1 mice were treated with antibodies generated against the extracellular portion of the VEGF receptor 2 (DC101), the area of the receptor where VEGF binding occurs. Mice developed renal cysts between 2 and 3 weeks. The DC101-treated mice also had increased cell proliferation in the renal tubule epithelium. In addition, mice receiving DC101 developed abnormal glomeruli, proteinuria, and patchy cellular infiltrates. Early disruption of VEGFR-2 signaling during the perinatal period results in renal cyst formation, impaired glomerulogenesis, and inflammation. VEGF could be a key link between vascular and cystic changes in kidney cyst formation.


Subject(s)
Kidney Diseases, Cystic , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Animals, Newborn , Antibodies, Monoclonal/pharmacology , Immunohistochemistry , Kidney Diseases, Cystic/etiology , Kidney Diseases, Cystic/metabolism , Kidney Diseases, Cystic/pathology , Mice , Mice, Inbred Strains , Signal Transduction/drug effects , Time Factors
4.
Gut ; 52(9): 1347-54, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12912869

ABSTRACT

BACKGROUND: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. AIMS: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. METHODS: A model of CCl(4) induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl(4), and indices of fibrosis were assessed at eight weeks. RESULTS: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of alpha-smooth muscle actin positive cells and alpha1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased alpha1(I)-procollagen mRNA expression in activated HSC. CONCLUSIONS: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.


Subject(s)
Endothelial Growth Factors/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Liver Cirrhosis/etiology , Lymphokines/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Hepatocytes/metabolism , Hepatocytes/pathology , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred BALB C , Neovascularization, Pathologic/metabolism , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , von Willebrand Factor/metabolism
5.
Br J Cancer ; 88(5): 796-802, 2003 Mar 10.
Article in English | MEDLINE | ID: mdl-12618892

ABSTRACT

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.


Subject(s)
Endothelial Growth Factors/genetics , Epidermal Growth Factor/physiology , Gene Expression Regulation, Neoplastic/physiology , Intercellular Signaling Peptides and Proteins/genetics , Lymphokines/genetics , Neuropilin-1/genetics , Stomach Neoplasms/metabolism , Base Sequence , Blotting, Northern , Blotting, Western , DNA Primers , Humans , Immunohistochemistry , Phosphorylation , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Stomach Neoplasms/pathology , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
6.
Eur J Cancer ; 38(8): 1133-40, 2002 May.
Article in English | MEDLINE | ID: mdl-12008203

ABSTRACT

We hypothesised that the combination of anti-angiogenic and anti-epidermal growth factor (EFG)-receptor (R) therapies would more effectively inhibit gastric cancer growth than single-agent therapy. TMK-1 gastric cancer cells were injected into the gastric wall of nude mice to generate tumours. After 4 days, mice were randomly assigned to the following groups: control, DC101 ([vascular endothelial growth factor (VEGF)-receptor (R)-2 antibody], C225 (EGF-R antibody), or a combination of DC101 and C225. The combination therapy significantly inhibited gastric tumour growth compared with the control group, whereas the decrease in tumour growth in mice treated with DC101 or C225 alone did not reach statistical significance. All mice administered DC101 demonstrated decreased tumour vascularity and increased endothelial cell apoptosis. C225 alone did not affect angiogenesis, but inhibited tumour cell proliferation. The combination therapy led to a further decrease in tumour cell proliferation. The combination of anti-VEGF-R and anti-EGF-R therapies was effective in inhibiting gastric cancer growth. These findings support the hypothesis that inhibiting multiple biological pathways that mediate tumour growth may be an effective therapeutic strategy.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , ErbB Receptors/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Stomach Neoplasms/drug therapy , Animals , Apoptosis , Cell Division , ErbB Receptors/immunology , Immunohistochemistry , Mice , Mice, Nude , Neovascularization, Pathologic/prevention & control , Random Allocation , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Growth Factor/immunology , Receptors, Vascular Endothelial Growth Factor , Stomach Neoplasms/blood supply , Tumor Cells, Cultured
7.
Proc Natl Acad Sci U S A ; 98(19): 10857-62, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11553814

ABSTRACT

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.


Subject(s)
Autocrine Communication , Endothelial Growth Factors/metabolism , Leukemia, Promyelocytic, Acute/physiopathology , Lymphokines/metabolism , Paracrine Communication , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , Animals , Cell Division , Cells, Cultured , Coculture Techniques , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute/metabolism , Mice , Neoplasm Invasiveness , Neoplasm Transplantation , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/physiopathology , Neovascularization, Pathologic/physiopathology , Receptors, Vascular Endothelial Growth Factor , Time Factors , Transplantation, Heterologous , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
8.
Br J Cancer ; 85(4): 584-9, 2001 Aug 17.
Article in English | MEDLINE | ID: mdl-11506500

ABSTRACT

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.


Subject(s)
Carcinoma/immunology , Colonic Neoplasms/immunology , Endothelial Growth Factors/pharmacology , ErbB Receptors/biosynthesis , Lymphokines/pharmacology , Peritoneal Neoplasms/immunology , Animals , Apoptosis , Ascites/immunology , Ascites/physiopathology , Carcinoma/secondary , Cell Survival , Colonic Neoplasms/pathology , Endothelial Growth Factors/biosynthesis , ErbB Receptors/physiology , Lymphokines/biosynthesis , Male , Mice , Neovascularization, Pathologic , Peritoneal Neoplasms/secondary , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
9.
J Exp Med ; 193(9): 1005-14, 2001 May 07.
Article in English | MEDLINE | ID: mdl-11342585

ABSTRACT

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.


Subject(s)
Endothelial Growth Factors/physiology , Hematopoiesis/physiology , Hematopoietic Stem Cells/physiology , Lymphokines/physiology , Membrane Glycoproteins/physiology , Proto-Oncogene Proteins , Signal Transduction/physiology , Adenoviridae , Angiopoietin-1 , Animals , Bone Marrow Cells , Endothelial Growth Factors/administration & dosage , Endothelial Growth Factors/blood , Endothelial Growth Factors/metabolism , Female , Genetic Vectors , Hematopoietic Stem Cell Mobilization , Leukocytes/physiology , Lymphokines/administration & dosage , Lymphokines/blood , Lymphokines/metabolism , Male , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/blood , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Neoplasm Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Spleen/cytology , Time Factors , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
10.
Nat Med ; 7(5): 575-83, 2001 May.
Article in English | MEDLINE | ID: mdl-11329059

ABSTRACT

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.


Subject(s)
Capillary Permeability , Endothelial Growth Factors/physiology , Lymphokines/physiology , Neoplasms, Experimental/blood supply , Neovascularization, Pathologic , Pregnancy Proteins/physiology , Animals , Base Sequence , DNA Primers , Embryonic and Fetal Development , Mice , Placenta Growth Factor , Plasma , Pregnancy Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Wound Healing/physiology
11.
Nat Genet ; 28(2): 131-8, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11381259

ABSTRACT

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.


Subject(s)
Cell Hypoxia/genetics , Endothelial Growth Factors/genetics , Lymphokines/genetics , Motor Neurons/pathology , Nerve Degeneration/genetics , Response Elements/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Axons/physiology , Binding Sites , Electrophysiology , Endothelial Growth Factors/metabolism , Humans , Lymphokines/metabolism , Mice , Mice, Knockout , Motor Neurons/physiology , Muscle Contraction , Muscle Fibers, Skeletal/pathology , Muscular Atrophy/genetics , Muscular Atrophy/pathology , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuropilin-1 , Peripheral Nerves/pathology , Promoter Regions, Genetic , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/genetics , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Sequence Deletion , Spinal Cord/physiology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
12.
Drug Discov Today ; 6(10): 517-528, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11369291

ABSTRACT

Antibodies represent a unique class of therapeutics because of their high specificity towards a defined target antigen. Recent clinical success with antibody-based cancer therapeutics has led to an increase in the clinical development of these agents. Antibody therapeutics offer a promising approach for inhibiting new blood vessel formation (angiogenesis), which is associated with a variety of diseases, including cancer. In this review we will focus on angiogenesis-related mechanisms targeted by antibody-based therapeutics, with an emphasis on those studies where pre-clinical in vivo data is available.

13.
Hepatology ; 33(4): 841-7, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11283848

ABSTRACT

The vascular endothelial growth factor-A (VEGF-A), also known as the vascular permeability factor (VPF), has been shown to play an important role in malignant ascites formation. The effects of VEGF-A are mediated through flt-1 and kinase insert domain-containing receptor/fetal liver kinase (KDR/Flk-1) receptors. It has been shown that KDR/Flk-1 is a predominant receptor in solid hepatocellular carcinoma (HCC) development, but the role of this receptor in hepatic ascites formation has not yet been elucidated. In this study, we examined the role of KDR/Flk-1 in the murine MH134 hepatic malignant ascites formation by means of VEGF-A- and KDR/Flk-1-specific neutralizing antibodies (VEGF-A nAb and KDR/Flk-1 nAb, respectively). The mean volume of ascites, number of tumor cells in ascites, and the peritoneal capillary permeability were significantly suppressed by VEGF-A nAb and KDR/Flk-1 nAb treatment. These inhibitory effects of KDR/Flk-1 nAb were more potent than those of VEGF-A nAb. The autophosphorylation of KDR/ Flk-1 in the peritoneal wall was almost completely abolished by KDR/ Flk-1 nAb, whereas a certain level of activation was still shown by VEGF-A nAb treatment. Another VEGF-family, VEGF-C, which also binds KDR/Flk-1, was detected in the ascites. Furthermore, in the therapeutic experiment, although both VEGF-A nAb and KDR/Flk-1 nAb prolonged the survival rate of ascites-bearing mice, the latter showed a more significant impact on the survival of animals. These results suggest that KDR/Flk-1 is a major regulator of malignant hepatic ascites formation, and that in addition to VEGF-A, VEGF-C may also be involved in the malignant ascites formation via KDR/ Flk-1 activation.


Subject(s)
Ascites/etiology , Carcinoma, Hepatocellular/complications , Liver Neoplasms/complications , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Growth Factor/physiology , Animals , Antibodies/pharmacology , Antibodies/therapeutic use , Ascites/metabolism , Ascites/pathology , Capillary Permeability/drug effects , Carcinoma, Hepatocellular/pathology , Cell Division/drug effects , Endothelial Growth Factors/immunology , Endothelial Growth Factors/metabolism , Liver Neoplasms/pathology , Mice , Mice, Inbred C3H , Peritoneum/blood supply , Peritonitis/drug therapy , Peritonitis/etiology , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Growth Factor/immunology , Receptors, Vascular Endothelial Growth Factor , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor C
14.
Blood ; 97(6): 1679-84, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11238107

ABSTRACT

Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific cadherin that plays an important role in the control of vascular organization. Blocking VE-cadherin antibodies strongly inhibit angiogenesis, and inactivation of VE-cadherin gene causes embryonic lethality due to a lack of correct organization and remodeling of the vasculature. Hence, inhibitors of VE-cadherin adhesive properties may constitute a tool to prevent tumor neovascularization. In this paper, we tested different monoclonal antibodies (mAbs) directed to human VE-cadherin ectodomain for their functional activity. Three mAbs (Cad 5, BV6, BV9) were able to increase paracellular permeability, inhibit VE-cadherin reorganization, and block angiogenesis in vitro. These mAbs could also induce endothelial cell apoptosis in vitro. Two additional mAbs, TEA 1.31 and Hec 1.2, had an intermediate or undetectable activity, respectively, in these assays. Epitope mapping studies show that BV6, BV9, TEA 1.31, and Hec 1.2 bound to a recombinant fragment spanning the extracellular juxtamembrane domains EC3 through EC4. In contrast, Cad 5 bound to the aminoterminal domain EC1. By peptide scanning analysis and competition experiments, we defined the sequences TIDLRY located on EC3 and KVFRVDAETGDVFAI on EC1 as the binding domain of BV6 and Cad 5, respectively. Overall, these results support the concept that VE-cadherin plays a relevant role on human endothelial cell properties. Antibodies directed to the extracellular domains EC1 but also EC3-EC4 affect VE-cadherin adhesion and clustering and alter endothelial cell permeability, apoptosis, and vascular structure formation.


Subject(s)
Antibodies, Monoclonal/pharmacology , Cadherins/immunology , Capillary Permeability/drug effects , Endothelium, Vascular/cytology , Antibodies, Monoclonal/metabolism , Apoptosis/drug effects , Binding Sites , Cadherins/chemistry , Cell Adhesion/drug effects , Dimerization , Endothelium, Vascular/metabolism , Endothelium, Vascular/ultrastructure , Epitope Mapping , Humans , Intercellular Junctions/drug effects , Neovascularization, Physiologic/drug effects , Protein Structure, Tertiary , Umbilical Veins
15.
Int J Oncol ; 18(2): 221-6, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11172585

ABSTRACT

Vascular endothelial growth factor (VEGF) is the predominant regulator of colon cancer angiogenesis and is associated with a poor prognosis and the development of metastases. We hypothesized that DC101, an antibody against the VEGF receptor-2 (flk-1), may be efficacious in the therapy of colon cancer peritoneal carcinomatosis in a murine model. BALB/c mice underwent intraperitoneal injection of CT-26 colon cancer cells to generate peritoneal metastases. Mice received control solvent or DC101 for up to 60 days. In parallel studies, mice were sacrificed at sequential time points to determine the effect of DC101 on tumor angiogenesis, tumor cell proliferation and apoptosis, and endothelial cell apoptosis. Mice treated with DC101 demonstrated a 30% increase in mean survival. In addition, DC101 also led to a significant decrease in tumor vascularity, growth and tumor cell proliferation. In sequential studies, anti-VEGF-R therapy led to a progressive increase in endothelial cell apoptosis followed by an increase in tumor cell apoptosis. These findings suggest that anti-flk-1 therapy may prolong survival in patients with colon cancer carcinomatosis. The temporal studies demonstrating that anti-flk-1 therapy lead to an increase in endothelial cell apoptosis that in turn lead to an increase in tumor cell apoptosis confirms the role of VEGF as an endothelial cell survival factor.


Subject(s)
Antibodies/pharmacology , Carcinoma/drug therapy , Colonic Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Peritoneal Neoplasms/drug therapy , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Animals , Antibodies/therapeutic use , Apoptosis/drug effects , Apoptosis/physiology , Carcinoma/mortality , Cell Division/drug effects , Cell Division/physiology , Colonic Neoplasms/blood supply , Colonic Neoplasms/mortality , Endothelium, Vascular/drug effects , Male , Mice , Mice, Inbred BALB C , Peritoneal Neoplasms/mortality , Receptors, Vascular Endothelial Growth Factor , Tumor Cells, Cultured
16.
Cancer Res ; 61(1): 39-44, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11196192

ABSTRACT

Antiangiogenic therapy can enhance radiation-induced tumor growth inhibition. However, the effects of combined antiangiogenic and radiation therapy on long-term tumor control and normal tissue response have not been reported. We treated mice bearing two different human tumor xenografts with anti-vascular endothelial growth factor receptor-2 antibody (DC101) and five dose fractions of local radiation and followed them for at least 6 months. DC101 significantly decreased the dose of radiation necessary to control 50% of tumors locally. The decrease was 1.7- and 1.3-fold for the moderately radiosensitive small cell lung carcinoma 54A and the highly radioresistant glioblastoma multiforme U87, respectively. In contrast to tumors, no increase in skin radiation reaction by the antibody was detected. Surprisingly, 44% of mice bearing 54A tumor developed clear ascites after DC101 treatment at its highest dose; this was fatal to 20% of mice. This adverse effect was seen only in mice that received whole-body irradiation 1 day before tumor implantation. The encouraging results on two human tumor xenografts suggest that vascular endothelial growth factor receptor-2 blockade merits further investigation to assess its potential as an enhancer of radiation therapy in the clinic.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies, Monoclonal/pharmacology , Carcinoma, Small Cell/therapy , Glioblastoma/therapy , Lung Neoplasms/therapy , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Growth Factor/antagonists & inhibitors , Receptors, Growth Factor/immunology , Angiogenesis Inhibitors/toxicity , Animals , Antibodies, Monoclonal/toxicity , Body Weight/immunology , Body Weight/radiation effects , Carcinoma, Small Cell/blood supply , Carcinoma, Small Cell/radiotherapy , Cell Division/immunology , Cell Division/radiation effects , Combined Modality Therapy , Dose Fractionation, Radiation , Dose-Response Relationship, Immunologic , Dose-Response Relationship, Radiation , Glioblastoma/blood supply , Glioblastoma/radiotherapy , Humans , Intestinal Diseases/etiology , Intestinal Diseases/immunology , Lung Neoplasms/blood supply , Lung Neoplasms/radiotherapy , Male , Mice , Mice, Nude , Oxygen/metabolism , Radiation Tolerance/immunology , Radiation Tolerance/physiology , Receptors, Vascular Endothelial Growth Factor , Skin/radiation effects , Whole-Body Irradiation/adverse effects , Xenograft Model Antitumor Assays
17.
Angiogenesis ; 4(1): 53-60, 2001.
Article in English | MEDLINE | ID: mdl-11824379

ABSTRACT

Different forms of vascular endothelial growth factor (VEGF) and their cellular receptors (VEGFR) are associated with angiogenesis, as demonstrated by the lethality of VEGF-A, VEGFR-1 or VEGFR-2 knockout mice. Here we have used an in vitro angiogenesis model, consisting of human microvascular endothelial cells (hMVEC) cultured on three-dimensional (3D) fibrin matrices to investigate the roles of VEGFR-1 and VEGFR-2 in the process of VEGF-A and VEGF-C-induced tube formation. Soluble VEGFR-1 completely inhibited the tube formation induced by the combination of VEGF-A and TNF alpha (VEGF-A/TNF alpha). This inhibition was not observed when tube formation was induced by VEGF-C/TNF alpha or bFGF/TNF alpha. Blocking monoclonal antibodies specific for VEGFR-2, but not antibodies specifically blocking VEGFR-1, were able to inhibit the VEGF-A/TNF alpha-induced as well as the VEGF-C/TNF alpha-induced tube formation in vitro. P1GF-2, which interacts only with VEGFR-1, neither induced tube formation in combination with TNF alpha, nor inhibited or stimulated by itself the VEGF-A/TNF alpha-induced tube formation in vitro. These data indicate that VEGF-A or VEGF-C activation of the VEGFR-2, and not of VEGFR-1, is involved in the formation of capillary-like tubular structures of hMVEC in 3D fibrin matrices used as a model of repair-associated or pathological angiogenesis in vitro.


Subject(s)
Endothelial Growth Factors/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/growth & development , Neovascularization, Physiologic/drug effects , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Growth Factor/physiology , Animals , Antibodies, Monoclonal/pharmacology , Cells, Cultured , Endothelium, Vascular/physiology , Humans , In Vitro Techniques , Mice , Models, Cardiovascular , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor C , Vascular Endothelial Growth Factor Receptor-1
18.
Mamm Genome ; 11(11): 1030-3, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11063261

ABSTRACT

Murine vascular endothelial cadherin-2 (VE-cad-2) is a cellular adhesion molecule that is distinct from vascular endothelial cadherin 1 (VE-cad-1) in that it does not interact with catenins and does not appear to affect cell migration or growth. In this study, we have cloned a full-length cDNA of the human homolog of VE-cad-2 and used it to map the chromosomal locus of the VE-cad-2 gene. Human VE-cad-2 maps to Chromosome (Chr) 5q31. The cDNA of human VE-cad-2 is highly homologous to mouse VE-cad-2, except for a C-terminal tail. The genomic structure of VE-cad-2 is strikingly similar to that reported for a large family of neuronal protocadherin genes mapped to Chr 5q, yet the amino acid sequences between VE-cad-2 and the protocadherins are substantially divergent. The promoter of human VE-cad-2 contains two TATA boxes and transcription initiates from a single site 3' to these elements. Similar to mouse VE-cad-2, the human gene is expressed primarily in highly vascularized tissues.


Subject(s)
Cadherins/genetics , Cadherins/metabolism , Chromosomes, Human, Pair 5 , Endothelium, Vascular/physiology , 5' Untranslated Regions , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Exons , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Protocadherins , TATA Box
19.
Cancer Res ; 60(16): 4556-60, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10969807

ABSTRACT

Tumor oxygenation is critical for tumor survival as well as for response to therapy, e.g., radiation therapy. Hormone ablation therapy in certain hormone-dependent tumors and antiangiogenic therapy lead to vessel regression and have also shown beneficial effects when combined with radiation therapy. These findings are counterintuitive because vessel regression should reduce oxygen tension (pO2) in tumors, decreasing the effectiveness of radiotherapy. Here we report on the dynamics of pO2 and oxygen consumption in a hormone-dependent tumor following hormone ablation and during treatment with an anti-VEGFR-2 monoclonal antibody (mAb) or a combination of doxorubicin and cyclophosphamide; the latter combination is not known to cause vessel regression at doses used clinically. Androgen-dependent male mouse mammary carcinoma (Shionogi) was implanted into transparent dorsal skin-fold chambers in male severe combined immunodeficient mice. Thirteen days after the tumors were implanted, mice were treated with antiangiogenic therapy (anti-VEGFR-2 mAb, 1.4 mg/30 g body weight), hormone ablation by castration, or doxorubicin (6.5 mg/kg every 7 days) and cyclophosphamide (100 mg/kg every 7 days). A non-invasive in vivo method was used to measure pO2 profiles and to calculate oxygen consumption rates (Q(O2)) in tumors. Tumors treated with anti-VEGFR-2 mAb exhibited vessel regression and became hypoxic. Initial vessel regression was followed by a "second wave" of angiogenesis and increases in both pO2 and Q(O2). Hormone ablation led to tumor regression followed by an increase in pO2 coincident with regrowth. Chemotherapy led to tumor growth arrest characterized by constant Q(O2) and elevated pO2. The increased pO2 during anti-VEGFR-2 mAb and hormone ablation therapy may explain the observed beneficial effects of combining antiangiogenic or hormone therapies with radiation treatment. Thus, understanding the microenvironmental dynamics is critical for optimal scheduling of these treatment modalities.


Subject(s)
Androgens/physiology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Mammary Neoplasms, Experimental/metabolism , Neoplasms, Hormone-Dependent/metabolism , Orchiectomy , Oxygen/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Angiogenesis Inhibitors/pharmacology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Cyclophosphamide/administration & dosage , Doxorubicin/administration & dosage , Luminescent Measurements , Male , Mammary Neoplasms, Experimental/blood supply , Mammary Neoplasms, Experimental/therapy , Mice , Mice, SCID , Microscopy, Fluorescence/methods , Neoplasm Transplantation , Neoplasms, Hormone-Dependent/blood supply , Neoplasms, Hormone-Dependent/therapy , Oxygen/blood , Oxygen Consumption , Partial Pressure , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Growth Factor/immunology , Receptors, Vascular Endothelial Growth Factor
20.
Biochim Biophys Acta ; 1475(3): 216-24, 2000 Jul 26.
Article in English | MEDLINE | ID: mdl-10913819

ABSTRACT

A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation.


Subject(s)
3T3 Cells/drug effects , Fabaceae/chemistry , Lectins/pharmacology , Mannose-Binding Lectins , Plants, Medicinal , 3T3 Cells/cytology , 3T3 Cells/metabolism , Animals , Antigens, CD34/analysis , Cell Differentiation , Cell Division , Cell Survival , Culture Media, Conditioned , Fetal Blood , Humans , Interleukin-3/antagonists & inhibitors , Iodine Radioisotopes , Lectins/genetics , Lectins/isolation & purification , Macrophage Colony-Stimulating Factor , Mice , Monocytes/drug effects , Monocytes/immunology , Plant Lectins , Protein Binding , Protein Sorting Signals , Seeds/chemistry , Transfection
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