ABSTRACT
The molecular mechanism underlying gastric cancer (GC) invasion and metastasis is still poorly understood. In this study, we tried to investigate the roles of CXCR4 and CXCR2 signalings in gastric cancer metastasis. A highly invasive gastric cancer cell model was established. Chemokines receptors were profiled to search for the accountable ones. Then the underlying molecular mechanism was investigated using both in vitro and in vivo techniques, and the clinical relevance of CXCR4 and CXCR2 expression was studied in gastric cancer samples. CXCR4 and CXCR2 were highly expressed in a high invasive gastric cancer cell model and in gastric cancer tissues. Overexpression of CXCR4 and CXCR2 was associated with more advanced tumor stage and poorer survival for GC patients. CXCR4 and CXCR2 expression strongly correlated with each other in the way that CXCR2 expression changed accordingly with the activity of CXCR4 signaling and CXCR4 expression also changed in agreement with CXCR2 activity. Further studies demonstrated CXCR4 and CXCR2 can both activated NF-κB and STAT3 signaling, while NF-κBp65 can then transcriptionally activate CXCR4 and STAT3 can activate CXCR2 expression. This crosstalk between CXCR4 and CXCR2 contributed to EMT, migration and invasion of gastric cancer. Finally, Co-inhibition of CXCR4 and CXCR2 is more effective in reducing gastric cancer metastasis. Our results demonstrated that CXCR4 and CXCR2 cross-activate each other to promote the metastasis of gastric cancer.
Subject(s)
Cell Movement , Gene Expression Regulation, Neoplastic , Receptors, CXCR4/metabolism , Receptors, Interleukin-8B/metabolism , Stomach Neoplasms/pathology , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation , Female , Humans , Lymphatic Metastasis , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Middle Aged , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Receptors, CXCR4/genetics , Receptors, Interleukin-8B/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.
Subject(s)
Apoptosis/physiology , Communicable Diseases/pathology , Neoplasms/pathology , Phosphatidylserines/metabolism , Animals , Antibodies/immunology , Antibodies/therapeutic use , Apoptosis Regulatory Proteins/metabolism , Autoimmunity , Communicable Diseases/immunology , Communicable Diseases/metabolism , Humans , Membrane Glycoproteins/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Phosphatidylserines/immunology , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD(+) synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD(+) pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, ß-lapachone (ß-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. ß-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD(+) consumption. Synergy with FK866+ß-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (â¼85%) of PDA cases. This treatment strategy simultaneously decreases NAD(+) synthesis while increasing NAD(+) consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)(+) depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, µ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD(+)-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD(+) synthesis and ß-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD(+) pools in cancer cells to sensitize them to ROS-mediated cell death by ß-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.
Subject(s)
Naphthoquinones/pharmacology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Poly Adenosine Diphosphate Ribose/metabolism , Acrylamides/pharmacology , Cell Death/drug effects , Cell Line, Tumor , DNA Breaks, Double-Stranded/drug effects , Drug Synergism , Energy Metabolism/drug effects , Glycolysis/drug effects , Humans , NAD(P)H Dehydrogenase (Quinone)/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Pancreatic Neoplasms/enzymology , Piperidines/pharmacology , Reactive Oxygen Species/metabolism , Pancreatic NeoplasmsABSTRACT
Opiates, such as morphine, decrease neurogenesis in the postnatal hippocampal subgranular zone (SGZ) by inhibiting progenitor proliferation and maturation. However, it is not known how morphine influences the growth factors and vasculature that encompass the neurogenic SGZ microenvironment. We examined morphine's effect on pro- and anti-proliferative factors in the dentate gyrus (DG; Experiment 1) as well as the DG neurovasculature itself (Experiment 2). For Experiment 1, mice were implanted with subcutaneous sham or morphine pellets (0 and 48 h) and were decapitated 24 or 96 h later. One brain hemisphere was postfixed to examine proliferation by immunohistochemistry, and a DG-enriched sample was dissected from the other hemisphere to examine the neurogenic microenvironment via immunoblotting for known pro- and anti-proliferative factors. Consistent with previous results, morphine decreased the number of proliferating cells in the SGZ, as the number of Ki67-immunoreactive (IR) cells was decreased at 96 h. Morphine did not alter DG levels of the pro-proliferative factor brain-derived neurotrophic factor, anti-proliferative factor interleukin-1 beta, or their receptors TrkB and IL1R1 at either time point. However, morphine increased the pro-proliferative factor vascular endothelial growth factor (VEGF) at 96 h. Given that VEGF is also a potent angiogenic factor, Experiment 2 examined whether the morphine-induced increase in VEGF correlated with altered DG neurovasculature. Mice were implanted with morphine pellets as in Experiment 1, and 2 h before perfusion (24 or 96 h) were administered bromodeoxyuridine (BrdU; intraperitoneal, 150 mg/kg). Tissue was co-stained for BrdU and the endothelial cell marker endoglin to enable examination of DG vessels and proximity of BrdU-IR cells to endoglin-IR vessels. At 96 h, endoglin-IR vessel area and perimeter were increased, but proximity of BrdU-IR cells to endoglin-IR vessels remained unchanged. These data suggest that following chronic morphine exposure, factors within the neurogenic microenvironment are maintained or upregulated to compensate for decreased SGZ proliferation.
Subject(s)
Analgesics, Opioid/toxicity , Dentate Gyrus/blood supply , Dentate Gyrus/drug effects , Morphine/toxicity , Neurogenesis/drug effects , Animals , Brain-Derived Neurotrophic Factor/metabolism , Dentate Gyrus/physiology , Endoglin , Hippocampus/blood supply , Hippocampus/drug effects , Hippocampus/physiology , Immunoblotting , Immunohistochemistry , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Ki-67 Antigen/metabolism , Mice , Mice, Inbred C57BL , Neurogenesis/physiology , Neurons/drug effects , Neurons/physiology , Receptor, trkB/metabolism , Receptors, Interleukin-1 Type I/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Several studies have implicated the angiogenic cytokine vascular endothelial growth factor (VEGF) in the development of diabetic nephropathy, but no data are available about its local activity during human disease. Glomeruli from 52 archival biopsies from type II diabetics were evaluated and compared to 10 renal biopsies without kidney disease (controls). Glomerulosclerosis, capillary rarefaction, glomerular and endothelial cell proliferation, apoptosis, VEGF expression, as well as receptor-bound VEGF indicating local VEGF activity, and phosphorylation of the signal transduction molecule Akt were investigated. Owing to substantial heterogeneity of glomerular lesions in individual biopsies, these parameters were correlated with the degree of injury in individual glomeruli rather than biopsies. Severe glomerular capillary rarefaction was linked to the degree of glomerulosclerosis. While cellular apoptosis was detected independent of the stage of injury, endothelial cell proliferation indicating capillary repair was markedly increased only in mildly/moderately injured glomeruli. In controls, VEGF was predominantly expressed in podocytes, whereas receptor-bound VEGF was confined to the glomerular endothelium. VEGF expression was increased in all diabetic glomeruli by many different cell types. In contrast, VEGF receptor activation was increased predominantly in the endothelium of only mildly injured glomeruli, but significantly decreased in more severely injured glomeruli. Diabetic nephropathy is associated with glomerular capillary rarefaction. Despite overall increased glomerular VEGF, the decreased receptor-bound VEGF on the endothelium may be an indicator of an insufficient capillary repair reaction.
Subject(s)
Diabetic Nephropathies/diagnosis , Endothelium, Vascular/chemistry , Kidney Glomerulus/blood supply , Kidney Glomerulus/chemistry , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/metabolism , Aged , Capillaries/pathology , Capillaries/physiology , Cell Proliferation , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Endothelial Cells/chemistry , Endothelial Cells/pathology , Endothelium, Vascular/pathology , Female , Humans , Kidney Glomerulus/pathology , Male , Middle Aged , Phosphorylation , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Vascular Endothelial Growth Factor/analysis , Receptors, Vascular Endothelial Growth Factor/metabolism , RegenerationSubject(s)
Antigens, CD34/immunology , Awards and Prizes , Bone Marrow/immunology , Bone Marrow/metabolism , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Glioblastoma/immunology , Glioblastoma/metabolism , Internship and Residency , Neurosurgery/education , Vascular Endothelial Growth Factor Receptor-2/immunology , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Disease Models, Animal , Fluorescent Antibody Technique , Humans , Mice , Microscopy, ConfocalABSTRACT
The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(121) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing vascularization and growth of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(121) only showed enhancement of i.c. tumor growth but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neovascularization, whereas VEGF(121) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. Endothelial cell migration stimulated by VEGF(121) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.
Subject(s)
Brain Neoplasms/blood supply , Endothelial Growth Factors/physiology , Glioma/blood supply , Lymphokines/physiology , Neovascularization, Pathologic/metabolism , Animals , Brain Neoplasms/metabolism , Cell Movement/drug effects , Endothelial Growth Factors/biosynthesis , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Glioma/metabolism , Humans , Lymphokines/biosynthesis , Lymphokines/pharmacology , Neovascularization, Pathologic/pathology , Protein Isoforms , RNA, Messenger/metabolism , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/biosynthesis , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Vitronectin/pharmacologyABSTRACT
Vascular endothelial growth factor (VEGF) is a primary stimulant of the vascularization of solid tumors and has therefore been the focus of intense research aimed at blocking its activity in solid tumors. VEGF production by tumor cells is induced by oncogenic gene mutations and hypoxic conditions inside the tumor mass. VEGF receptor expression on endothelial cells lining blood vessels in the tumor is also induced by hypoxia and the increased local concentration of VEGF. Therefore in the tumor microenvironment there is an upregulation of both VEGF and its receptor leading to a high concentration of occupied receptor on tumor vascular endothelium. The VEGF-VEGF receptor complex (VEGF-VEGFR) presents an attractive target for the specific delivery of drugs or other effectors to tumor endothelium. Herein we review the development of monoclonal antibodies that selectively bind to the VEGF-VEGFR and their use as targeting agents that selectively bind to VEGF activated blood vessels. Additionally, we summarize the properties of 2C3, a novel monoclonal anti-VEGF antibody that blocks VEGF from binding to VEGFR2 but not VEGFR1. 2C3 may be utilized as both an anti-angiogenic agent by inhibiting VEGFR2 activity and potentially as a vascular targeting agent by binding to blood vessels that express the VEGF-VEGFR1 complex.
Subject(s)
Endothelial Growth Factors/metabolism , Endothelium, Vascular/metabolism , Lymphokines/metabolism , Neoplasms/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Animals , Biomarkers, Tumor , Endothelial Growth Factors/immunology , Humans , Lymphokines/immunology , Mice , Neoplasms/blood supply , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Growth Factor/immunology , Receptors, Vascular Endothelial Growth Factor , Regional Blood Flow , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Active angiogenesis, together with an up-regulation of angiogenic factors, is evident in the synovium of both rheumatoid arthritis (RA) and osteoarthritis (OA). The present study assessed, by immunohistochemistry, the microvessel density in the synovium of these arthritides and in normal controls, in relation to the expression of the angiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) and the apoptosis-related proteins bcl-2 and p53. More importantly, using the novel 11B5 MAb, the activated "VEGF/flk-1(KDR)-receptor" microvessel density was assessed. VEGF expression in fibroblasts was diffuse in both RA and OA. Diffuse PD-ECGF expression of fibroblasts was noted in all cases of RA, while fibroblast reactivity was focal in the OA material. The standard microvessel density (sMVD), as assessed with the anti-CD31 monoclonal antibody (MAb), was higher in RA (64+/-12) and in OA (65+/-16) than in normal tissues (52+/-8; p=0.008 and 0.0004, respectively). The activated microvessel density (aMVD), assessed with the 11B5 MAb, was significantly higher in RA (29+/-10) than in OA (17+/-4; p<0.0001) and than in normal tissues (14+/-2; p<0.0001). The "activation ratio" (aMVD/sMVD) was statistically higher in RA (0.46+/-0.17) than in OA and normal synovial tissues, the latter two having a similar ratio (0.28+/-0.08 and 0.26+/-0.03, respectively). Cytoplasmic bcl-2 expression was frequent in the synovial cells of OA, but rare in RA. Nuclear p53 protein accumulation was never observed. It is suggested that the angiogenic pathway VEGF/flk-1(KDR) may play an important role in the pathogenesis of RA and OA. Thus, failure of VEGF/flk-1(KDR) activation, in the presence of increased VEGF expression, may indicate a synovium with an impaired capacity to establish a viable vasculature, consistent with the degenerative nature of OA. On the other hand, the activated angiogenesis in RA shows a functional, still pathologically up-regulated VEGF/flk-1(KDR) pathway. Whether restoration of an impaired VEGF/flk-1(KDR) pathway in OA, or inhibition of this in RA, would prove of therapeutic importance requires further investigation.
Subject(s)
Arthritis, Rheumatoid/metabolism , Neovascularization, Pathologic/metabolism , Osteoarthritis/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Synovial Membrane/blood supply , Adult , Aged , Arthritis, Rheumatoid/pathology , Endothelial Growth Factors/metabolism , Female , Fibroblasts/metabolism , Humans , Immunoenzyme Techniques , Lymphokines/metabolism , Male , Microcirculation/pathology , Middle Aged , Osteoarthritis/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Mitogen/metabolism , Receptors, Vascular Endothelial Growth Factor , Synovial Membrane/metabolism , Tumor Suppressor Protein p53/metabolism , Up-Regulation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
SPARC is a multifunctional glycoprotein that belongs to the matricellular group of proteins. It modulates cellular interaction with the extracellular matrix (ECM) by its binding to structural matrix proteins, such as collagen and vitronectin, and by its abrogation of focal adhesions, features contributing to a counteradhesive effect on cells. SPARC inhibits cellular proliferation by an arrest of cells in the G1 phase of the cell cycle. It also regulates the activity of growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF). The expression of SPARC in adult animals is limited largely to remodeling tissue, such as bone, gut mucosa, and healing wounds, and it is prominent in tumors and in disorders associated with fibrosis. The crystal structure of two of the three domains of the protein has revealed a novel follistatin-like module and an extracellular calcium-binding (EC) module containing two EF-hand motifs. The follistatin-like module and the EC module are shared by at least four other proteins that comprise a family of SPARC-related genes. Targeted disruption of the SPARC locus in mice has shown that SPARC is important for lens transparency, as SPARC-null mice develop cataracts shortly after birth. SPARC is a prototypical matricellular protein that functions to regulate cell-matrix interactions and thereby influences many important physiological and pathological processes.
Subject(s)
Osteonectin/physiology , Animals , Extracellular Matrix/metabolism , Extracellular Matrix/physiology , Gene Expression Regulation , Growth Substances/metabolism , Humans , Osteonectin/chemistry , Osteonectin/genetics , Osteonectin/metabolism , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
The expression of SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is elevated in endothelial cells participating in angiogenesis in vitro and in vivo. SPARC acts on endothelial cells to elicit changes in cell shape and to inhibit cell cycle progression. In addition, SPARC binds to and diminishes the mitotic activity of vascular endothelial growth factor. To determine the effect(s) of SPARC on angiogenic responses in vivo, we implanted polyvinyl alcohol sponges subcutaneously into wild-type and SPARC-null mice. On days 12 and 20 following implantation, SPARC-null mice showed increased cellular invasion of the sponges in comparison to wild-type mice. Areas of the sponge with the highest cell density exhibited the highest numbers of vascular profiles in both wild-type and SPARC-null animals. The endothelial component of the vessels was substantiated by immunoreactivity with three different markers specific for endothelial cells. Although sponges from SPARC-null relative to wild-type mice were populated by significantly more cells and blood vessels, an increase in the ratio of vascular to nonvascular cells was not apparent. No differences in the percentage of proliferating cells within the sponge were detected between wild-type and SPARC-null sections. However, elevated levels of vascular endothelial growth factor were associated with sponges from SPARC-null versus wild-type mice. An increase in vascular endothelial growth factor production was also observed in SPARC-null primary dermal fibroblasts relative to those of wild-type cells. In conclusion, we have shown that the fibrovascular invasion of polyvinyl alcohol sponges is enhanced in mice lacking SPARC, and we propose that increased levels of vascular endothelial growth factor account, at least in part, for this response.
Subject(s)
Neovascularization, Physiologic/physiology , Osteonectin/physiology , Animals , Endothelial Growth Factors/metabolism , Fibroblasts/metabolism , Immunohistochemistry , Lymphokines/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Polyvinyl Alcohol/administration & dosage , Skin/cytology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Vascular targeting agents, which selectively destroy tumor blood vessels, are attractive agents for the treatment of solid tumors. They differ from anti-angiogenic agents in that they target the mature, blood-conducting vessels of the tumors. They are better suited for larger tumors where angiogenesis can occur less frequently. For application in man, target molecules are needed that are selectively expressed on the vascular endothelium of tumors. Such markers include the complexes that are formed when vascular endothelial growth factor (VEGF) binds to its receptors (VEGFR). VEGF production by tumor cells is induced by oncogenic gene mutations and by the hypoxic conditions within the tumor mass. The receptors, VEGFR1 (FLT-1) and VEGFR2 (KDR/Flk-1), are upregulated on vascular endothelial cells in tumors by hypoxia and by the increased local concentration of VEGF. Consequently, there is a high concentration of occupied receptors on tumor vascular endothelium. Here, we review the concept of vascular targeting and the development of monoclonal antibodies that bind to VEGF: VEGFR complexes and their use as tumor vascular targeting agents. A promising monoclonal antibody is 2C3, which blocks VEGF from binding to VEGFR2 but not VEGFR1. We conclude that 2C3 might have dual activity as an anti-angiogenic agent by inhibiting VEGFR2 activity and as a vascular targeting agent for selective drug delivery to tumor vessels.
Subject(s)
Endothelial Growth Factors/antagonists & inhibitors , Lymphokines/antagonists & inhibitors , Neoplasms/blood supply , Neoplasms/therapy , Neovascularization, Pathologic/therapy , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Endothelial Growth Factors/immunology , Endothelial Growth Factors/metabolism , Humans , Lymphokines/immunology , Lymphokines/metabolism , Neovascularization, Pathologic/immunology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
SPARC is a multifunctional glycoprotein that belongs to the matricellular group of proteins. It modulates cellular interaction with the extracellular matrix (ECM) by its binding to structural matrix proteins, such as collagen and vitronectin, and by its abrogation of focal adhesions, features contributing to a counteradhesive effect on cells. SPARC inhibits cellular proliferation by an arrest of cells in the G1 phase of the cell cycle. It also regulates the activity of growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF). The expression of SPARC in adult animals is limited largely to remodeling tissue, such as bone, gut mucosa, and healing wounds, and it is prominent in tumors and in disorders associated with fibrosis. The crystal structure of two of the three domains of the protein has revealed a novel follistatin-like module and an extracellular calcium-binding (EC) module containing two EF-hand motifs. The follistatin-like module and the EC module are shared by at least four other proteins that comprise a family of SPARC-related genes. Targeted disruption of the SPARC locus in mice has shown that SPARC is important for lens transparency, as SPARC-null mice develop cataracts shortly after birth. SPARC is a prototypical matricellular protein that functions to regulate cell-matrix interactions and thereby influences many important physiological and pathological processes.
Subject(s)
Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Osteonectin/metabolism , Animals , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/physiology , Gene Expression Regulation , Growth Substances/metabolism , Humans , Osteonectin/chemistry , Osteonectin/genetics , Osteonectin/physiology , Protein Structure, Secondary , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic growth factor that is a primary stimulant of the development and maintenance of a vascular network in embryogenesis and the vascularization of solid tumors. At the present time there are two well-characterized receptors for VEGF that are selectively expressed on endothelium. VEGF receptor 2 [VEGFR2 (KDR/Flk-1)] mediates endothelial cell mitogenesis and permeability increases, whereas the role of VEGF receptor 1 [VEGFR1 (Flt-1)] has not been clearly defined. In the present study, a monoclonal antibody, 2C3, is shown to block the interaction of VEGF with VEGFR2 but not with VEGFR1 through ELISA, receptor binding assays, and receptor activation assays. 2C3 blocks the VEGF-induced vascular permeability increase in guinea pig skin. 2C3 has potent antitumor activity, inhibiting the growth of newly injected and established human tumor xenografts in mice. These findings demonstrate the usefulness of 2C3 in dissecting the pathways that are activated by VEGF in cells that express both VEGFR1 and VEGFR2, as well as highlighting the dominant role of VEGFR2 in mediating VEGF-induced vascular permeability increase and tumor angiogenesis.
Subject(s)
Antibodies, Monoclonal/pharmacology , Endothelial Growth Factors/immunology , Growth Inhibitors/pharmacology , Lymphokines/immunology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , Capillary Permeability/drug effects , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/therapy , Cell Division/drug effects , Endothelial Growth Factors/antagonists & inhibitors , Endothelial Growth Factors/metabolism , Enzyme-Linked Immunosorbent Assay , Fibrosarcoma/pathology , Fibrosarcoma/therapy , Growth Inhibitors/immunology , Guinea Pigs , Humans , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Lymphokines/antagonists & inhibitors , Lymphokines/metabolism , Male , Mice , Neoplasm Transplantation , Phosphorylation/drug effects , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Rhabdomyosarcoma/pathology , Rhabdomyosarcoma/therapy , Skin/blood supply , Substrate Specificity , Transplantation, Heterologous , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth FactorsABSTRACT
Vascular endothelial growth factor (VEGF) is an important angiogenic factor, linked to poor outcome in human malignancies including non-small cell lung carcinoma (NSCLC). We used the 11B5 monoclonal antibody recognizing the VEGF/KDR complex (R. A. Brekken et al., Cancer Res., 58: 1952-1959, 1998) to assess the VEGF expression in cancer cells and the VEGF/KDR activated microvessel density (aMVD) in early operable NSCLC. The JC70 anti-CD31 monoclonal antibody was used to assess the standard MVD (sMVD). The aMVD was significantly higher in the invading front of the tumors and in the normal lung adjacent to the tumors as compared with normal lung distant to the tumor or to inner tumor areas (P < 0.0002). The sMVD was higher in the normal lung and decreased from the invading front to inner tumor areas (P < 0.0001). However, the vascular activation (aMVD:sMVD) was 4-6 times higher in the tumor areas as compared with lung from normal individuals (36-58% versus 9%; P < 0.0001). Fibroblast 11B5 reactivity, noted in 25% of cases, correlated with high aMVD and sMVD in the inner tumor areas. Multivariate analysis showed that aMVD was the most potent and independent prognostic factor (P = 0.001; t-ratio, 3.28). It is concluded that intense VEGF/KDR angiogenic pathway activation is a tumor-specific feature in more than 50% of NSCLC cases and is associated with poor postoperative outcome. Clinical trials involving targeting of the VEGF/KDR-positive vasculature with specific antibodies, such as 11B5, are, therefore, encouraged.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Endothelial Growth Factors/biosynthesis , Lung Neoplasms/metabolism , Lymphokines/biosynthesis , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptors, Growth Factor/biosynthesis , Adenocarcinoma/metabolism , Adult , Aged , Antibodies, Monoclonal/metabolism , Carcinoma, Non-Small-Cell Lung/blood supply , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Squamous Cell/metabolism , Female , Fibroblasts/metabolism , Humans , Immunohistochemistry , Lung/metabolism , Lung Neoplasms/blood supply , Lung Neoplasms/mortality , Male , Microcirculation , Middle Aged , Necrosis , Prognosis , Receptors, Vascular Endothelial Growth Factor , Time Factors , Up-Regulation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.
Subject(s)
Capillary Permeability , Genetic Vectors/metabolism , Kidney/metabolism , Mammary Neoplasms, Experimental/blood supply , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Adenoviridae/genetics , Animals , Capillaries/metabolism , Endothelium, Vascular/metabolism , Female , Kidney/blood supply , Kidney/ultrastructure , Kidney Glomerulus/blood supply , Kidney Glomerulus/metabolism , Kidney Glomerulus/ultrastructure , Kidney Tubules/blood supply , Kidney Tubules/metabolism , Kidney Tubules/ultrastructure , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Nude , Microscopy, Electron , Neovascularization, Pathologic , Receptors, Vascular Endothelial Growth FactorABSTRACT
Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 and flk-1(KDR), constitute an important angiogenic pathway which, under hypoxic conditions, is up-regulated in many solid tumours. We used the monoclonal antibody 11B5, specific for recognizing VEGF expression and the 'VEGF/flk-1(KDR) complex' on tumour endothelium, to assess free VEGF protein expression and VEGF/receptor activated microvessel density (aMVD) in a series of 104 inoperable locally advanced squamous cell carcinomas of the head and neck, treated with chemo-radiotherapy. High VEGF expression in cancer cells was strongly associated with high VEGF/receptor expression in the vasculature. The high VEGF expression and the aMVD were not associated with the standard microvessel density (sMVD), as assessed with the monoclonal antibody anti-CD31 and, were not detected in normal tissue. An increased sMVD, however, was significantly related with the expression thymidine phosphorylase (TP), and also with the nuclear accumulation of the oncoprotein p53, but neither p53 nor TP was associated with VEGF expression by cancer cells or VEGF/receptor complex aMVD. In 35% of cancer cases examined, more than 20% of the microvessels assessed with anti-CD31 also expressed the VEGF/KDR complex. The vasculature of the normal head and neck mucosa did not express the VEGF/KDR complex. There was no association between VEGF expression or VEGF/receptor complex aMVD and response to chemo-radiotherapy or patient's survival. It is concluded that activation of the angiogenic pathway VEGF/flk-1(KDR) is tumor specific in a subgroup of locally advanced squamous cell carcinomas of the head and neck. Selective destruction of this type of vasculature, using immunoconjugates directed against the VEGF/receptor complex, may prove therapeutically useful for patients with a high tumoral VEGF/flk-1(KDR) activated microvessel fraction.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Endothelial Growth Factors/metabolism , Head and Neck Neoplasms/metabolism , Lymphokines/metabolism , Neoplasm Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Adult , Aged , Antibodies, Monoclonal , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/pathology , Female , Head and Neck Neoplasms/blood supply , Head and Neck Neoplasms/pathology , Humans , Linear Models , Male , Microcirculation , Middle Aged , Mucous Membrane/metabolism , Receptors, Vascular Endothelial Growth Factor , Survival Analysis , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that induces angiogenesis in several pathological conditions. To block angiogenesis, soluble VEGF receptor can be used. In this study, we describe a method for high yield expression of soluble VEGF receptor 2 (sFlk-1) in a baculovirus expression system (30 mg purified sFlk-1 per L of insect cell supernatant). We also determined the binding constants for both human and mouse VEGF to the recombinant receptor by surface plasmon resonance. In this cell-free assay, under the given experimental conditions, the on-rate ka was 0.5-2.2 x 10(6) M-1s-1 and the off-rate kd was 2-4 x 10(-4) s-1 (KD = 2-6 x 10(-10) M). To our knowledge this is the first study to report on- and off-rates for the VEGF:sFlk-1 interaction. Heparin was not required for the binding of VEGF to sFlk-1 in this assay. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptor.
Subject(s)
Receptor Protein-Tyrosine Kinases/biosynthesis , Receptors, Growth Factor/biosynthesis , Receptors, Mitogen/biosynthesis , Animals , Binding Sites , Biosensing Techniques , Cell Line , Electrophoresis, Polyacrylamide Gel , Endothelial Growth Factors/metabolism , Humans , Ligands , Lymphokines/metabolism , Mice , Protein Binding , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Growth Factor/genetics , Receptors, Mitogen/genetics , Receptors, Vascular Endothelial Growth Factor , Recombinant Proteins/metabolism , Solubility , Spodoptera , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. VEGF production is induced by oncogenic gene mutations in the tumor cells and by hypoxic conditions inside the tumor mass. Hypoxia and the locally increased concentration of VEGF lead to an up-regulation of VEGF receptor expression on tumor endothelial cells. Therefore, in the tumor microenvironment, there is an up-regulation of both VEGF and its receptor, leading to a high concentration of occupied receptor on tumor vascular endothelium. The VEGF:receptor complex presents an attractive target for the specific delivery of drugs or other effectors to tumor endothelium. In the present study, several hybridomas that secrete monoclonal antibodies against the VEGF:receptor (Flk-1) complex or against VEGF itself have been raised. Three of the antibodies (3E7, GV39M, and 11B5) bind with high affinity to the VEGF:Flk-1 complex in ELISA and to tumor endothelium in frozen sections of human tumors, rodent tumors, and human tumor xenografts. 3E7 and GV39M localize selectively to tumor endothelium after i.v. injection into mice bearing human tumor xenografts. Additionally, one antibody (2C3) was raised that blocks the interaction between VEGF and KDR/Flk-1. 2C3 inhibits VEGF-mediated growth of endothelial cells in vitro and localizes strongly to connective tissue in tumors after injection into mice bearing human tumor xenografts. These findings suggest that 3E7, GV39M, and 2C3 are candidates for targeting and imaging the vasculature or connective tissue of tumors.