PURPOSE: This study investigated the importance of alphavbeta5 function during vascular endothelial growth factor (VEGF) induced corneal angiogenesis by examining the effects of antibody to alphavbeta5 that blocks alphav 5-mediated cell adhesion to vitronectin. METHODS: A hydrogel disk containing 500 ng of VEGF was implanted into the superior corneal stroma of each of sixteen New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent to the first, randomized to contain either 40 g of antibody to alphavbeta5 (n=8) or phosphate-buffered saline (PBS)(n=8). Both disks were positioned 1.2 mm apart from the superior limbus. Eyes were examined daily under a stereomicroscope by two observers and assigned an angiogenesis score based on number and length of new blood vessels. RESULTS: On days 3 through 7 postimplantation, angiogenesis scores were significantly lower in eyes treated with antibody to alphavbeta5 (averaged score=16.33) as compared to eyes treated with PBS (averaged score=26.52)(P<0.05, Wilcoxon signed rank test). CONCLUSIONS: In a rabbit corneal micropocket assay, antibody to alphavbeta5 inhibits corneal angiogenesis induced by VEGF. Substances that target the integrin alphavbeta5 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.
Blood Vessels ; Cell Adhesion ; Corneal Neovascularization* ; Corneal Stroma ; Hydrogel ; New Zealand ; Vascular Endothelial Growth Factor A ; Vitronectin

Both fibronectin and vitronectin bind to Pneumocystis carinii (P. carinii) and mediate the attachment of the organisms to respiratory epithelial cells. Surfactant A and D play a role in the interaction between P. carinii and host cells. In this study we examined the expression of fibronectin, vitronectin, surfactant-A and D in the interaction between P. carinii and alveolar epithelial cells by immunohistochemistry and pre-embedding immunoelectron microscopy. The experimental rat model of P. carinii pneumonia was induced by administration of low protein diet (8%) and drinking water containing dexamethasone (2 mg/liter) for 6 to 8 weeks. The primary antibodies for light and electron microscopic immunohistochemistries were monoclonal antibodies including fibronectin (1:100) and vitronectin (1:100), and polyclonal antibodies including surfactant A (1:50) and D (1:50), respectively. Light microscopic immunohistochemistry for the fibronectin, vitronectin, surfactant-A and D showed strong expressions on the P. carinii and surface linings of type I alveolar epithelial cells. The electron microscopic immunohistochemistry of the fibronectin and vitronectin showed a strong immunoexpression along the surface pellicles and tubular extensions of P. carinii trophozoites, and surface membranes of the type I epithelial cells. The surfactant-A and D proteins showed a strong expression on the pellicles of P. carinii and surface membranes of the type I epithelial cells, but a weak expression on the free-floating surfactant materials. In conclusions, the trophozoites of P. carinii were mostly attached to type I epithelial cells. The fibronectin, vitronectin, surfactant-A and D were strongly expressed, and played an enhancing role in the binding between the P. carinii organisms and the type I alveolar epithelial cells.
Antibodies ; Antibodies, Monoclonal ; Dexamethasone ; Diet, Protein-Restricted ; Drinking Water ; Epithelial Cells* ; Fibronectins* ; Immunohistochemistry ; Membranes ; Microscopy, Immunoelectron ; Models, Animal ; Pneumocystis carinii* ; Pneumocystis* ; Pneumonia ; Pneumonia, Pneumocystis* ; Trophozoites ; Vitronectin*

BACKGROUND: Pneumocystis carinii (P. carinii) attaches to alveolar cells and causes injury to the epithelial cells by direct toxic effects or inhibition of epithelial growth and replication. Although respiratory cell damage or death is a common feature in P. carinii pneumonia, there has been little reports about expression of apoptosis of the lung tissue in the literatures. METHODS: We examined expression of fibronectin and vitronectin in the interaction between P. carinii and alveolar cells, and in situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) expression of apoptosis in the respiratory cells by immunohistochemistry and pre-embedding immunoelectron microscopy. RESULTS: Light microscopic (LM) and electron microscopic (EM) immunohistochemical stains for the fibronectin and vitronectin showed strong expressions on the pellicles and tubular extensions of P. carinii and weak expression along the surfaces of type I alveolar cells. LM and EM TUNEL stains showed positive expression in the nuclei of alveolar cells, apoptotic bodies in the cytoplasm of alveolar macrophages and cellular debris in alveolar spaces. CONCLUSIONS: P. carinii induces injury and apoptosis of alveolar cells after attachment of the organisms to host cells, and alveolar macrophages enhance the clearance of apoptotic bodies of alveolar cells as well as phagocytosis and degradation of P. carinii.
Apoptosis* ; Biotin ; Coloring Agents ; Cytoplasm ; Epithelial Cells ; Fibronectins ; Immunohistochemistry ; In Situ Nick-End Labeling ; Lung ; Macrophages, Alveolar ; Microscopy ; Microscopy, Immunoelectron ; Phagocytosis ; Pneumocystis carinii* ; Pneumocystis* ; Pneumonia ; Pneumonia, Pneumocystis* ; Vitronectin

BACKGROUND: We determined the changes of complement regulator gene expression in the amyloid-beta1-42(A beta1-42) and interferon-gamma(IFN-gamma)-stimulated human astrocytoma cell line. METHODS: The human astrocytoma cell line, U373MG, was stimulated with IFN-gamma(62.5-1,000U/ml) in the presence or absence of aggregated A beta1-42(1-20micrometer) for 24 hours. Messenger RNA expression of C1 inhibitor(C1-INH), complement factor I(CFI), clusterin, vitronectin, decay accelerating factor(DAF), membrane cofactor protein(MCP), and CD59 was measured by quantitative real-time reverse transcriptase-PCR. RESULTS: IFN-gamma(final concentration, 500U/ml) markedly increased the expression of mRNA for C1-INH in a time dependent fashion. A beta1-42(final concentration, 2micrometer) induced a slight increase in the expression of C1-INH. Messenger RNAs for CFI and clusterin were minimally increased, but other regulators were unchanged or decreased by either A beta1-42 or IFN-gamma. IFN-gamma overrode A beta1-42-induced mRNA expression of C1-INH when the cells were treated with these two reagents together. CONCLUSION: Among the complement regulator genes in the human astrocytoma cell line, U373MG, only C1-INH was significantly up-regulated by IFN-gamma with or without A beta1-42 administration.
Alzheimer Disease ; Aminopeptidases ; Amyloid beta-Peptides ; Astrocytoma ; Cell Line ; Clusterin ; Complement Factor I ; Complement System Proteins ; Genes, Regulator ; Humans ; Indicators and Reagents ; Interferon-gamma ; Interferons ; Membranes ; RNA, Messenger ; Vitronectin

Vascular endothelial cell expression of avb3, and integrin receptor that binds von Willebrand factor, vitronectin, and fibrinogen, increases in response to angiogenic stimuli such as basic fibroblast growth factor(bFGF) and during capillary proliferation in vivo. We investigated the importance of avb3 function during bFGF-induced corneal angiogenesis by examining the effects of 9D491, a monoclnal natibody against b3 that blocks avb3-mediated cell adhesion to vitronectin and fibrinogen in vitro. A hydrogel disk containing 500ng of bFGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the firtt, randomized to contain either 3ug of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and counted an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 post-implantation, angiogenesis scores were significantly lower in eyes treated with avb3 mAb(averaged score=3.3) as compared to eyes treated with 6E10(averaged score=8.0)(p<0.002, Wilcoxon rank sum test). In a rabbit corneal pocket model of angiogenesis, neutralizing monoclonal antibody to b3 inhibits corneal angiogenesis induced by bFGF. Substances that target the integrin b3 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.
Blood Vessels ; Capillaries ; Cell Adhesion ; Corneal Neovascularization* ; Corneal Stroma ; Endothelial Cells ; Fibrinogen ; Fibroblasts ; Hydrogel ; Masks ; New Zealand ; Vitronectin ; von Willebrand Factor

Vascular endothelial cell expression of avb3, and integrin receptor that binds von Willebrand factor, vitronectin, and fibrinogen, increases in response to angiogenic stimuli such as basic fibroblast growth factor(bFGF) and during capillary proliferation in vivo. We investigated the importance of avb3 function during bFGF-induced corneal angiogenesis by examining the effects of 9D491, a monoclnal natibody against b3 that blocks avb3-mediated cell adhesion to vitronectin and fibrinogen in vitro. A hydrogel disk containing 500ng of bFGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the firtt, randomized to contain either 3ug of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and counted an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 post-implantation, angiogenesis scores were significantly lower in eyes treated with avb3 mAb(averaged score=3.3) as compared to eyes treated with 6E10(averaged score=8.0)(p<0.002, Wilcoxon rank sum test). In a rabbit corneal pocket model of angiogenesis, neutralizing monoclonal antibody to b3 inhibits corneal angiogenesis induced by bFGF. Substances that target the integrin b3 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.
Blood Vessels ; Capillaries ; Cell Adhesion ; Corneal Neovascularization* ; Corneal Stroma ; Endothelial Cells ; Fibrinogen ; Fibroblasts ; Hydrogel ; Masks ; New Zealand ; Vitronectin ; von Willebrand Factor

Epstein-Barr virus (EBV) establishes a latent infection in greater than 90% of the world's adult population and associates with various tumors. EBV primarily infects epithelial cells and B cell in vivo. Mechanism of EBV infection in B cells is known to involve binding of EBV glycoprotein gp350 to CD21 on B cell surface. Epithelial cells are infected with EBV even though most of epithelial cells do not express CD21. Recently, integrin alphavbeta5, alphavbeta6 and alphavbeta8 on epithelial cells were reported to facilitate EBV infection by interacting with gHgL complex. We examined the expression profile of integrins known to be expressed on epithelial cells. Integrin alphavbeta5 and alphavbeta6, but not alphavbeta8 were detected in a gastric epithelial cell line, AGS. We then tested whether siRNAs specific to beta6 can inhibit EBV infection of epithelial cells. One among the four tested siRNAs significantly reduced beta6 expression and suppressed transfer infection of EBV to AGS cells. Our data suggest that siRNAs to integrins might be useful to control EBV infection to epithelial cells.
Adult ; B-Lymphocytes ; Epithelial Cells ; Epstein-Barr Virus Infections ; Glycoproteins ; Herpesvirus 4, Human ; Humans ; Integrin beta Chains ; Integrins ; Receptors, Vitronectin ; RNA, Small Interfering

Collagen Type I ; biosynthesis ; Hepatocyte Growth Factor ; biosynthesis ; Hepatocytes ; cytology ; metabolism ; physiology ; Liver Cirrhosis ; metabolism ; pathology ; physiopathology ; Liver Regeneration ; Receptors, Vitronectin ; biosynthesis

Vascular cells respond to multiple cytokines, they also express a variety of integrin adhesion receptor. A number of the vascular cell integrins are functionally and structurally homologous, suggesting some level of biologic redundancy. We investigated the importance of alphavbeta3 function during vascular endothelial growth factor(VEGF) induced corneal angiogenesis by examining the effects of 9D491, a monoclonal antibody against beta3 that blocks alphavbeta3-mediated cell adhesion to vitronectin and fibrinogen. A hydrogel disk containing 500ng of VEGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the first, randomized to contain either 2.6microgram of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and assigned an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 postimplantation, angiogenesis scores were not significantly lower in eyes treated with anti-alphavbeta3 mAb (averaged score=21.6) as compared to eyes treated with 6E10 (averaged score=24.0) (p<0.2, Wilcoxon rank sum test). In a rabbit corneal pocket assay, monoclonal antibody to beta3 could not inhibit corneal angiogenesis induced by VEGF.
Blood Vessels ; Cell Adhesion ; Corneal Neovascularization* ; Corneal Stroma ; Cytokines ; Fibrinogen ; Hydrogel ; Integrin beta3 ; Integrins ; Masks ; New Zealand ; Vascular Endothelial Growth Factor A* ; Vitronectin

BACKGROUND AND OBJECTIVES: Recent studies have examined the structure-function relationship of high-density lipoprotein (HDL). This study aimed to identify and rank HDL-associated proteins involved in several biological function of HDL.METHODS: HDLs isolated from 48 participants were analyzed. Cholesterol efflux capacity, effect of HDL on nitric oxide production, and vascular cell adhesion molecule-1 expression were assessed. The relative abundance of identified proteins in the highest vs. lowest quartile was expressed using the normalized spectral abundance factor ratio.RESULTS: After adjustment by multiple testing, six proteins, thyroxine-binding globulin, alpha-1B-glycoprotein, plasma serine protease inhibitor, vitronectin, angiotensinogen, and serum amyloid A-4, were more abundant (relative abundance ratio ≥2) in HDLs with the highest cholesterol efflux capacity. In contrast, three proteins, complement C4-A, alpha-2-macroglobulin, and immunoglobulin mu chain C region, were less abundant (relative abundance ratio <0.5). In terms of nitric oxide production and vascular cell adhesion molecule-1 expression, no proteins showed abundance ratios ≥2 or <0.5 after adjustment. Proteins correlated with the functional parameters of HDL belonged to diverse biological categories.CONCLUSIONS: In summary, this study ranked proteins showing higher or lower abundance in HDLs with high functional capacities and newly identified multiple proteins linked to cholesterol efflux capacity.
Amyloid ; Angiotensinogen ; Atherosclerosis ; Cardiovascular Diseases ; Cholesterol ; Complement System Proteins ; Immunoglobulin mu-Chains ; Lipoproteins ; Nitric Oxide ; Plasma ; Proteomics ; Serine Proteases ; Thyroxine-Binding Globulin ; Vascular Cell Adhesion Molecule-1 ; Vitronectin