Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C.

Objective: These experiments were designed to elucidate mechanisms mediating vascular dysfunction induced by advanced glycation end products (AGEs).

Methods: Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 week later, granulation tissue that formed in the bottom of the chamber was exposed twice daily for 7 days to glycated rat serum albumin in the presence and absence of inhibitors of reactive oxygen intermediates, nitric oxide synthase and guanylate cyclase, protein kinase C (PKC), and a neutralizing vascular endothelial growth factor (VEGF) antibody. Vascular (125)I-albumin clearance and blood flow were quantified by use of a double isotope-dilution technique and radiolabeled microspheres, respectively.

Vascular endothelial growth factor and basic fibroblast growth factor differentially modulate early postnatal coronary angiogenesis.

The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged.

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells.

Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor.

We tested the hypothesis that selectin inhibition with blocking antibodies or a small-molecular-weight inhibitor of L-, P-, and E-selectin, methoxybenzoylpropionic acid (MBPA), prevents thrombus formation in a canine coronary Folts' model. Cyclic flow variations (CFVs) were induced by crush injury and constriction of the left anterior descending coronary artery in dogs. Systemic infusion of antibodies to P- and L-selectin abolished CFVs, respectively, in 50% and 17% of treated dogs [P = not significant (NS)].

A novel protein with homology to the junctional adhesion molecule. Characterization of leukocyte interactions.

We have cloned a novel cDNA belonging to the Ig superfamily that shows 44% similarity to the junctional adhesion molecule (JAM) and maps to chromosome 21q21.2. The open reading frame of JAM2 predicts a 34-kDa type I integral membrane protein that features two Ig-like folds and three N-linked glycosylation sites in the extracellular domain.

Bradycardia-induced coronary angiogenesis is dependent on vascular endothelial growth factor.

A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily.

Development of novel monoclonal antibodies for the analysis of functional sites in FGF-2.

Fibroblast growth factor 2 (FGF-2) can function as a potent mitogen, as well as a survival factor for a variety of mammalian cell types. The biological effects of FGF-2 are mediated by its interaction with two types of cellular binding sites: (1) high affinity tyrosine kinase receptors; and (2) low affinity heparan sulfate proteoglycans (HSPGs) on the cell surface. Although numerous FGF-2 antibodies have been used previously to analyze its biological actions, few studies have utilized antibodies to analyze domains within FGF-2 involved in its interactions with the two binding sites.

Selectin blockade prevents antigen-induced late bronchial responses and airway hyperresponsiveness in allergic sheep.

Antigen challenge can elicit an allergic inflammatory response in the airways that involves eosinophils, basophils, and neutrophils and that is expressed physiologically as a late airway response (LAR) and airway hyperresponsiveness (AHR). Although previous studies have suggested that E-selectin participates in these allergic airway responses, there is little information concerning the role of L-selectin. To address this question, we examined the effects of administering an L-selectin-specific monoclonal antibody, DU1-29, as well as three small molecule selectin binding inhibitors, on the development of early airway responses (EAR), LAR and AHR in allergic sheep undergoing airway challenge with Ascaris suum antigen.

Vascular remodeling in response to altered blood flow is mediated by fibroblast growth factor-2.

Vascular structures adapt to changes in blood flow by adjusting their diameter accordingly. The factors mediating this process are only beginning to be identified. We have recently established a mouse model of arterial remodeling in which flow in the common carotid artery is interrupted by ligation of the vessel near the carotid bifurcation, resulting in a dramatic reduction in vessel diameter as a consequence of inward remodeling and intimal lesion formation.

KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells.

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions.

Role for heparin-binding growth factors in glucose-induced vascular dysfunction.

Vascular hyperpermeability and excessive neovascularization are hallmarks of early and late vascular endothelial cell dysfunction induced by diabetes. Vascular endothelial growth factor (VEGF) appears to be an important mediator for these early and late vascular changes. We reported previously, using skin chambers mounted on backs of SD rats, that neutralizing antibodies directed against VEGF blocked vascular permeability and blood flow changes induced by elevated tissue glucose and sorbitol levels in a dosage-dependent manner.

Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor.

The purpose of these experiments was to investigate a potential role for vascular endothelial growth factor (VEGF) in mediating vascular dysfunction induced by increased glucose flux via the sorbitol pathway. Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 wk later, granulation tissue in the chamber was exposed twice daily for 7 d to 5 mM glucose, 30 mM glucose, or 1 mM sorbitol in the presence and absence of neutralizing VEGF antibodies. Albumin permeation and blood flow were increased two- to three-fold by 30 mM glucose and 1 mM sorbitol; these increases were prevented by coadministration of neutralizing VEGF antibodies.

A cyclic hexapeptide is a potent antagonist of alpha 4 integrins.

The alpha 4 integrins mediate leukocyte adhesion to specific counter-receptors, including vascular cell adhesion molecule-1 (VCAM-1), the fibronectin splice variant containing connecting segment 1 (CS1), and mucosal addressin cell adhesion molecule-1. A series of cyclized peptides based on the LDV sequence of CS1 were synthesized and assayed for inhibition of alpha 4 integrin binding. The most potent peptide, C*WLDVC* (where * indicates disulfide-linked residues), inhibited alpha 4 beta 1-dependent binding of lymphocytes to VCAM-1 and CS1 with half-maximal inhibition achieved at 1 to 3 microM of peptide.

Brain nicotinic receptors isolated by a monospecific antibody against a synthetic alpha-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody.

Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain.

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM.

Characterization of nicotinic receptors on cultured cortical neurons using anti-idiotypic antibodies and ligand binding.

Monoclonal anti-idiotypic antibodies that represent the internal image of nicotine's natural isomer, L-nicotine, were used in conjunction with L-[3H]nicotine binding to characterize nicotinic receptors on neurons cultured from fetal rat cortex. Of the antibodies tested, two (422F11 and 420G11) were found that recognized a class of high affinity [3H]nicotine binding sites present on neuronal cells, but not on glia. The binding properties and pharmacological specificity of these sites compared well with those determined previously for putative nicotinic cholinergic receptors in adult rat brain.

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies.

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.

Idiotype-anti-idiotype hapten immunoassays: assay for cotinine.

Practical application of the idiotype-anti-idiotype reaction to hapten immunoassays has been demonstrated with cotinine as an example. The assay relies on the ability of cotinine, a major nicotine metabolite, to inhibit binding between a monoclonal anti-cotinine antibody (the idiotype) and a second monoclonal antibody (the anti-idiotype) specific for the antigen combining region on the idiotype. A solid phase enzyme-linked immunoadsorbent assay (ELISA) format was adopted in which fluid phase anti-cotinine and cotinine present either as a standard or in a test sample were incubated in microtiter plate wells coated with F(ab')2 fragments of the anti-idiotype.

Anti-idiotypic antibody probes of neuronal nicotinic receptors.

Monoclonal anti-idiotypic antibodies specific for the combining site on a monoclonal antinicotine were used in immunocytochemistry to localize nicotine binding sites on rat brain cortical sections and in immunoaffinity chromatography to isolate receptor from solubilized brain tissue. The receptor, which consists of two subunits with Mr values of 43 and 50 kDa, was eluted from the antiidiotype column with either pH3 citrate buffer or 25 mM (-)-nicotine, but was not present in eluates from immobilized anti-Electrophorus acetylcholine receptor or anti-methotrexate. The anti-idiotypes specifically inhibited [3H]nicotine binding to rat brain homogenate and (-)-nicotine inhibited anti-idiotype binding to brain sections based on abrogation of immunofluorescence staining.

Monoclonal antibody ELISA for cotinine in saliva and urine of active and passive smokers.

The value of a monoclonal antibody-based ELISA for measuring cotinine in saliva and urine of active and passive smokers was assessed. Cotinine (mean +/- SEM) was detected in all 26 saliva (392 +/- 74 ng/ml) and 27 urine (4264 +/- 508 ng/mg creatinine; 2566 +/- 364 ng/ml) samples from smoking parents, but in only two of 36 salivas and one of 37 urines from nonsmokers (P less than 0.001).

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies.

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [3H]nicotine binding to the purified material. An enantiomeric analogue of nicotine, (-)-6-hydroxymethyl-nicotine, was used to prepare the affinity column. In addition, with the use of anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa.

Monoclonal anti-idiotypic antibodies that recognize the binding site for nicotine on rat brain receptor.

Anti-idiotypic monoclonal antibodies have been prepared that represent the internal image of nicotine and are specific for the nicotine binding site on rat brain receptor. Specificity of these antibodies for the combining site on anti-nicotine was demonstrated by their ability to inhibit binding of monoclonal anti-nicotine to immobilized nicotine-polylysine. Furthermore, purified rat brain nicotine receptor but not acetylcholine receptor from fish electric organ effectively competed with anti-nicotine for immobilized nicotine and for immobilized anti-idiotype.

Comparison of monoclonal and polyclonal antibodies to cotinine in nonisotopic and isotopic immunoassays.

Monoclonal antibodies (McAb) were used to develop nonisotopic and radioimmunoassays (RIA) for quantitative determination of the major nicotine metabolite, cotinine, in physiological fluids. ELISAs and fluorescence immunoassays were carried out in microtiter plate wells coated with a conjugate of cotinine 4'-carboxylic acid bound covalently to poly-L-lysine. The detection systems were horseradish peroxidase (HRP)-labeled staphylococcal protein A, HRP-streptavidin-biotin, and biotinylated alkaline phosphatase-4-methylumbelliferyl phosphate.

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.