Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

Toward stable N4-modified neurotensins for NTS1-receptor-targeted tumor imaging with 99mTc

A series of Gly-neurotensin(8-13) analogues modified at the N-terminus by acyclic tetraamines (Demotensin 1-4) were obtained by solid-phase peptide synthesis techniques. Strategic replacement of amino acids and/or reduction of sensitive peptide bonds were performed to enhance conjugate resistance against proteolytic enzymes. During 99mTc-labeling, single species radiopeptides, [99mTc]Demotensin 1-4, were easily obtained in high yields and typical specific activities of 1 Ci/micromol. Peptide conjugates displayed a high affinity binding to the human neurotensin subtype 1 receptor (NTS1-R) expressed in colon adenocarcinoma HT-29 or WiDr cells and/or in human tumor sections. [99mTc]Demotensin 1-4 internalized very rapidly in HT-29 or WiDr cells by a NTS1-R-mediated process. [99mTc]Demotensin 3 and 4, which remained stable during 1 h incubation in murine plasma, were selectively studied in nude mice bearing human HT-29 and WiDr xenografts. After injection, [99mTc]Demotensin 3 and 4 effectively and specifically localized in the experimental tumors and were rapidly excreted via the kidneys into the urine, exhibiting overall biodistribution patterns favorable for NTS1-R-targeted tumor imaging in man.

Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity

Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

Use of concatamers to study GABAA receptor architecture and function: application to delta-subunit-containing receptors and possible pitfalls

Many membrane proteins, including the GABA(A) [GABA (gamma-aminobutyric acid) type A] receptors, are oligomers often built from different subunits. As an example, the major adult isoform of the GABA(A) receptor is a pentamer built from three different subunits. Theoretically, co-expression of three subunits may result in many different receptor pentamers. Subunit concatenation allows us to pre-define the relative arrangement of the subunits. This method may thus be used to study receptor architecture, but also the nature of binding sites. Indeed, it made possible the discovery of a novel benzodiazepine site. We use here subunit concatenation to study delta-subunit-containing GABA(A) receptors. We provide evidence for the formation of different functional subunit arrangements in recombinant alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors. As with all valuable techniques, subunit concatenation has also some pitfalls. Most of these can be avoided by carefully titrating and minimizing the length of the linker sequences joining the two linked subunits and avoiding inclusion of the signal sequence of all but the N-terminal subunit of a multi-subunit construct. Maybe the most common error found in the literature is that low expression can be overcome by simply overloading the expression system with genetic information. As some concatenated constructs result by themselves in a low level of expression, this erroneous assembly leading to receptor function may be promoted by overloading the expression system and leads to wrong conclusions.

Major histocompatibility complex and other allergy-related candidate genes associated with insect bite hypersensitivity in Icelandic horses

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of insects. IBH is a multifactorial disease with contribution of genetic and environmental factors. Candidate gene association analysis of IBH was performed in a group of 89 Icelandic horses all born in Iceland and imported to Europe. Horses were classified in IBH-affected and non-affected based on clinical signs and history of recurrent dermatitis, and on the results of an in vitro sulfidoleukotriene (sLT)-release assay with Culicoides nubeculosus and Simulium vittatum extract. Different genetic markers were tested for association with IBH by the Fisher's exact test. The effect of the major histocompatibility complex (MHC) gene region was studied by genotyping five microsatellites spanning the MHC region (COR112, COR113, COR114, UM011 and UMN-JH34-2), and exon 2 polymorphisms of the class II Eqca-DRA gene. Associations with Eqca-DRA and COR113 were identified (p < 0.05). In addition, a panel of 20 single nucleotide polymorphisms (SNPs) in 17 candidate allergy-related genes was tested. During the initial screen, no marker from the panel was significantly (p < 0.05) associated with IBH. Five SNPs associated with IBH at p < 0.10 were therefore used for analysis of combined genotypes. Out of them, SNPs located in the genes coding for the CD14 receptor (CD14), interleukin 23 receptor (IL23R), thymic stromal lymphopoietin (TSLP) and transforming growth factor beta 3 (TGFB3) molecules were associated with IBH as parts of complex genotypes. These results are supported by similar associations and by expression data from different horse populations and from human studies.

NO-dependent CaMKII activation during -adrenergic stimulation of cardiac muscle

AIMS:During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. METHODS AND RESULTS:Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. CONCLUSIONS:β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

Diversity of structure and function of alpha1alpha6beta3delta GABAA receptors: comparison with alpha1beta3delta and alpha6beta3delta receptors

delta subunit-containing gamma-aminobutyric acid, type A (GABA(A))receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with alpha(1) and/or alpha(6) subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA(A) receptor pentamers by concatenation. These receptors composed of alpha(1), alpha(6), beta(3), and delta subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that delta can assume multiple positions in a receptor pentamer made up of alpha(1), alpha(6), beta(3), and delta subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of delta subunits between two alpha subunits in alpha(1)alpha(6)beta(3)delta receptors. This property is shared by alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors, but there are differences in the additionally expressed isoforms.

Evolution of bombesin conjugates for targeted PET imaging of tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel (64/67)Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC(50): 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle(14) by Met, and deletion of D-Tyr(6), further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6(th), 11(th), and 14(th) position of BN induced only slight influences on affinity to mouse GRPr. [Cu(II)]-CPTA-[βAla(11)] BN(7-14) ([Cu(II)]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [(64/67)Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [(64/67)Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [(64/67)Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.

Differential modulation of ROS signals and other mitochondrial parameters by the antioxidants MitoQ, resveratrol and curcumin in human adipocytes

Abstract Mitochondrial reactive oxygen species (ROS) have been demonstrated to play an important role as signaling and regulating molecules in human adipocytes. In order to evaluate the differential modulating roles of antioxidants, we treated human adipocytes differentiated from human bone marrow-derived mesenchymal stem cells with MitoQ, resveratrol and curcumin. The effects on ROS, viability, mitochondrial respiration and intracellular ATP levels were examined. MitoQ lowered both oxidizing and reducing ROS. Resveratrol decreased reducing and curcumin oxidizing radicals only. All three substances slightly decreased state III respiration immediately after addition. After 24 h of treatment, MitoQ inhibited both basal and uncoupled oxygen consumption, whereas curcumin and resveratrol had no effect. Intracellular ATP levels were not altered. This demonstrates that MitoQ, resveratrol and curcumin exert potent modulating effects on ROS signaling in human adipocyte with marginal effects on metabolic parameters.

Secretin receptors in the human liver: expression in biliary tract and cholangiocarcinoma, but not in hepatocytes or hepatocellular carcinoma

BACKGROUND/AIMS: Gut hormone receptors are over-expressed in human cancer and allow receptor-targeted tumor imaging and therapy. A novel promising receptor for these purposes is the secretin receptor. The secretin receptor expression was investigated in the human liver because the liver is a physiological secretin target and because novel diagnostic and treatment modalities are needed for liver cancer. METHODS: Nineteen normal livers, 10 cirrhotic livers, 35 cholangiocarcinomas, and 45 hepatocellular carcinomas were investigated for secretin receptor expression by in vitro receptor autoradiography using (125)I-[Tyr(10)] rat secretin and, in selected cases, for secretin receptor mRNA by RT-PCR. RESULTS: Secretin receptors were present in normal bile ducts and ductules, but not in hepatocytes. A significant receptor up-regulation was observed in ductular reaction in liver cirrhosis. Twenty-two (63%) cholangiocarcinomas were positive for secretin receptors, while hepatocellular carcinomas were negative. RT-PCR revealed wild-type receptor mRNA in the non-neoplastic liver, wild-type and spliced variant receptor mRNAs in cholangiocarcinomas found receptor positive in autoradiography experiments, and no receptor transcripts in autoradiographically negative cholangiocarcinomas. CONCLUSIONS: The expression of secretin receptors in the biliary tract is the molecular basis of the secretin-induced bicarbonate-rich choleresis in man. The high receptor expression in cholangiocarcinomas may be used for in vivo secretin receptor-targeting of these tumors and for the differential diagnosis with hepatocellular carcinoma.

Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.

Integrins and cardiovascular disease

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor alpha IIb beta 3, has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of alpha IIb beta 3 inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS: The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS: For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS: This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE: Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.