A neutralizing antibody against DKK1 does not reduce plaque formation in classical murine models of atherosclerosis: Is the therapeutic potential lost in translation?

BACKGROUND AND AIMS: Clinical interventions targeting nonlipid risk factors are needed given the high residual risk of atherothrombotic events despite effective control of dyslipidemia. Dickkopf-1 (DKK1) plays a lipid-independent role in vascular pathophysiology but its involvement in atherosclerosis development and its therapeutic attractiveness remain to be established. METHODS: Patient data, in vitro studies and pharmacological intervention in murine models of atherosclerosis were utilized. RESULTS: In patients' material (n = 127 late stage plaque specimens and n = 10 control vessels), DKK1 mRNA was found to be higher in atherosclerotic plaques versus control arteries. DKK1 protein was detected in the luminal intimal area and in the necrotic core of plaques. DKK1 was released from isolated primary human platelets (~12 - 21-fold) and endothelial cells (~1.4-2.5-fold) upon stimulation with different pathophysiological stimuli. In ApoE-/- and Ldlr-/- mice, plasma DKK1 concentrations were similar to those observed in humans, whereas DKK1 expression in different atheroprone arterial segments was very low/absent. Chronic treatment with a neutralizing DKK1 antibody effectively reduced plasma concentrations, however, plaque lesion area was not reduced in ApoE-/- and Ldlr-/- mice fed a western diet for 14 and 16 weeks. Anti-DKK1 treatment increased bone volume and bone mineral content. CONCLUSIONS: Functional inhibition of DKK1 with an antibody does not alter atherosclerosis progression in classical murine models. This may reflect the absence of DKK1 expression in plaques and more advanced animal disease models could be needed to evaluate the role and therapeutic attractiveness of DKK1 in late stage complications such as plaque destabilization, calcification, rupture and thrombosis.

A Membrane-Bound Diacylglycerol Species Induces PKCϵ-Mediated Hepatic Insulin Resistance.

Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR.

Cross-species reactive monoclonal antibodies against the extracellular domains of the insulin receptor and IGF1 receptor.

Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples. Using these strategies, we were able to identify highly specific insulin receptor monoclonal antibodies that lack cross-reactivity to the IGF1 receptor using the Adimab platform and a highly specific IGF1 receptor monoclonal antibody that lacks cross-reactivity to the insulin receptor using the rabbit antibody platform. Unlike earlier monoclonal antibodies reported in the literature, these antibodies show cross-species reactivity to the extracellular domains of mouse, rat, pig, and human receptors, indicating that they bind conserved epitopes. Furthermore, the antibodies work well in several different assay formats, including ELISA, flow cytometry, and immunoprecipitation, and therefore provide new tools to study insulin and IGF1 receptor biology with translation across several species and experimental model systems.

Activation of insulin receptors and IGF-1 receptors in COLO-205 colon cancer xenografts by insulin and insulin analogue X10 does not enhance growth under normo- or hypoglycaemic conditions.

AIMS/HYPOTHESIS: Recent studies with normal rats and mouse allograft models have reported that insulin and insulin analogues do not activate the IGF-1 receptor in vivo, and that this characteristic therefore cannot be responsible for the increased incidence of mammary tumours observed for the insulin analogue X10 in chronic toxicity studies with Sprague Dawley rats. This is in clear contrast to reports of insulin and insulin analogues in vitro. Clarification of this is important for understanding the mechanisms behind possible growth-promoting effects of insulin analogues, and will have implications for the development of novel insulin analogues. METHODS: We established a xenograft model in BALB/c nude mice with the human colon cancer cell line COLO-205, which expresses human insulin and IGF-1 receptors, and explored the acute and chronic effects of treatment with supra-pharmacological doses of human insulin, insulin analogue X10 and human IGF-1. With a novel antibody, acute IGF-1 receptor activation was also examined in various tissues from normal rats treated with human insulin, insulin analogue X10 or human IGF-1. Finally, the effects of pharmacologically relevant doses of human insulin and insulin analogue X10 on receptor activation and growth of COLO-205 xenograft were explored in BALB/c nude mice with alloxan-induced hyperglycaemia. RESULTS: In normal rats and in BALB/c nude mice bearing a COLO-205 cell xenograft, treatment with supra-pharmacological doses of human insulin, insulin analogue X10 or human IGF-1 resulted in activation of insulin receptors as well as IGF-1 receptors. Treatment of diabetic nude mice with pharmacologically relevant doses of human insulin or insulin analogue X10, which decreased blood glucose from hyperglycaemic levels to the normoglycaemic range, did not increase IGF-1 receptor activation. Furthermore, repeated treatment with supra-pharmacological as well as pharmacological doses of human insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts. CONCLUSIONS/INTERPRETATION: This study demonstrates that activation of IGF-1 receptors in cancer cells by insulin and insulin analogues cannot be considered as a purely in vitro phenomenon. It does occur in vivo in animal models, although only after treatment with supra-pharmacological doses. Furthermore, treatment with insulin or insulin analogue X10 did not influence the growth of COLO-205 xenografts under normo- or hypoglycaemic conditions. Further studies are needed before a conclusion can be reached on whether IGF-1 receptor activation by insulin analogues correlates with increased growth in vivo.

Data analysis and uncertainty estimation in supercontinuum laser absorption spectroscopy.

A set of algorithms is presented that facilitates the evaluation of super continuum laser absorption spectroscopy (SCLAS) measurements with respect to temperature, pressure and species concentration without the need for simultaneous background intensity measurements. For this purpose a non-linear model fitting approach is employed. A detailed discussion of the influences on the instrument function of the spectrometer and a method for the in-situ determination of the instrument function without additional hardware are given. The evaluation procedure is supplemented by a detailed measurement precision assessment by applying an error propagation through the non-linear model fitting approach. While the algorithms are tailored to SCLAS, they can be transferred to other spectroscopic methods, that similarly require an instrument function. The presented methods are validated using gas cell measurements of methane in the near infrared region at pressures up to 8.7 bar.

PolySpec: polynomial spectrum models for fast and light-weight spectroscopic evaluation.

For the analysis of spectroscopic data, model fitting approaches are commonly applied. The spectrum model applied in these fitting processes significantly influences the performance of the spectroscopic evaluation, which can be critical in real-time process diagnostics and control. In this work a spectrum model is introduced that uses a polynomial description of absorbances, transmittances, or similar in dependence on parameters such as temperature, pressure, and mole fraction. Using this approach, either experimental spectra or spectrum databases can be compressed into a matrix of polynomial coefficients. The evaluation of this model consists of a single matrix multiplication and, with a slight modification, derivatives with regard to specific parameters can be calculated in the same way. Both these points are important to model fitting methods for spectroscopic data, as the simple evaluation method allows for a fast analysis and the direct calculation of derivatives simplifies the application of gradient-based fitting methods. Additionally, the easy parallelizability of the matrix multiplication promotes the application of this method in real-time evaluations on programmable logic devices.

Novel time-of-flight fiber dispersion measurement technique using supercontinuum light sources and acousto-optical tunable filters.

Long-distance fiber links require precise knowledge of fiber dispersion characteristics. Similar dispersion characteristics are necessary for supercontinuum broadband laser absorption spectroscopy (SCLAS) to allow proper data evaluation and species concentration determination, as well as numerous other applications. In this work, a time-of-flight approach to measuring the dispersion characteristic of fibers with supercontinuum laser light sources (SCLs) and acousto-optical tunable filters (AOTFs) is presented. Broadband emission of the SCL is filtered with a narrowband AOTF and dispersed in time by the fiber under test. By using the wavelength-specific delay, the dispersion characteristic can be calculated. The technique is especially suited for longer fibers and was verified against a state-of-the-art phase-shift-based dispersion measurement system. Advantages of the new approach include solely utilizing SCLAS system components, as well as a high level of automation and wide spectral coverage, ranging from 1100 to 1700 nm in a single measurement setup.

Broadband supercontinuum laser absorption spectrometer for multiparameter gas phase combustion diagnostics.

We report on the development and application of a broadband absorption spectrometer utilizing a pulsed supercontinuum laser light source and dispersion compensating fiber with a single-pass absorption path to obtain absolute methane mole fractions in a laminar nonpremixed CH(4)/air flame supported on a Wolfhard-Parker burner. The basic principle of supercontinuum broadband absorption spectroscopy (SCLAS) provides advantageous means of combustion diagnostics since the broad spectral coverage allows for use in high-pressure high-temperature environments. Furthermore, a previously validated tunable diode laser absorption spectroscopy fitting algorithm was applied to the recorded spectra and found to be applicable to SCLAS measurements as well, by comparison of fitted methane gas concentrations to reference measurements on the Wolfhard-Parker burner. The spectrometer reached spectral resolutions of up to 0.152 cm(-1), while providing a spectral coverage of over 110 cm(-1), with an absorption path length of only 41 mm. First measurements of absolute CH(4) mole fractions showed the suitability of SCL-based spectroscopy for combustion diagnostics with short absorption path lengths in the nIR spectral region. Here, we achieved in-flame methane mole fraction resolutions of 3%(Vol.) (1210 ppm·m) and optical resolutions of up to 1.1×10(-2). Based on this first validation, this method can now be extended to other species and combustion parameters such as temperature and pressure.

C-Terminally PEGylated hGH-derivatives.

A two-step strategy was used for the preparation of C-terminally PEGylated hGH-derivatives. In a first step a CPY-catalyzed transpeptidation was performed on hGH-Leu-Ala, introducing reaction handles, which were used in the second step for the ligation of PEG-moieties. Both oxime-ligation and copper(I) catalyzed [2+3]-cycloaddition reactions were used for the attachment of PEG-moieties. The biological data show a dependency of the potency of the hGH-derivatives on both size as well as shape of the PEG-group.

Stimulation of pancreatic beta-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways.

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as beta-cell growth factors and may therefore be of critical importance for the maintenance of a proper beta-cell mass. We have investigated the molecular mechanism of incretin-induced beta-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, liraglutide, increased beta-cell replication 50-80% at 10-100 nM upon a 24 h stimulus, whereas glucagon at a similar concentration had no significant effect. The stimulatory effect of GLP-1 and GIP was efficiently mimicked by the adenylate cyclase activator, forskolin, at 10 nM (approximately 90% increase) and was additive (approximately 170-250% increase) with the growth response to human growth hormone (hGH), indicating the use of distinct intracellular signalling pathways leading to mitosis by incretins and cytokines, respectively. The response to both GLP-1 and GIP was completely blocked by the protein kinase A (PKA) inhibitor, H89. In addition, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, both inhibited GLP-1- and GIP-stimulated proliferation. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, had no inhibitory effect on either GLP-1 or GIP stimulated proliferation. Cyclin Ds act as molecular switches for the G0/G1-S phase transition in many cell types and we have previously demonstrated hGH-induced cyclin D2 expression in the insulinoma cell line, INS-1. GLP-1 time-dependently induced the cyclin D1 mRNA and protein levels in INS-1E, whereas the cyclin D2 levels were unaffected. However, minor effect of GLP-1 stimulation was observed on the cyclin D3 mRNA levels. Transient transfection of a cyclin D1 promoter-luciferase reporter construct into islet monolayer cells or INS-1 cells revealed approximately a 2-3 fold increase of transcriptional activity in response to GLP-1 and GIP, and a 4-7 fold increase in response to forskolin. However, treatment of either cell type with hGH had no effect on cyclin D1 promoter activity. The stimulation of the cyclin D1 promoter by GLP-1 was inhibited by H89, wortmannin, and PD98059. We conclude that incretin-induced beta-cell replication is dependent on cAMP/PKA, p42 MAPK and PI3K activities, which may involve transcriptional induction of cyclin D1. GLP-1, GIP and liraglutide may have the potential to increase beta-cell replication in humans which would have significant impact on long-term diabetes treatment.

Skin changes induced by a zinc oxide dressing compared with a hydrocolloid dressing in healthy individuals.

BACKGROUND/PURPOSE: Incidence of skin complications in ostomy patients constitutes a well-known and well-described problem. The reasons are, however, very difficult to describe because of the many factors contributing to the problem. This article describes the skin changes derived exclusively from the adhesives used in a carefully controlled, long-term study using two fundamentally different types of adhesives: a hydrocolloid adhesive and a zinc oxide adhesive. METHODS: The adhesives were changed daily on the volar forearm of 11 volunteers for a 4-week period. Once a week, transepidermal water-loss (TEWL), water content of the skin, erythema and the peel force applied for removal of the adhesives were measured. On the last day of the study, a replica of the skin surface was obtained to determine changes in the skin topography, and a biopsy was taken to study changes at the cellular level. RESULTS AND CONCLUSION: We found increased TEWL and decreased water content in skin treated with the zinc oxide adhesive, but increased water-loss and water content when the hydrocolloid adhesive was used. In addition, the area treated with zinc oxide adhesive showed significant increase of epidermal thickness, scaly appearance and parakeratosis with similarities to pathological dry skin diseases such as psoriasis and atopic dermatitis, changes that were not found when using the hydrocolloid adhesive. The skin response seems to be the result of the content of zinc oxide and the mechanical interaction of the zinc oxide adhesive. We conclude that the nature of the adhesive plays an important role in the skin response to repeated application of adhesives, as seen in peristomal skin.

The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro.

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the beta-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining beta-cell mass in both type 1 and type 2 diabetic patients.