Hermann Dietrich

Interleukin-6 stimulation of growth of prostate cancer in vitro and in vivo through activation of the androgen receptor

It is hypothesized that ligand-independent activation of the androgen receptor is one of the mechanisms implicated in tumour progression. However, supportive evidence is limited to the effect of HER-2/neu that stimulates prostate cancer progression through activation of the androgen receptor. In the present study, we have asked whether the proinflammatory cytokine interleukin-6 (IL-6), which is known to stimulate androgen receptor activity and expression of its downstream target genes, may also induce growth of androgen-sensitive cells. We have found that IL-6 differentially regulates proliferation of LAPC-4 and MDA PCa 2b cells. In MDA PCa 2b cells, growth stimulation by IL-6 was reversed by administration of either the non-steroidal anti-androgen bicalutamide or the inhibitor of the mitogen-activated protein kinase pathway PD98059. Neither cell line was found to express endogenous IL-6. Interestingly, the treatment of those prostate cancer cells did not increase phosphorylation of STAT3. The effect of IL-6 on stimulation of androgen receptor activity in MDA PCa 2b cells was lower than that of androgen, comparable with findings reported by other researchers. However, growth of MDA PCa 2b xenografts in castrated animals treated with IL-6 was similar to that in non-castrated animals. In addition, bicalutamide showed an inhibitory effect on IL-6-regulated growth in vivo. Taken together, data in the present study demonstrate that IL-6 may cause growth of androgen receptor-positive tumours in vitro and in vivo through activation of the androgen receptor.

Design, synthesis, physical and chemical characterisation, and biological interactions of lectin-targeted latex nanoparticles bearing Gd–DTPA chelates: an exploration of magnetic resonance molecular imaging (MRMI)

The physical and chemical parameters involved in the design and synthesis of biospecifically targeted nanoparticulate contrast media for magnetic resonance molecular imaging (MRMI) were explored in this pilot investigation. Latex nanoparticles 100, 400 and 900xa0nm in diameter were doubly derivatised, first with tomato lectin and then with gadoliniumIII-diethylenetriamine pentaacetic acid (Gd-chelates) to target them to epithelial and endothelial glycocalyceal N-glycans and to generate contrast enhancement in magnetic resonance imaging (MRI). After intravenous injection into mice, human placental cotyledons or human Vena saphena magna, contrasty images of the vascular structures were obtained in 1.5xa0T MRI with spatial resolution 0.1xa0mm in the imaging plane and 0.6xa0mm in the z axis, persisting >60xa0min and resistant to washing out by buffer rinses. Ultrastructural analysis of the nanoparticles revealed the targeting groups at the nanoparticle surfaces and the distribution of the Gd-chelates within the nanoparticles and enabled counts for use in determining relaxivity. The relaxivity values revealed were extremely high, accounting for the strong MR signals observed. Occasionally, nanoparticles larger than 100xa0nm were seen in close spatial association with disrupted regions of cell membrane or of collagen fibrils in the extracellular matrix. The data suggest that 100-nm nanoparticles generate adequate contrast for MRMI and cause least disruption to endothelial cell surfaces.

In vivo imaging of the effect of LPS on arterial endothelial cells: molecular imaging of heat shock protein 60 expression

Bacterial endotoxins are known as stress factors for endothelial cells. In 20 normocholesterolemic New Zealand White (NZW) rabbits, endothelial stress was induced by intravenous (i.v.) injection of lipopolysaccharide (LPS), while eight NZW rabbits were sham-treated or served as untreated controls. In vivo molecular imaging was performed using co-registered computer tomography and positron emission tomography 24xa0h after i.v. injection of 124I-labeled monoclonal anti-HSP60 or 124I-radiolabelled isotype control antibodies. Compared to control animals, in vivo images of rabbit aortae revealed significantly increased endothelial binding of 124I-labeled anti-HSP60 antibodies upon LPS, especially at sites of aortal branching. This was confirmed by immunohistochemistry and autoradiography data. Our results showed, as proof-of-principle, that HSP60-expression in normocholesterolemic rabbits is significantly increased after induction of endothelial stress and that non-invasive in vivo molecular imaging of early aortal HSP60-expression using 124I-labeled anti-HSP60 monoclonal antibodies is possible.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides

OBJECTIVEnThe aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis.nnnMETHODSnApoE(-/-) mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay.nnnRESULTSnDecreased lesion size was accompanied by (a) increased splenic Treg numbers; (b) increased interleukin (IL)-10 mRNA levels in the aorta; (c) increased levels of anti-mbHSP65 and anti-mouse HSP60 antibodies pointing to pro-eukaryotic HSP60 humoral crossreaction, not curtailed by oral tolerization; (d) most importantly, we identified and functionally characterized novel atherogenic and atheroprotective mbHSP65 epitopes.nnnCONCLUSIONnAtheroprotective mbHSP65 peptides may be considered as potential candidates for the development of a tolerizing vaccine to prevent and treat atherosclerosis, while keeping protective immunity to non-atherogenic domains of mbHSP65 intact.

Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits

Highlights ► Small hairpin RNA specific for SR-BI nucleotides 214-232 reduces SR-BI expression in vitro. ► SiRNA treatment reduces liver expression of SR-BI in New Zealand White rabbits. ► Liver specific inhibition of SR-BI leads to a 50% reduction of atherosclerosis in cholesterol fed rabbits. ► The role of SR-BI in rabbits may be different from the one found in rodents.

Mapping QTL affecting a systemic sclerosis-like disorder in a cross between UCD-200 and red jungle fowl chickens

Systemic sclerosis (SSc) or scleroderma is a rare, autoimmune, multi-factorial disease characterized by early microvascular alterations, inflammation, and fibrosis. Chickens from the UCD-200 line develop a hereditary SSc-like disease, showing all the hallmarks of the human disorder, which makes this line a promising model to study genetic factors underlying the disease. A backcross was generated between UCD-200 chickens and its wild ancestor - the red jungle fowl and a genome-scan was performed to identify loci affecting early (21 days of age) and late (175 days of age) ischemic lesions of the comb. A significant difference in frequency of disease was observed between sexes in the BC population, where the homogametic males were more affected than females, and there was evidence for a protective W chromosome effect. Three suggestive disease predisposing loci were mapped to chromosomes 2, 12 and 14. Three orthologues of genes implicated in human SSc are located in the QTL region on chromosome 2, TGFRB1, EXOC2-IRF4 and COL1A2, as well as CCR8, which is more generally related to immune function. IGFBP3 is also located within the QTL on chromosome 2 and earlier studies have showed increased IGFBP3 serum levels in SSc patients. To our knowledge, this study is the first to reveal a potential genetic association between IGFBP3 and SSc. Another gene with an immunological function, SOCS1, is located in the QTL region on chromosome 14. These results illustrate the usefulness of the UCD-200 chicken as a model of human SSc and motivate further in-depth functional studies of the implicated candidate genes.

Knockout of Apolipoprotein E in rabbit promotes premature intervertebral disc degeneration: A new in vivo model for therapeutic approaches of spinal disc disorders

Intervertebral disc (IVD) degeneration that accelerates the loss of disc structural and functional integrities is recognized as one of the major factors of chronic back pain. Cardiovascular risk factors, such as deficits of apolipoproteins that elevate the levels of cholesterol and triglycerides, are considered critical for the progress of atherosclerosis; notably in the abdominal aorta and its lumbar branching arteries that supply lumbar vertebrae and IVDs. Obstruction of the lumbar arteries by atherosclerosis is presumed to promote lumbar disc degeneration and low back pain. APOE-knockout rabbits have recently been shown to generate hyperlipidemia with increased levels of cholesterol and triglycerides that mimic the symptoms of atherosclerosis in humans. Here, we analysed IVD degeneration in the lumbar spines of ten homozygous APOE-knockout and four wild-type New Zealand White rabbits of matching age to prove accelerated IVD degeneration in APOE-knockout rabbits, since APOE-knockout rabbits could be a beneficial model for therapeutic approaches of degenerative IVD disorders. Experiments were performed using T1/T2-weighted magnetic resonance imaging, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, glucose-oxidase assay, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot. APOE-knockout lumbar spines showed more advanced IVD degeneration, obstructed lumbar arteries and lower enhancement of contrast agent in IVDs. Moreover, lower concentration of glucose, lower number of viable cells and lower concentrations of aggrecan, collagen II and higher concentration of collagen I were detected in APOE-knockout IVDs (p < 0.0001). APOE-knockout in rabbits could induce structurally deteriorating premature IVD degeneration that mimics the symptoms of accelerated IVD degeneration in humans. APOE-knockout rabbits could be used as beneficial model, as they can bypass the standard surgical interventions that are commonly applied in research animals for the induction of enhanced IVD degeneration. Their parallel use in therapeutic approaches of IVD disorders and atherosclerosis could reduce the number of research animals to be used and contribute to the principles of 3Rs (Replacement, Reduction and Refinement).

Efficient therapy of ischaemic lesions with VEGF121-fibrin in an animal model of systemic sclerosis.

Background In systemic sclerosis (SSc), chronic and uncontrolled overexpression of vascular endothelial growth factor (VEGF) results in chaotic vessels, and intractable fingertip ulcers. Vice versa, VEGF is a potent mediator of angiogenesis if temporally and spatially controlled. We have addressed this therapeutic dilemma in SSc by a novel approach using a VEGF121 variant that covalently binds to fibrin and gets released on demand by cellular enzymatic activity. Using University of California at Davis (UCD)-206 chickens, we tested the hypothesis that cell-demanded release of fibrin-bound VEGF121 leads to the formation of stable blood vessels, and clinical improvement of ischaemic lesions. Methods Ninety-one early and late ischaemic comb and neck skin lesions of UCD-206 chickens were treated locally with VEGF121-fibrin, fibrin alone, or left untreated. After 1u2005week of treatment the clinical outcome was assessed. Angiogenesis was studied by immunofluorescence staining of vascular markers quantitatively analysed using TissueQuest. Results Overall, 79.3% of the lesions treated with VEGF121-fibrin showed clinical improvement, whereas 71.0% of fibrin treated controls, and 93.1% of untreated lesions deteriorated. This was accompanied by significantly increased growth of stable microvessels, upregulation of the proangiogenic VEGFR-2 and its regulator TAL-1, and increase of endogenous endothelial VEGF expression. Conclusions Our findings in the avian model of SSc suggest that cell-demanded release of VEGF121 from fibrin matrix induces controlled angiogenesis by differential regulation of VEGFR-1 and VEGFR-2 expression, shifting the balance towards the proangiogenic VEGFR-2. The study shows the potential of covalently conjugated VEGF-fibrin matrices for the therapy of ischaemic lesions such as fingertip ulcers.

AB0206 Altered Expression of Angiopoietins and Tie-2 in Ischemic Lesions of UCD-206 Chickens, an Animal Model for Systemic Sclerosis

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease affecting skin and internal organs. It is characterized by vascular damage, inflammation, and fibrosis. Microvascular injury and insufficient angiogenesis lead to tissue ischemia and clinical manifestations such as fingertip ulcers. UCD-206 chickens are the only animal model showing all hallmarks of the human disease, including microvascular damage and insufficient angiogenesis. The major inducer of angiogenesis is vascular endothelial growth factor (VEGF). Uncontrolled expression of VEGF and its receptors VEGFR-1 and VEGFR-2 has been shown in both, human and avian SSc. Another set of molecules that critically regulate angiogenesis are the endothelial cell-specific receptor tyrosine kinase 2 (Tie-2) and its ligands angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). Although abnormal plasma levels of soluble sTie-2, sAng-1, and sAng-2 have been reported in SSc patients, expression in lesional tissues has not been examined. Objectives To study the expression of Tie-2, Ang-1 and Ang-2 in lesional and non-lesional skin of UCD-206 chickens. Methods The expression of Tie-2, Ang-1 and Ang-2 was studied by indirect immunofluorescence tests on frozen tissue sections from lesional and non-lesional neck skins from 5 weeks old UCD-206 chickens. Age matched H.B15 chickens were used as healthy controls. Samples were double stained with monoclonal anti-Tie-2 antibody and the endothelial cell marker anti-von Willebrand factor (vWF), monoclonal anti-Ang-1 antibody and anti-alpha smooth muscle actin antibody, as a marker for mural cells, or with monoclonal anti-Ang-2 antibody and anti-vWF. The tissue cytometry software TissueQuest® was used for quantitative analyses. Statistical significance was calculated using the Mann-Whitney-U test. Results The number of Tie-2 expressing endothelial cells was significantly increased in lesional skin compared to non-lesional UCD-206 skin and healthy controls. Expression of Ang-2 was significantly decreased in lesional compared to non-lesional UCD-206 skin and to healthy H.B15 skin. Whereas no significant difference was seen in the numbers of Ang-1 expressing mural cells, other Ang-1 expressing cells were reduced in lesional skin compared to non-lesional and healthy controls. The altered endothelial expression of Ang-2 and Tie-2 resulted in significantly decreased Ang-2:Tie-2 ratios in lesional (median 0.9, IQR 0.3 – 1.8) compared to non-lesional UCD-206 skin (median 6.9, IQR 3.4 – 14.2, p≤0.0005) and healthy controls. (median 11.2, IQR 2.4 – 25.2, p≤0.0005). The Ang-2:Ang-1 ratio was also significantly diminished in lesional (median 0.4, IQR 0.1 – 0.5) compared to non-lesional UCD-206 skin (median 0.7, IQR 0.4 – 1.3, p≤0.01) and healthy controls. (median 1.0, IQR 0.6 – 1.6, p≤0.002). Conclusions Altered expression of Ang-1, Ang-2 and Tie-2 might contribute to the vasculopathy in SSc. Acknowledgements This work was funded by the Austrian Science Fund (FWF): P23230-B13. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2338

OP0300 Effect of VEGF121-Fibrin on the Expression of VEGF Receptors in Ischemic Lesions of UCD-206 Chickens, an Animal Model for Systemic Sclerosis

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vascular alterations, perivascular mononuclear cell infiltration, and fibrosis of skin and viscera. Microvascular injury, disturbed vascular repair and insufficient angiogenesis result in an overall reduced capillary density, and clinical manifestations such as fingertip ulcers. Expression of vascular endothelial cell growth factor (VEGF), the most potent angiogenic factor, and its receptors VEGFR-1 and VEGFR-2 is highly upregulated in SSc skin, but seems to be uncontrolled and chronic. However, a very high VEGF expression is associated with the lack of fingertip ulcers. Therefore, local administration of VEGF in a form that allows controlled release might be a promising therapeutic approach. Objectives Based on a proof of concept study in UCD-206 chickens, an animal model showing all hallmarks of the human disease, where we showed that cell demanded release of locally applied exogenous matrix-bound VEGF121-fibrin leads to the formation of stable blood vessels, and clinical improvement of ischemic lesions, we analysed the effect of this therapeutic approach on VEGFR-1 and VEGFR-2 expression. Methods Early and late ischemic comb and neck skin lesions of UCD-206 chickens were treated locally with VEGF121-fibrin or as a placebo control with fibrin alone. After 7 days the clinical outcome was assessed, and skin biopsies were taken for further analyses. To study the expression of VEGFR-1 and VEGFR-2 on endothelial cells, frozen tissue sections were double stained with the respective VEGF-receptor specific antibody and anti-van Willebrand factor (vWF) antibody by indirect immunofluorescence tests. The numbers of VEGFR-1 positive and VEGFR-2 positive endothelial cells were quantified using Tissuequest. Statistical significance was calculated using the Mann-Whitney-U test. Results Seven days after treatment with VEGF121-fibrin approximately 80% of the lesions showed clear clinical improvement, whereas 82% of placebo controls and 97% of untreated lesions had deteriorated. Angiogenesis was significantly increased after treatment with VEGF121-fibrin compared to controls. VEGF121-fibrin treatment increased the expression of the pro-angiogenic receptor VEGFR-2, and strongly reduced the endothelial expression of the modulatory or anti-angiogenic receptor VEGFR-1. Conclusions In the avian SSc model, the cell demanded release of VEGF121 from fibrin seems to result in controlled angiogenesis with formation of morphological normal blood vessels. The released VEGF differentially regulates the expression of VEGFR-1 and VEGFR-2 shifting the balance towards the pro-angiogenic VEGFR-2, thus increasing the angiogenic response. Long term effects of VEGF121-fibrin on vessel stability, the expression of endogenous VEGF, and its receptors will be investigated in a follow up study. Disclosure of Interest None Declared