The Effects of Endurance Exercise and Diet on Atherosclerosis in Young and Aged ApoE-/- and Wild-Type Mice.

BACKGROUND: Atherosclerosis is the leading cause of death worldwide. The disease development is by and large driven by old age and lifestyle factors, such as diet, physical activity, and smoking. In the present study, we have investigated the effect of exercise and diet on the development of atherosclerosis in young and aged mice. OBJECTIVE: This study aimed at comparing multiple age-dependent factors that may influence atherosclerosis in a transgenic mouse model. METHODS: Young (14 weeks) and aged (49-52 weeks) C57BL/6 wild-type (WT) and atherosclerosis-prone ApoE-/- mice were subjected to physical endurance exercise on a treadmill, with or without a high-fat diet. Five weeks later, the frequencies of regulatory T cells (TREGs) in lymph nodes were assessed by flow cytometry, plasmatic cytokines (interleukin [IL]-1ß, IL-6, IL-10, IL-17, interferon-γ, tumor necrosis factor-α, and transforming growth factor [TGF]-ß1) levels were determined by Luminex assay. Lipids (cholesterol and triglycerides) and anti-heat shock protein 60 (HSP60) autoantibodies were measured by ELISA. Aortic lesion sizes were assessed by en face imaging. Microarray analysis and qPCR of skeletal muscle gene expression were also performed. RESULTS: Exercise leads to a reduction of aortic lesions in young ApoE-/- and aged WT mice independent of diet. In most groups, this reduction was followed by an increased proportion of TREGs and TGF-ß1 levels. Moreover, gene expression analysis showed that exercise seems to affect the AMPK signaling pathway. In particular, PGC-1α1 mRNA was induced in aged WT mice, whereas it was reduced in young ApoE-/- mice. In addition, GSEA analysis showed a marked reduction in the insulin signaling pathway in aged ApoE-/- mice. CONCLUSION: Practicing endurance exercise seems to be enough for reducing early aortic lesion formation, independent of diet. However, this was only true in mice with smaller aortic lesions, since mice with large, advanced, complicated atherosclerotic plaques did not show any reduction in lesion size with exercise training.

Oral Tolerization with Mycobacterial Heat Shock Protein 65 Reduces Chronic Experimental Atherosclerosis in Aged Mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the artery wall where both innate and adaptive immunity play important roles. Modulation of the immune response against the stress protein antigen, heat shock protein (HSP) 60, by administration of mycobacterial HSP65 (mbHSP65) orally and/or nasally shows promising therapeutic results in young animals in the sense of less severe experimental atherosclerosis; however, the case of aged animals with already established atherosclerosis has so far never been investigated. OBJECTIVE: To investigate if mbHSP65 immunization would further accelerate atherosclerotic progression in aged ApoE-/- mice (18 months old) with already long-established atherosclerosis and if these mice could be orally tolerized against mbHSP65. METHODS: Aged wild-type (WT) and ApoE-/- mice (65 weeks) were immunized and/or orally treated with mbHSP65 and then either kept on normal chow or changed to high-cholesterol diet (HCD). Atherosclerosis was assessed by en face analysis and the number of CD4+CD25+FoxP3+ T regulatory cells (Tregs) was assessed by flow cytometry in lymph node and spleen cells. Total cholesterol and triglyceride levels were determined. Soluble mammalian HSP60 and anti-mouse HSP60 (mHSP60) and anti-mbHSP65 antibodies were detected by enzyme-linked immunosorbent assay. RESULTS: As expected, aged WT mice had only minor lesions in the aorta, which did not change under HCD for 14 weeks. Aged ApoE-/- mice already had large complicated plaques, which increased in size under HCD. mbHSP65 immunization led to a significant aggravation of atherosclerosis in both WT and ApoE-/- mice irrespective of the nature of their diet. This increase was accompanied by increased titers of both anti-mHSP60 and anti-mbHSP65 antibodies in the circulation. The increased plaque formation could be significantly diminished with oral mbHSP65 tolerization. An increased number of Tregs and lower or unchanged levels of cholesterol and triglycerides were associated with the reduced size of aortal lesions. CONCLUSION: Oral tolerization against mbHSP65 could be used both to prevent and to treat chronic atherosclerosis in aged individuals.

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).

Searching for a good model for systemic sclerosis: the molecular profile and vascular changes occurring in UCD-200 chickens strongly resemble the early phase of human systemic sclerosis.

INTRODUCTION: Vascular injury and endothelial cell (EC) apoptosis are the earliest events in systemic sclerosis (SSc), before the onset of fibrosis, and stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGFA), endothelin-1 (ET-1) and platelet-derived growth factors (PDGF-BB) represent the key molecules to study the link between vascular injury and fibrosis during SSc. The University of California at Davis line 200 (UCD-200) chickens display the same hallmarks of human SSc: vascular occlusion, perivascular lymphocytic infiltration and fibrosis of skin and internal organs. In this study we assessed both cytokines and growth factors involved in the early phases of the UCD-200 chickens' skin lesions, to determine whether these animals might represent an appropriate experimental model to study the pathogenesis of SSc. MATERIAL AND METHODS: Immunofluorescence analysis was performed on human SSc skin, human healthy control (hHC) skin, UCD-200 combs and HC H.B15 chicken (cHC) combs, using anti-SDF-1, CXCR4, VEGFA, VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), ET-1, ET receptor A (ETAR), ET receptor B (ETBR), PDGF-BB, and PDGF receptor (PDGFR) antibodies. The plasma concentrations of SDF-1, VEGFA, ET-1 and PDGF-BB were determined by ELISA. RESULTS: All the molecules analyzed showed higher levels in SSc patients and UCD-200 chickens than in hHC and cHC. Furthermore, the levels of the assessed molecules paralleled the severity of comb involvement. CONCLUSIONS: The molecular similarities between avian and human SSc, observed in this study, suggest that the UCD-200 chickens are an interesting model for translational approaches to SSc.

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 1 week 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.

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

OBJECTIVE: The 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. METHODS: ApoE(-/-) 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. RESULTS: Decreased 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. CONCLUSION: Atheroprotective 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.

New insight into amphotericin B resistance in Aspergillus terreus.

Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.

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.

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

OBJECTIVE: Scavenger receptor-class B type I (SR-BI), the receptor for HDL-cholesterol, plays a key role in HDL metabolism, whole body cholesterol homeostasis, and reverse cholesterol transport. We investigated the in vivo impact of hepatic SR-BI inhibition on lipoprotein metabolism and the development of atherosclerosis employing RNA interference. METHODS: Small hairpin RNA plasmid specific for rabbit SR-BI was complexed with galactosylated poly-l-lysine, allowing an organ-selective, receptor-mediated gene transfer. Rabbits were fed a cholesterol-rich diet, and were injected with plasmid-complexes once a week. RESULTS: After 2 weeks of treatment hepatic SR-BI mRNA levels were reduced by 80% accompanied by reduced SR-BI protein levels and a modulation of the lipoprotein profile. Rabbits treated with SR-BI-specific plasmid-complexes displayed higher cholesteryl ester transfer from HDL to apoB-containing lipoproteins, lower HDL-cholesterol, and higher VLDL-cholesterol levels, when compared to controls. In a long-term study, this gene therapeutic intervention led to a similar modulation of the lipoprotein profile, to lower total cholesterol levels, and most importantly to a 50% reduction of the relative atherosclerotic lesion area. CONCLUSION: Our results are another indication that the role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits--a CETP-expressing animal model displaying a manlike lipoprotein profile may be different from the one found in rodents.

68Ga-siderophores for PET imaging of invasive pulmonary aspergillosis: proof of principle.

UNLABELLED: The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult and lacks specificity and sensitivity. In the pathophysiology of Aspergillus fumigatus, iron plays an essential role as a nutrient during infection. A. fumigatus uses a specific and highly efficient iron uptake mechanism based on iron-complexing ferric ion Fe(III) siderophores, which are a requirement for A. fumigatus virulence. We aimed to evaluate the potential of siderophores radiolabeled with (68)Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA. METHODS: (68)Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) was performed at high specific activity. Stability, protein binding, and log P values were determined. In vitro uptake in A. fumigatus cultures was tested under varying conditions. Biodistribution was studied in healthy noninfected BALB/c mice, and uptake was studied in a model of A. fumigatus infection using immunosuppressed Lewis rats. RESULTS: High-specific-activity (68)Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties ((68)Ga-TAFC, log P = -2.59; (68)Ga-FC, log P = -3.17) with low values of protein binding for (68)Ga-TAFC (<2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 microM) of siderophore or NaN(3), indicating specific, energy-dependent uptake. In noninfected mice, (68)Ga-TAFC showed rapid renal excretion and low blood values (1.6 +/- 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact (68)Ga-TAFC was detected. In contrast, (68)Ga-FC revealed high retention in blood (16.1 +/- 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of (68)Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n = 5) and 0.29 +/- 0.11 %ID/g in mildly infected (n = 3) and 0.95 +/- 0.37 %ID/g in severely infected (n = 4) rats. PET showed focal accumulation in infected lung tissue. CONCLUSION: Both siderophores bound (68)Ga with high affinity, and (68)Ga-TAFC, especially, showed high stability. (68)Ga-TAFC displayed highly selective accumulation by A. fumigatus subspecies in vitro and in vivo. The high and specific uptake by A. fumigatus proves the potential of (68)Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.

Secretoneurin and the tachykinins substance P and neurokinin-A/B in NMDA-induced excitotoxicity in the rat retina.

In a recent investigation using the NMDA-excitotoxicity model in the rat retina, we found that, whereas, following intravitreal injection of NMDA, a time-dependent decrease of the levels of a neuropeptide, namely vasoactive intestinal polypeptide (VIP), was fully counteracted by topical treatment with flunarizine eye drops, the levels of pituitary adenylate-cyclase activating peptide-38 (PACAP-38), another neuropeptide, remained unchanged. The aim of the present study was to find out if NMDA causes reduction in the levels of other neuropeptides such as secretoneurin (SN), neurokinin-A/B (NKA/NKB) and substance P (SP), and if so, whether flunarizine has the ability to counteract this effect or prevent such reduction. The reduction of the levels of SN and NKA/NKB 14 days after intravitreal injection of 2 µl of 100 nmol NMDA into one eye was more pronounced than after 7 days; topical flunarizine had a slight counteracting effect, but could not prevent the decrease in the levels of these peptides. Reduction in SP levels after 28 and 56 days was fully counteracted by flunarizine. By enabling a pronounced influx of Ca²+ ions into peptide-expressing cells, NMDA leads to cell death. Since each of these peptides exerts neuroprotective properties in the central nervous system, the drop in their levels caused by acute insult (e.g. NMDA excitotoxicity) or chronic insult (e.g. glaucoma) may cause a breakdown of endogenous neuroprotection in the retina given that these peptides feature neuroprotective properties in the retina as well.

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.

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 24 h after i.v. injection of (124)I-labeled monoclonal anti-HSP60 or (124)I-radiolabelled isotype control antibodies. Compared to control animals, in vivo images of rabbit aortae revealed significantly increased endothelial binding of (124)I-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 (124)I-labeled anti-HSP60 monoclonal antibodies is possible.

Regulation of growth of prostate cancer cells selected in the presence of interleukin-6 by the anti-interleukin-6 antibody CNTO 328.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional regulator of cellular events in prostate cancer. LNCaP-IL-6+ cells selected in the presence of IL-6 were taken for assessment of effects of the chimeric monoclonal anti-IL-6 antibody CNTO 328. METHODS: Cell viability was assessed after treatment with CNTO 328 by the ATP assay. Expression of Bcl-2 and Bax and activation of signaling pathways were evaluated by Western analysis. Nude mice were inoculated with LNCaP-IL-6+ cells and treated with CNTO 328. The tumors were analyzed by immunohistochemistry for expression of Ki-67, tissue transglutaminase, and vascular endothelial growth factor. RESULTS: CNTO 328 caused a statistically significant inhibition of cell viability. The protein levels of Bcl-2 and the phosphorylation of ERK1/2 mitogen-activated protein kinases were decreased by the anti-IL-6 antibody. Treatment with CNTO 328 yielded an increase in the phosphorylation of signal transducers and activators of transcription factor 3. The mean tumor volume in animals inoculated with LNCaP-IL-6+ cells and treated with CNTO 328 was insignificantly lower than that in animals treated with the control antibody. There was a statistically significant decrease in the percentage of Ki-67-positive cells in CNTO 328-treated tumors. CONCLUSION: CNTO 328 has a potential in prostate cancer therapy and could be further tested in various combination experimental treatments.

Accelerated arteriosclerosis of vein grafts in inducible NO synthase(-/-) mice is related to decreased endothelial progenitor cell repair.

Inducible NO synthase (iNOS) is expressed by macrophages and smooth muscle cells in atherosclerotic lesions. Previously, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of iNOS in the development of vein graft arteriosclerosis in iNOS(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in iNOS(-/-) mice was increased 2-fold compared with that of wild-type controls. Neointimal lesions contained mainly MAC-1+ macrophages and alpha-actin+ smooth muscle cells (SMCs) in both vein grafts of iNOS(-/-) and iNOS(+/+) mice. Immunofluorescence analysis revealed that increased iNOS expression in neointimal macrophages and SMCs of wild-type, but not iNOS(-/-), mice coincided with increased vascular endothelial growth factor (VEGF) expression in vein grafts. When vein grafts were performed in iNOS(-/-)/TIE2-LacZ transgenic mice expressing LacZ gene only in endothelial cells, the number of beta-galactosidase+ cells in iNOS(-/-) vein grafts were significantly decreased. Furthermore, treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester resulted in delayed endothelial progenitor cell attachment, whereas L-arginine intake through drinking water enhanced endothelial repair. Interestingly, local application of VEGF to iNOS(-/-) vein grafts restored endothelial progenitor homing and reduced neointimal lesions, whereas the VEGF receptor inhibitor SU1498 increased the lesion formation. Additionally, iNOS-deficient SMCs showed a low level of VEGF production in response to interleukin 1beta stimulation. Thus, iNOS deficiency accelerates neointima formation by abrogating VEGF production and endothelial progenitor cell attachment and differentiation.

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 900 nm in diameter were doubly derivatised, first with tomato lectin and then with gadolinium(III)-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 1.5 T MRI with spatial resolution 0.1 mm in the imaging plane and 0.6 mm in the z axis, persisting >60 min 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 100 nm 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.

Investigations for retinopathy in an avian model for systemic sclerosis.

Systemic Sclerosis (SSc) is a connective tissue disease affecting the skin and internal organs. Retinopathy has been described in patients with SSc, but cannot be distinguished from secondary changes due to concomitant hypertension. UCD 200 chickens, a well-established animal model for SSc, were used in this study to investigate the posterior ocular segment for manifestations of SSc. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling was applied to detect endothelial cell (EC) apoptosis, a condition previously shown to represent the first step in SSc pathogenesis in humans and to be present in the skin and the involved internal organs of UCD 200 chickens in the acute stage. Our study showed a complete absence of EC apoptosis in the pecten and choroidal vessels of UCD 200 chickens in the acute stage. Ophthalmoscopy, biomicroscopy and histology revealed normal structures of the pecten, retina and choroid in the chronic stage. In summary, we showed that there is no primary involvement of the posterior ocular segment in avian SSc. SSc of UCD 200 chickens closely mimics human SSc, presenting all the clinical, serological and histological disease manifestations seen in the human counterpart. Therefore, our data raise serious doubts about primary posterior ocular involvement in human SSc. However, fundal examinations in patients with SSc may have their justification for assessment of hypertensive retinopathy.

Phage-displayed recombinant single-chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP): cDNA cloning, characterization and CETP quantification.

Cholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), both known to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective KD values of 4.36 x 10(-9) M and 4.64 x 10(-9) M as determined by affinity sensor technology. Amino acid sequence comparison indicated the complementarity-determining regions of the respective heavy chains to be responsible for CETP high affinity binding. Fragment 1CL8 was successfully employed in clinical chemical quantification systems that uncovered an association in humans between plasma CETP concentration and total body fat mass (r=0.50, p<0.002). Because of the demonstrated superb CETP capturing capacity, combined with high binding affinity to CETP, ready access and unlimited supply, 1CL8 and 1CL10 are expected to prove powerful tools for studies on the role of CETP in atherogenesis.

In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti-endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis-inducing effect of AECAs in vivo. METHODS: The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD-200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA-positive serum samples from UCD-200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1-week-old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL. RESULTS: Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA-positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls. CONCLUSION: This study is the first to demonstrate the in vivo apoptosis-inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.

Progression of arteriovenous bypass restenosis in mice exposed to a 50 Hz magnetic field.

The controversy over whether magnetic fields (MF) produced by electrical wiring and appliances contribute to diseases such as cancer has been debated in the literature for more than 2 decades. These extremely low frequency fields at 50 or 60 Hz are omnipresent in the industrialized world and have been linked to various forms of cancer by epidemiological studies. Little has been published investigating any possible role of MF and cardiovascular disease, and this is the first study looking specifically at the effect of exposure to high-intensity MF on the development and progression of restenosis. A mouse arteriovenous bypass model was used, and mice were exposed to MF for periods of 1, 2, or 3 weeks. Neointima formation, infiltration of mononuclear cells, and heat shock protein 60 expression were all studied at the conclusion of the exposure regimen. Animals exposed to the MF for 1 week showed significantly smaller neointima formation compared with control mice exposed to a null field, although this difference was not observed in mice exposed for 2 or 3 weeks. No difference was found between mice exposed to MF and controls in any of the other parameters investigated.