Studies with the azoxymethane-rat preclinical model for assessing colon tumor development and chemoprevention

During recent years, multidisciplinary studies in epidemiology and molecular biology have contributed to our understanding of the etiology of colorectal cancer; more importantly they have enabled us to approach its prevention. An impressive body of epidemiological data suggests an inverse relationship between colorectal cancer risk and consumption of diets rich in omega (omega)-3 fatty acids (n-3 PUFAs) or the regular use of nonsteroidal antiinflammatory drugs (NSAIDs), including aspirin. The development of strategies for the chemoprevention of colorectal cancer have been facilitated by the use of relevant animal models mimicking the neoplastic processes that occur in humans, including similarities in histopathology and molecular and genetic lesions during both the early and promotion/progression stages of carcinogenesis. Studies with the azoxymethane-F344 rat model indicate that diets rich in n-3 PUFAs, NSAIDs, selective cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) inhibitors, and curcumin can reduce the incidence of colon cancer. Advances in the knowledge of the mechanisms by which chemopreventive agents act offer opportunities to use combinations of specific chemopreventive agents, having clinically beneficial aggregate activity with minimal toxicity. This approach is extremely important when a promising chemopreventive agent demonstrates apparent efficacy but may produce toxic effects at high doses. Our studies show that a combination of very low doses of piroxicam (NSAID) and difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, or very low doses of COX-2 and HMG-CoA reductase inhibitors are more effective in inhibiting colon carcinogenesis than administration of these compounds as single agents even at higher levels. The natural history of colorectal cancer, from dysplastic aberrant crypts to adenomas and adenocarcinomas, offers multiple opportunities for assessment and intervention. Of further importance is to identify whether the molecular targets that are critical in the growth and survival of the malignant colorectal cell are modulated by n-3 PUFAs, NSAIDs, or COX-2 and iNOS inhibitors.     

Lipocalin2 protects against airway inflammation and hyperresponsiveness in a murine model of allergic airway disease

Background Allergen‐induced bronchial asthma is a chronic airway disease that involves the interplay of various genes with environmental factors triggering different inflammatory pathways. Objective The aim of this study was to identify possible mediators of airway inflammation (AI) in a model of allergic AI via microarray comparisons and to analyse one of these mediators, Lipocalin2 (Lcn2), for its role in a murine model of allergic airway disease. Methods Gene microarrays were used to identify genes with at least a twofold increase in gene expression in the lungs of two separate mouse strains with high and low allergic susceptibility, respectively. Validation of mRNA data was obtained by Western blotting, followed by functional analysis of one of the identified genes, Lcn2, in mice with targeted disruption of specific gene expression. Epithelial cell cultures were undertaken to define induction requirements and possible mechanistic basis of the results observed in the Lcn2 knock‐out mice. Results Lcn2 was up‐regulated upon allergen sensitization and airway challenges in lung tissues of both mouse strains and retraced on the protein level in bronchoalveolar lavage fluids. Functional relevance was assessed in mice genetically deficient for Lcn2, which showed enhanced airway resistance and increased AI associated with decreased apoptosis of lung inflammatory cells, compared with wild‐type controls. Similarly, application of Lcn2‐blocking antibodies before airway challenges resulted in increased inflammation and reduced apoptosis. Conclusion These data indicate a protective role for Lcn2 in allergic airway disease, suggesting a pro‐apoptotic effect as the underlying mechanism. Cite this as: A. M. Dittrich, M. Krokowski, H.‐A. Meyer, D. Quarcoo, A. Avagyan, B. Ahrens, S. M. Kube, M. Witzenrath, C. Loddenkemper, J. B. Cowland and E. Hamelmann, Clinical & Experimental Allergy, 2010 (40) 1689–1700.     

NOD1 in the modulation of host–microbe interactions and inflammatory bone resorption in the periodontal disease model

Periodontitis is a chronic inflammatory condition characterized by destruction of non‐mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram‐negative microorganisms. Nucleotide‐binding oligomerization domain containing 1 (NOD1) is a member of the Nod‐like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram‐negative bacteria. This study assessed the role of NOD1 in inflammation‐mediated tissue destruction in the context of host–microbe interactions. We used mice with whole‐genome deletion of the NOD1 gene in a microbe‐induced periodontitis model using direct injections of heat‐killed Gram‐negative or Gram‐negative/Gram‐positive bacteria on the gingival tissues. In vitro experiments using primary bone‐marrow‐derived macrophages from wild‐type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram‐negative and Gram‐negative/Gram‐positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram‐negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram‐positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat‐killed Gram‐negative bacteria induced the same biological processes in wild‐type and NOD1‐deficient cells; however, expression of pro‐inflammatory mediators was increased in NOD1‐deficient cells. These results suggest a bone‐sparing role for NOD1 in this model.     

Circulating platelets as a source of the damage-associated molecular pattern HMGB1 in patients with systemic sclerosis

The link between platelet activation and vascular injury in Systemic Sclerosis (SSc) is poorly characterized. Here we report that platelet activation results in i) the translocation from the cytoplasm to the surface of HMGB1, a prototypical DAMP signal associated with tissue regeneration and ii) the release of platelet derived microparticles (PDμP) expressing HMGB1. Decreased HMGB1 content (334.6 ± 21.2 vs 587.1 ± 11.1 AUF, P < 0.001) and HMGB1 translocation to the outer leaflet of the plasma membrane (17.8 ± 3.5 vs 4.5 ± 0.5%, P < 0.001) characterize circulating platelets of SSc patients (n = 29) when compared with age-matched healthy controls (HC, n = 20). Conversely, a significantly higher fraction of PDμP in the blood of SSc patients, but not of HC, consistently expose (HMGB1 (MFI 62.8 ± 3.95 vs 4.3 ± 0.7). Platelet HMGB1 depletion is significantly associated in SSc patients with degranulation and with expression of P-selectin and of tissue factor as well as with fibrinogen binding to their plasma membrane. These findings indicate that platelets represent a source of HMGB1, an ancestral signal of necrosis, in the vasculature of SSc patients, possible contributing to persistent microvascular injury and endothelial cell activation.     

Isolated crypts form spheres prior to full intestinal differentiation when grown as xenografts: an in vivo model for the study of intestinal differentiation and crypt neogenesis, and for the abnormal crypt architecture of juvenile polyposis coli

We describe a model system in which single crypts, isolated from newborn rats, were embedded in a type I collagen gel and subcutaneously grafted to the flanks of nude mice, whereupon they underwent full intestinal morphogenesis. Small fragments of small intestine and colon were incubated with the divalent cation chelator EDTA, resulting in the release of crypts and villi. Released crypts were then suspended sparsely in type I collagen gel. Segments of gel containing a single crypt were grafted subcutaneously into a nude mouse. Grafts were harvested at weekly intervals. By 2 days, the mouth of the crypts had joined to seal the crypt and, within 1 week, the structure ballooned to form a spherical cystic structure lined by flattened epithelial cells showing no evidence of cytodifferentiation. After 2 weeks, host stromal cells had invaded the collagen and settled around this spherical crypt. At points where stromal cells appeared in contact with the crypt, the epithelium exhibited a more columnar phenotype. By 4 weeks, the 'crypt sphere' was surrounded by stroma expressing alpha-smooth muscle actin and, at this time, multiple buds appeared that gave rise to new crypts. By 5 weeks, villi had formed and cell lineages associated with the small intestine and colon were present; the original single crypt had transformed into a functional intestinal unit. Therefore, we have shown that a single crypt has the potential to grow, give rise to other crypts and dependent structures such as villi. This model has considerable potential for use in gene transfer experiments in the study of intestinal differentiation, and for the analysis of crypt neogenesis via crypt fission. Moreover, the appearances showed a close resemblance to those seen in juvenile polyposis syndrome (JPS), where the budding and fission of single crypts isolated by stromal overgrowth offers an alternative explanation for the histogenesis of JPS.     

Increased plasma concentration of surfactant protein D in chronic periodontitis independent of SFTPD genotype: potential role as a biomarker

Surfactant protein (SP) D belongs to the family of collectins, which are humoral molecules of the innate immune system. Collectins belong to pattern recognition receptors and are present in plasma and on mucosal surfaces and recognize several microbial components, the pathogen-associated molecular patterns (PAMPs). While SP-A is primarily expressed in the lung, expression of SP-D is more widely detected including different mucosal surfaces and in serum. Therefore, SP-D is considered a functional candidate in chronic periodontitis. The present study sought to investigate whether plasma concentration of SP-D is altered in chronic periodontitis and whether polymorphisms within the SFTPD gene (Met11Thr, Ala160Thr and Ser270Thr) are associated with chronic periodontitis. The study population comprised 105 patients with chronic periodontitis and 122 healthy, unrelated control individuals. SP-D Plasma concentrations were determined using enzyme-linked immunosorbent assay test. Genotyping of SFTPD polymorphisms was performed by polymerase chain reaction and restriction fragment length polymorphism analysis. Plasma concentrations were significantly increased in patients with chronic periodontitis compared with the controls. The median plasma concentrations were 81.6 ng/ml in the patients and 52.6 ng/ml in the controls (P = 0.00051). In contrast, the three SFTPD polymorphisms displayed no significant association with chronic periodontitis; thus, the increased plasma concentrations were independent on the genotype. The study showed significantly increased SP-D plasma concentrations in patients with chronic periodontitis compared with healthy controls. Thus, SP-D can potentially be used as a biomarker for chronic periodontitis. As no significant associations of SFTPD gene polymorphisms could be detected, other mechanisms influencing SP-D serum/plasma expression might exist.     

Blockade of integrin VLA-4 prevents inflammation and matrix metalloproteinase expression in a murine model of accelerated collagen-induced arthritis

DBA/1LacJ mice were immunized with type II collagen and boosted with bacterial lipopolysaccharide (LPS) 17 days later to induce accelerated arthritis. Clinical signs of inflammation were observed as early as Day 20. Matrix metalloproteinases MMP-2, -3, -9, and -13, but not MMP-12, mRNA levels were increased on Day 24. Administration of anti-VLA-4 antibody (mAb; 8 mg/kg/day for 3 days) at the time of LPS treatment strikingly inhibited arthritis-induced paw inflammation and histological scores, but not the increase in MMP expression. A higher dose of mAb (20 mg/kg/day for 4 days) inhibited pathology and normalized the levels of MMP mRNAs. In conclusion, the pathophysiology of this accelerated model of arthritis is VLA-4-dependent, and VLA-4-mediated events have a role in inflammation-induced MMP expression. Inhibition of arthritis-induced increases in MMP expression is not necessary to reduce pathology. This model is well suited for identifying agents that block integrin VLA-4 in vivo.     

Altered expression of CKs 14/20 is an early event in a rat model of multistep bladder carcinogenesis

Cytokeratins (CKs) 14 and 20 are promising markers for diagnosing urothelial lesions and for studying their prognosis and histogenesis. This work aimed to study the immunohistochemical staining patterns of CK14/20 during multistep carcinogenesis leading to papillary bladder cancer in a rat model. Thirty female Fischer 344 rats were divided into three groups: group 1 (control); group 2, which received N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) for 20 weeks plus 1 week without treatment; and group 3, which received BBN for 20 weeks plus 8 weeks without treatment. Bladder lesions were classified histologically. CK14 and CK20 immunostaining was assessed according to its distribution and intensity. In control animals, 0–25% of basal cells and umbrella cells stained positive for CK14 and CK20 respectively. On groups 2 and 3, nodular hyperplastic lesions showed normal CK20 and moderately increased CK14 staining (26–50% of cells). Dysplasia, squamous metaplasia, papilloma, papillary tumours of low malignant potential and low‐ and high‐grade papillary carcinomas showed increased CK14 and CK20 immunostaining in all epithelial layers. Altered CK14 and CK20 expression is an early event in urothelial carcinogenesis and is present in a wide spectrum of urothelial superficial neoplastic and preneoplastic lesions.     

Inflammation-induced modulation of cellular galectin-1 and -3 expression in a model of rat peritonitis

Objective and design: To investigate the effect of galectin-1 (Gal-1) and −3 (Gal-3) on leukocyte migration and analyze the expression of both galectins in inflammatory cells using a model of rat peritonitis. Material or Subjects: Sprague-Dawley rats (n = 4 per group). Treatment: Peritonitis was induced in animals through intraperitoneal injection of carrageenin (1.5 mg/kg) and rat mesenteries were analyzed at different time points (0, 4, 24 and 48h). For pharmacological treatment, rats received intravenous injection of Gal-1 or -3 (3μg/kg) followed by carrageenin. Methods: Western blotting and immunoelectron microscopy analysis. Statistical analysis was performed using ANOVA followed by Bonferroni test. Results: Pharmacological treatment with Gal-1, but not Gal-3, inhibited (~50 %) leukocyte recruitment into the peritoneal cavity at 4 h time-point. In this early phase, immunogold staining of mesenteries showed a diminished Gal-3 expression in degranulated mast cells and Gal-1 in transmigrated neutrophils (~20 % reduction compared to intravascular cells). In the later phases (24 and 48h), leukocyte turnover was associated with augmented Gal-1 expression in neutrophils and macrophages and Gal-3 in mast cells and macrophages. Conclusions: These results point to a balanced expression of cell-associated-Gal-1/Gal-3 and might impact on the development of new therapeutic strategies for inflammatory diseases. Received 2 September 2005; returned for revision 28 October 2005; returned for final revision 22 November 2005; accepted by M. Parnham 23 November 2005     

Analysis of gene expression in PTHrP−/− mammary buds supports a role for BMP signaling and MMP2 in the initiation of ductal morphogenesis

Parathyroid hormone–related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP^?/? mammary buds, and in WT and K14‐PTHrP ventral skin at E15.5. By cross‐referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds. Developmental Dynamics 238:2713–2724, 2009. © 2009 Wiley‐Liss, Inc.     

The involvement of matrix metalloproteinases in basement membrane injury in a murine model of acute allergic airway inflammation

BACKGROUND: Airway remodelling in asthma such as subepithelial fibrosis is thought to be the repair process that follows the continuing injury as of chronic airway inflammation. However, how acute allergic inflammation causes tissue injury in the epithelial basement membrane in asthmatic airways remains unclear. Matrix metalloproteinases (MMPs) capable of degrading almost all of the extracellular matrix components have been demonstrated to be involved in cell migration through the basement membrane in vivo and in vitro. OBJECTIVE: We investigated the alterations of matrix construction and the role of MMPs in matrix degradation in the subepithelium during acute allergic airway inflammation. METHODS: Airway inflammation, the ultrastructure of the subepithelium and injury of types III and IV collagen in tracheal tissues from ovalbumin (OVA)-sensitized mice after OVA inhalation with or without the administration of tissue inhibitor of metalloproteinase-2 (TIMP-2) and dexamethasone were evaluated by cell counting in bronchoalveolar lavage (BAL) fluids, electron microscopy and immunohistochemistry, respectively. RESULTS: The disruption of the lamina densa and matrix construction and the decrease of the immunoreactivity for type IV collagen in subepithelium were observed in association with the accumulation of inflammatory cells in airways 3 days after OVA inhalation. This disorganization of the matrix components in the subepithelium, as well the cellular accumulation, was abolished by the administration of TIMP-2 and dexamethasone. The immunoreactivity for type IV collagen in the subepithelium in OVA-inhaled mice returned to the level of that in saline-inhaled mice 10 days after inhalation in association with a decrease of the cell numbers in the BAL fluid. The immunoreactivity for type III collagen was changed neither 3 nor 10 days after OVA inhalation. CONCLUSION: These results suggest that epithelial basement membrane gets injured by, at least in part, MMPs as a consequence of cell transmigration through the membrane during acute allergic airway inflammation.     

脱细胞近交系微型猪肌腱修复兔跟腱缺损

背景：异体肌腱移植是目前修复肌腱缺损的理想方法,但移植后的排斥反应是其使用受到限制的主要原因。 目的：观察脱细胞处理的版纳近交系微型猪肌腱移植修复兔跟腱缺损的疗效,以及其作为异种肌腱移植支架材料的可行性。 方法：将40只日本大白兔制作双后肢跟腱缺损实验动物模型后,随机均分为2组,脱细胞猪肌腱组用脱细胞版纳近交系微型猪肌腱修复,自体肌腱组用自体肌腱修复,术后用3-0肌腱线改良HEMI-KESSLER法进行端端原位吻合。 结果与结论：①移植后2周内,脱细胞猪肌腱组与自体肌腱组白细胞计数、C-反应蛋白测量结果差异无显著性意义(P > 0.05)。②两组移植后局部反应小,伤口一期愈合,屈踝功能恢复正常。③组织学检查均未见明显淋巴细胞浸润,肌腱缝合处胶原纤维相互衔接。结果说明脱细胞版纳近交系微型猪肌腱能成功修复兔跟腱缺损,且具有组织相容性好、移植排斥反应轻的优点。     

Zebrafish as a model for long QT syndrome: the evidence and the means of manipulating zebrafish gene expression

Congenital long QT syndrome (LQT) is a group of cardiac disorders associated with the dysfunction of cardiac ion channels. It is characterized by prolongation of the QT-interval, episodes of syncope and even sudden death. Individuals may remain asymptomatic for most of their lives while others present with severe symptoms. This heterogeneity in phenotype makes diagnosis difficult with a greater emphasis on more targeted therapy. As a means of understanding the molecular mechanisms underlying LQT syndrome, evaluating the effect of modifier genes on disease severity as well as to test new therapies, the development of model systems remains an important research tool. Mice have predominantly been the animal model of choice for cardiac arrhythmia research, but there have been varying degrees of success in recapitulating the human symptoms; the mouse cardiac action potential (AP) and surface electrocardiograms exhibit major differences from those of the human heart. Against this background, the zebrafish is an emerging vertebrate disease modelling species that offers advantages in analysing LQT syndrome, not least because its cardiac AP much more closely resembles that of the human. This article highlights the use and potential of this species in LQT syndrome modelling, and as a platform for the in vivo assessment of putative disease-causing mutations in LQT genes, and of therapeutic interventions.     

Comparison of gene expression profiles in HepG2 cells exposed to arsenic,cadmium, nickel,and three model carcinogens for investigating the mechanisms of metal carcinogenesis

Carcinogenesis is an important chronic toxicity of metals and metalloids, although their mechanisms of action are still unclear. Comparison of gene expression patterns induced by carcinogenic metals, metalloids, and model carcinogens would give an insight into understanding of their carcinogenic mechanisms. In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2, after exposing to two metals (cadmium and nickel), a metalloid (arsenic), and three model carcinogenic chemicals N‐dimethylnitrosoamine (DMN), 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), and tetrachloroethylene (TCE) using DNA microarrays with 8,795 human genes. Of the genes altered by As, Cd, and Ni exposures, 31–55% were overlapped with those altered by three model carcinogenic chemical exposures in our experiments. In particular, the metals and metalloid shared certain characteristics with TPA and TCE in remarkable upregulations of the genes associated with progression of cell cycle, which might play a central role in As, Cd, and Ni carcinogenesis. This characteristic of gene expression alteration was partially counteracted by intracellular accumulation of vitamin C in As‐exposed cells, whereas the number of cell‐cycle associated genes was increased in Cd‐ and Ni‐exposed cells. In our experimental conditions, ROS might have an accelerative effect on the cell proliferation mechanisms of As, but have an inhibitory effect on those of other two heavy metals. Furthermore, based on the results of Q‐PCR, the oncogene PTTG1, which was upregulated by all carcinogenic chemical exposures in the array experiments, might be a useful biomarker for evaluation of the carcinogenesis of inorganic carcinogens. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.