Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

O papel da proteína supressora de tumor p53 na indução de ciclooxigenase-2 em Carcinoma de Pulmão de Células Não Pequenas: aspectos moleculares e clínicos

O Carcinoma de Pulmão de Células Não Pequenas (NSCLC) é uma doença freqüentemente letal e altamente resistente à terapia oncológica convencional, como por exemplo, o tratamento quimioterápico com cisplatina e paclitaxel. A superexpressão de Ciclooxigenase-2 (COX-2) é constantemente observada em pacientes com NSCLC, estando associada ao prognóstico ruim destes pacientes. Acredita-se que a alta expressão de COX-2 produz efeitos anti-apoptóticos, porém pouco é conhecido sobre os mecanismos de regulação desta enzima. Muitos sinais capazes de ativar COX-2 também induzem a proteína supressora de tumor p53, conhecida pelo seu papel fundamental no controle da proliferação celular e apoptose. Dados recentes indicam que a proteína p53 é um importante regulador da expressão de COX-2. O objetivo desta dissertação foi avaliar os efeitos da quimioterapia na expressão da enzima COX-2 em linhagens celulares com diferente status do gene TP53, e ainda, correlacionar a expressão de COX-2 e o status mutacional de TP53, com as características clínico-patológicas de pacientes com NSCLC. Como ferramentas experimentais foram usadas técnicas de biologia celular e molecular como interferência de RNA, PCR em tempo real, análise mutacional e imuno-histoquímica. Com os resultados obtidos, observamos que as linhagens celulares de câncer de pulmão que apresentam p53 na sua forma selvagem, quando expostas ao tratamento com cisplatina, apresentaram indução da expressão de COX-2 (RNAm e proteína), em adição ao aumento da síntese de Prostaglandina E2 (PGE2). Em contrapartida, a expressão de COX-2 não foi alterada após o tratamento com cisplatina nas linhagens celulares que apresentavam mutação no gene TP53. Ao avaliar o tratamento com paclitaxel, foi observado um aumento da expressão de COX-2 nas linhagens A549 e H460 (linhagens celulares do tipo selvagem para p53), entretanto não foi observada alteração nos níveis de PGE2. Em adição, o tratamento com paclitaxel induziu um aumento da expressão de COX-2 na linhagem com deleção em TP53, ACC LC-319. Em seguida, após silenciamento de p53 na linhagem celular A549, por interferência de RNA, a cisplatina passou a não ser mais capaz de induzir o aumento da expressão de COX-2. No tratamento com paclitaxel, o silenciamento de TP53 não mudou a expressão de COX-2, indicando assim um efeito independente de p53. Dessa maneira, sugerimos que a indução de COX-2, por cisplatina, em linhagens celulares NSCLC é dependente de p53. Na análise dos pacientes NSCLC, os resultados demonstram que 54% dos pacientes apresentam expressão positiva de COX-2. Mutações em TP53 foram observadas em 57% dos pacientes, incluindo 56% de fumantes correntes e 37% de ex-fumantes. Uma associação entre a expressão de COX-2 e o status selvagem de TP53 foi observada, entre os pacientes que apresentaram expressão positiva de COX-2, 80% apresentaram TP53 selvagem. Um número maior de pacientes é necessário para aumentar o poder estatístico e confirmar as tendências observadas nesse estudo

Identification and analysis of mechanisms involved in a broad-spectrum disease resistant mutant of the winter wheat cv. Guardian

M66 an X-ray induced mutant of winter wheat (Triticum aestivum) cv. Guardian exhibits broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. tritici), yellow rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia recondita f. sp. tritici), along with partial resistance to stagnonospora nodorum blotch (caused by the necrotroph Stagonosporum nodorum) and septoria tritici blotch (caused by the hemibiotroph Mycosphaerella graminicola) compared to the parent plant ‘Guardian’. Analysis revealed that M66 exhibited no symptoms of infection following artificial inoculation with Bgt in the glasshouse after adult growth stage (GS 45). Resistance in M66 was associated with widespread leaf flecking which developed during tillering. Flecking also occurred in M66 leaves without Bgt challenge; as a result grain yields were reduced by approximately 17% compared to ‘Guardian’ in the absence of disease. At the seedling stage, M66 exhibited partial resistance. M66, along with Tht mutants (Tht 12, Tht13), also exhibit increased tolerance to environmental stresses (abiotic), such as drought and heat stress at seedling and adult growth stages, However, adult M66 exhibited increased susceptibility to the aphid Schizaphis graminum compared to ‘Guardian’. Resistance to Bgt in M66 was characterized with increased and earlier H2O2 accumulation at the site of infection which resulted in increased papilla formation in epidermal cells, compared to ‘Guardian’. Papilla formation was associated with reduced pathogen ingress and haustorium formation, indicating that the primary cause of resistance in M66 was prevention of pathogen penetration. Heat treatment at 46º C prior to challenge with Bgt also induced partial disease resistance to Blumeria graminis f. sp. tritici in ‘Guardian’ and M66 seedlings. This was characterized by a delay in primary infection, due to increased production of ROS species, such as hydrogen peroxide, ROS-scavenging enzymes and Hsp70, resulting in cross-linking of cell wall components prior to inoculation. This actively prevented the fungus from penetrating the epidermal cell wall. Proteomics analysis using 2-D gel electrophoresis identified primary and secondary disease resistance effects in M66 including detection of ROS scavenging enzymes (4, 24 hai), such as ascorbate peroxidase and a superoxidase dismutase isoform (CuZnSOD) in M66 which were absent from ‘Guardian’. Chitinase (PR protein) was also upregulated (24 hai) in M66 compared to ‘Guardian’.Monosomic and ditelosomic analysis of M66 revealed that the mutation in M66 is located on the long arm of chromosome 2B (2BL). Chromosome 2BL is known to have key genes involved in resistance to pathogens such as those causing stripe rust and powdery mildew. The TaMloB1 gene, an orthologue of the barley Mlo gene, is also located on chromosome 2BL. Sanger sequencing of part of the coding sequence revealed no deletions in the TaMloB1 gene between ‘Guardian’ and M66.

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis

Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians which results in multiorgan dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR Delta F508 (Delta F508) mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1 beta. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type but not in Delta F508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-Delta F508 macrophages than in WT macrophages. An autophagosome is a compartment that engulfs nonfunctional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and Delta F508 macrophages. However, autophagy dysfunction is more pronounced in Delta F508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment

BACKGROUND: Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU) is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.

RESULTS: 5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.

CONCLUSION: Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.

Molecular bases of classic galactosemia : searching for new therapeutic strategies

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

Myeloperoxidase as Inflammatory Marker of Periodontal Disease: Experimental Study in Rats

Rationale: Previous studies have used myeloperoxidase (MPO) as an inflammatory marker to estimate the accumulation of neutrophils in inflamed regions. Objective: The aim of this experimental study was to quantify the levels of MPO related to experimental periodontal disease in rats. Methods: Periodontal disease was induced in a group of rats using placement of a ligature around molar teeth. A group of rats without ligature placement served as a control. Measurements were made on the 3rd, 7th, 15th and 30th day from baseline. Gingival tissues were taken for quantification of MPO levels by ELISA. Results: The rats with induced periodontal disease showed statistically higher MPO levels (p 0.05) when compared to control rats. A significant increase in the levels of MPO released on days 7 and 30 was observed, with higher levels in the group with induced periodontitis. Conclusion: The levels of MPO were found to be higher in rats with induced periodontal disease, confirming the hypothesis that MPO may serve as an inflammatory marker for periodontitis.

Periodontal Disease Decreases Insulin Sensitivity and Insulin Signaling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fluoxetine Inhibits Inflammatory Response and Bone Loss in a Rat Model of Ligature-Induced Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of photodynamic therapy on the development of ligature-induced periodontitis in rats

Background: the purpose of this study was to evaluate, histologically and radiographically, the effect of photodynamic therapy on the progression of experimentally induced periodontal disease in rats.Methods: Ligatures were placed at the first mandibular molar in rats. The animals were divided into four groups: group 1 (C) received no treatment; group 2 was treated topically with methylene blue (MB; 100 mu g/ml); group 3 was treated with low-level laser therapy (LLLT); and group 4 was treated topically with methylene blue followed by LLLT (4.5 J/cm(2)) (photodynamic therapy; PDT). Rats were sacrificed 5, 15, or 30 days postoperatively. Standardized radiographs were taken to measure bone loss around the mesial root surface of the first molar. Data were analyzed statistically (analysis of variance and Tukey test; P < 0.05). A scoring system was used to evaluate the connective tissue, periodontal ligament, and alveolar bone histologically. Data were analyzed statistically (Kruskal-Wallis test; P < 0.05).Results: Radiographic examination showed that there was significantly less bone loss in Group PDT compared to Group C at 5 and 15 days postoperatively. There was no significant difference in bone loss at 30 days. At 15 days, the histologic results showed significant differences in the extent of inflammatory reaction in the gingival tissue, with a greater extent of chronic inflammatory reaction in Group LLLT.Conclusion: PDT transiently reduced the periodontal tissue destruction.

Bone regeneration in cranioplasty and clinical complications in rabbits with alloxan-induced diabetes

This research evaluated the bone repair process in surgical defects created on the parietal bones of diabetic rabbits using the guided bone regeneration technique to observe the effects of alloxan in the induction of diabetes mellitus. Twenty-four adult rabbits were divided into three study groups: control (C), diabetic (D) and diabetic associated to polytetrafluoroethylene (PTFE) membrane (D-PTFE). For diabetes induction the animals received one dose of monohydrated alloxan (90 mg/kg) by intravenous administration in the auricular or femoral vein. In group D-PTFE the membrane covered both the floor and the surface of the bone defect. In groups D and C, the bone defect was filled up with blood clot. The specimens were fixed in 10% formol and prepared for histomorphometric analysis. The results showed that the 90 mg/kg dose of monohydrate alloxan was sufficient to promote diabetes mellitus when administered in the auricular vein. Bone regeneration was slower in the diabetic group when compared with the control and diabetic-PTFE groups, but there was no significant statistical difference between the two experimental groups (D and D-PTFE). The oral and general clinical complications among the diabetics were weight loss, polyuria, polyphagia and severe chronic gingivitis.

Low caloric value of ethanol itself increases alveolar bone loss in ligature-induced periodontitis in male rats

This study aimed at morphometrically evaluating the influence of variable caloric values of ethanol consumption on alveolar bone loss in periodontitis in male rats. Thirty-six male rats were randomized into four groups of nine rats each, as follows: Test group A (low) - rats were fed an ethanol-containing liquid diet (ethanol representing 22% of total caloric value); Control group A - rats were fed a pair-fed control diet (ethanol replaced by isocaloric amounts of carbohydrate); Test group B (high) - rats were fed an ethanol-containing liquid diet (ethanol representing 36% of total caloric value); Control group B - rats were fed a pair-fed control diet for Test B. Following anesthesia, cotton ligatures were placed around the cervix of the right upper second molar. At eight weeks, the maxillary bones were removed and alveolar bone loss was analyzed by measuring the distance between the cementoenamel junction and the alveolar bone crest at buccal and palatal sites of the upper second molar. The unligated groups showed no significant differences between the bone loss values observed for the low and high caloric values of ethanol (p > 0.05). In the ligated groups, the rats receiving low caloric values of ethanol showed significantly greater bone loss compared to the isocaloric rats (p < 0.05); however, the rats receiving high caloric values of ethanol showed no significant differences compared to the controls. Analysis of the results demonstrated that, in male rats, ethanol itself affected ligature-induced bone loss when representing a low value in the total caloric value.

SOCS3 expression correlates with severity of inflammation, expression of proinflammatory cytokines, and activation of STAT3 and p38 MAPK in LPS-induced inflammation in vivo

SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway, which is relevant in inflammatory conditions. We used a model of LPS-induced periodontal disease in rats to correlate SOCS3 expression with the inflammatory status. In vitro we used a murine macrophage cell line to assess the physical interaction between SOCS3 and STAT3 by coimmunoprecipitation. 30 ug of LPS from Escherichia coli were injected in the gingival tissues on the palatal aspect of first molars of the animals 3x/week for up to 4 weeks. Control animals were injected with the vehicle (PBS). The rats were sacrificed at 7, 15, and 30 days. Inflammation and gene expression were assessed by stereometric analysis, immunohistochemistry, RT-qPCR, and western blot. LPS injections increased inflammation, paralleled by an upregulation of SOCS3, of the proinflammatory cytokines IL-1β, IL-6, and TNF-and increased phosphorylation of STAT3 and p38 MAPK. SOCS3 expression accompanied the severity of inflammation and the expression of proinflammatory cytokines, as well as the activation status of STAT3 and p38 MAPK. LPS stimulation in a macrophage cell line in vitro induced transient STAT3 activation, which was inversely correlated with a dynamic physical interaction with SOCS3, suggesting that this may be a mechanism for SOCS3 regulatory function. © 2013 João Antônio Chaves de Souza et al.

Cimetidine Reduces Alveolar Bone Loss in Induced Periodontitis in Rat Molars

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microglial cells are involved in the susceptibility of NADPH oxidase knockout mice to 6-hydroxy-dopamine-induced neurodegeneration

We explored the impact of Nox-2 in modulating inflammatory-mediated microglial responses in the 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD) model. Nox1 and Nox2 gene expression were found to increase in striatum, whereas a marked increase of Nox2 expression was observed in substantia nigra (SN) of wild-type (wt) mice after PD induction. Gp91phox-/- 6-OHDA-lesioned mice exhibited a significant reduction in the apomorphine-induced rotational behavior, when compared to wt mice. Immunolabeling assays indicated that striatal 6-OHDA injections reduced the number of dopaminergic (DA) neurons in the SN of wt mice. In gp91phox-/- 6-OHDA-lesioned mice the DA degeneration was negligible, suggesting an involvement of Nox in 6-OHDA-mediated SN degeneration. Gp91phox-/- 6-OHDA-lesioned mice treated with minocycline, a tetracycline derivative that exerts multiple anti-inflammatory effects, including microglial inhibition, exhibited increased apomorphine-induced rotational behavior and degeneration of DA neurons after 6-OHDA injections. The same treatment also increased TNF-α release and potentiated NF-κB activation in the SN of gp91phox-/--lesioned mice. Our results demonstrate for the first time that inhibition of microglial cells increases the susceptibility of gp91phox-/- 6-OHDA lesioned mice to develop PD. Blockade of microglia leads to NF-κB activation and TNF-α release into the SN of gp91phox-/- 6-OHDA lesioned mice, a likely mechanism whereby gp91phox-/- 6-OHDA lesioned mice may be more susceptible to develop PD after microglial cell inhibition. Nox2 adds an essential level of regulation to signaling pathways underlying the inflammatory response after PD induction