Histology and histopathology Vol.28, nº 2 (2013)
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- PublicationOpen AccessA comparative study of extracellular matrix remodeling in two murine models of emphysema(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Lopes, F.D.T.Q.S.; Toledo, A.C.; Olivo, C.R.; Prado, C.M.; Leick, E.A.; Medeiros, M.C.; Santos, A.B.G.; Garippo, A.; Martins, M.A.; Mauad, T.A single instillation of porcine pancreatic elastase (PPE) results in significant airspace enlargement on the 28th day after instillation, whereas cigarette smoke (CS) exposure requires 6 months to produce mild emphysema in rodents. Considering that there are differences in the pathogenesis of parenchymal destruction in these different experimental models, it is likely that there may be different patterns of extracellular matrix (ECM) remodeling. To evaluate ECM remodeling, C57BL/6 mice were submitted to either a nasal drop of PPE (PPE 28 Days) or exposed for 6 months to cigarette smoke (CS 6 months). Control groups received either an intranasal instillation of saline solution (Saline 28 Days) or remained without any smoke inhalation for six months (Control 6 months). We measured the mean linear intercept and the volume proportion of collagen type I, collagen type III, elastin and fibrillin. We used emission-scanning confocal microscopy to verify the fiber distribution. Both models induced increased mean linear intercept in relation to the respective controls, being larger in the elastase model in relation to the CS model. In the CS model, emphysema was associated with an increase in the volume proportion of fibrillin, whereas in the PPE model there was an increase in the parenchymal elastin content. In both models, there was an increase in collagen type III, which was higher in the CS-exposed mice. We concluded that ECM remodeling is different in the two most used experimental models of emphysema.
- PublicationOpen AccessAberrant expression and association of VEGF and Dll4/Notch pathway molecules under hypoxia in patients with lung cancer(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Yu, Shuang; Sun, Jianhua Sun; Zhang, Jingru; Xu, Xingfang; Li, Hong; Shan, Baozhong; Tian, Tian; Wang, Hongchun; Ma, Daoxin; Ji, ChunyanTumor angiogenesis plays important roles in the pathogenesis and prognosis of lung cancer. Both vascular endothelial growth factor (VEGF) and Dll4/Notch pathways are critical for angiogenesis, whereas their relationship under hypoxia in lung cancer remains unknown. Thus, in the present study, we evaluated the expression of VEGF and Dll4/Notch signaling molecules, and assessed their association with the microvessel density (CD31) and hypoxia (HIF1a) in lung cancer and normal lung tissues using immunohistochemical and Real-time RT-PCR techniques. Then, we investigated the biological function of Dll4 by transfecting Dll4 into HUVECs. In lung cancer tissues, Notch pathway molecules (HES1) and VEGF pathway molecules (VEGFR1 and VEGFR2) were significantly up-regulated, while the ratio of VEGFR1/VEGFR2 was decreased. CD31 and HIF1a were also found to be elevated in lung cancer. VEGFR1 was negatively correlated with Notch1 while positively correlated with Dll4. CD31 was positively correlated with HIF1a but negatively correlated with VEGFR1. Moreover, HIF1a was nearly positively correlated with HES1 in lung cancer tissues. After transfection, Dll4, Notch1 and VEGFR1 were up-regulated while VEGF and VEGFR2 were down-regulated in Dll4-transfected HUVECs compared with controls. Also, our findings suggest that the expression of VEGF and VEGFR2 increased gradually with the disease progression of lung cancer. In summary, VEGF and Notch signaling pathway molecules were overexpressed in lung cancer, which positively correlates with hypoxia (HIF1a) and angiogenesis (CD31). There might be a negative feedback loop between VEGF and Dll4/Notch signaling pathway in lung tumor angiogenesis.
- PublicationOpen AccessCrosstalk between endothelial cell and thrombus in chronic thromboembolic pulmonary hypertension: perspective(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Sakao, Seiichiro; Tatsumi, KoichiroIt is generally accepted that chronic thromboembolic pulmonary hypertension (CTEPH) results from pulmonary emboli originating from deep vein thrombosis. However, this consensus opinion has been challenged, and the concept that some aspects of CTEPH exacerbation might result from a small-vessel disease leading to secondary thrombosis has been suggested. In addition to the effect of recurrent thromboembolism, a number of lines of clinical evidence indicate that progressive worsening is contributed to by remodeling in the small pulmonary arteries. Histopathological studies of the microvascular changes in CTEPH have identified vascular lesions similar to those seen in idiopathic pulmonary arterial hypertension (IPAH). Especially in in vitro and ex vivo experiments, pulmonary artery endothelial cells (ECs) in pulmonary hypertensive diseases are suggested to exhibit an unusual hyperproliferative potential with decreased susceptibility to apoptosis, indicating that dysfunctional ECs may contribute to the progression of the diseases. Although the degree and mechanisms of EC dysfunction as a contributor to CTEPH are unclear, EC dysfunction may occur in small arteries. Indeed, the cells stimulated by the microenvironment created by the unresolved clot may release substances that induce EC dysfunction. The EC dysfunctions in CTEPH may lead to disorders of the anti-coagulation properties in ECs and may result in additional clots in situ. Moreover, these may lead to the progression, not only of distal thrombus, but also of proximal clotting. This article reviews the pathobiological concepts of CTEPH and explains a crosstalk between EC dysfunction and in situ thrombi which may contribute to the vascular lesions of CTEPH.
- PublicationOpen AccessRat hair follicle-constituting cells labeled by a newly-developed somatic stem cell-recognizing antibody: a possible marker of hair follicle development(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Ichikawa, Chisa; Izawa, Takeshi; Juniantito, Vetnizah; Tanaka, Miyuu; Hori, Mayuka; Tanaka, Katsuhiro; Takenaka, Shigeo; Kuwamura, Mitsuru; Yamate, Jyoji. A3 was generated as an antibody recognizing somatic stem cells in rat tissues. We investigated the distribution of A3-positive cells in developing rat hair follicles by immunolabeling. A3-positive cells began to be seen in the hair germ and peg in fetuses and neonates; the positive cells were epithelial cells above basal cells. Furthermore, A3-positive cells were seen in the outer root sheath adjacent to the bulge in mature hair follicles. Double immunofluorescence revealed that these A3- positive epithelial cells reacted to E-cadherin (for all epithelial elements) but not to CK15 (for basal cells/epithelial stem cells) or to nestin (for stem cells), indicating that A3-positive epithelial cells are suprabasal cells in the developing epidermic hair follicle. Additionally, spindle-shaped mesenchymal cells surrounding the hair peg and mature hair follicle reacted to A3; in double immunofluorescence, the A3-positive cells were located outside collagen type IV-positive glassy membrane, and reacted to vimentin (for mesenchmal cells), Thy-1 (for immature mesenchymal cells), CD34 (for stem cells) and nestin, but not to α- smooth muscle actin (for myofibroblasts); the positive cells were regarded as immature mesenchymal cells with stem cell nature in the connective tissue sheath of developing hair follicles. A3-positive epithelial and mesenchymal cells did not show proliferating activity. Collectively, it is considered that A3-positive cells seen in developing rat hair follicles may be quiescent postprogenitor cells with the potential to differentiate into either highly-differentiated epithelial or mesenchymal cells. A3 would become a useful antibody to know the kinetics of rat hair follicle-constituting cells.
- PublicationOpen AccessRole of endothelial-mesenchymal transition in idiopathic portal hypertension(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Sato, Yasunori; Nakanuma, YasuniIdiopathic portal hypertension (IPH) is a condition of non-cirrhotic portal hypertension without a known cause of liver disease. Obliterative portal venopathy is regarded as the primary lesion, which is responsible for the pre-sinusoidal block of hepatic blood flow leading to the development of IPH. The disease pathogenesis of IPH seems to be heterogeneous, and the pathogenic mechanisms of obliterative portal venopathy have not been fully understood. Owing to the limited understanding of the disease pathogenesis, the treatment of IPH is still largely supportive. Recently, endothelial dysfunction has been documented during the development of portal hypertension, and its contribution to IPH is being analyzed. Endothelial-mesenchymal transition (EndMT) is a phenomenon whereby vascular endothelial cells acquire myofibroblastic features characterized by an ability to express mesenchymal cell products that are related to tissue fibrogenesis. In addition to cardiovascular development, there is increasing evidence showing that EndMT is likely to be involved in a variety of fibrotic diseases, such as cardiac, pulmonary, and renal fibrosis. This article reviews the recent progress in studies of the pathogenic mechanisms of IPH in terms of endothelial dysfunction of portal veins. In particular, the role of EndMT in obliterative portal venopathy of IPH is highlighted and discussed.
- PublicationOpen AccessThe effect of glucagon and cyclic adenosine monophosphate on acute liver damage induced by acetaminophen(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Kelava, Tomislav; Ćavar, Ivan; Vukojevic, Katarina; Saraga-Babić, Mirna; Čulo, FilipRecent investigations suggest that glucagon might have a potentially important hepatoprotective activity. We investigated the effect of glucagon in a model of acetaminophen-induced liver injury. CBA male mice were injected intraperitoneally with a lethal (300 mg/kg) or sublethal (150 mg/kg) dose of acetaminophen. The liver injury was assessed by observing the survival of mice, by liver histology and by measuring the concentration of alanine-aminotransferase (ALT). Inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB) protein expressions were determined immunohistochemically. Hepatic levels of reduced glutathione (GSH) and cyclic adenosine monophosphate (cAMP) were also measured. Results show that glucagon, dose and time dependently, protects against acetaminophen-induced hepatotoxicity. This protection was achieved with a dose of 0.5 mg/kg of glucagon given intraperitoneally 15 min before or 1 h after acetaminophen. Treatment of animals with acetaminophen elevated ALT and nitrite/nitrate concentration in the plasma, enhanced iNOS and NF-κB expression and reduced GSH and cAMP concentration in the liver. Animals treated with glucagon had higher hepatic cAMP level, lower ALT and nitrite/nitrate concentration in plasma and lower expression of iNOS in liver cells than animals in control group, whereas there was no difference in the expression of NF-κB. Glucagon did not prevent the loss of GSH content caused by acetaminophen. Our investigation indicates that glucagon has a moderately protective effect against acetaminophen-induced liver injury, which is, at least partially, mediated through the downregulation of iNOS and through the increase in hepatic cAMP content, but it is not mediated through the modulation of NF-κB activity.
- PublicationOpen AccessPhysical forces and non linear dynamics mould fractal cell shape. Quantitative morphological parameters and cell phenotype(2013) Bizzarri, M.; Pasqualato, A.; Cucina, A.; Pasta, V.Cell shape is mainly determined by biophysical constraints, interacting according to nonlinear dynamics upon the basic units provided by the genome. In turn, the specific configuration a cell acquires plays a fundamental, permissive role in modulating gene expression and many other complex biological functions. Cell shape is tightly connected to cell activity and can be considered the most critical determinant of cell function. As a consequence, measurable parameters describing shape could be considered as ‘omics’ descriptors of the specific level of observation represented by the cell-stroma system. Such an approach promises to formalize some of the underlying basic mechanisms and, ultimately, provide a holistic understanding of the biological processes.
- PublicationOpen AccessProangiogenic hematopoietic cells of monocytic origin: roles in vascular regeneration and pathogenic processes of systemic sclerosis(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Yamaguchi, Yukie; Kuwana, MasatakaNew blood vessel formation is critical, not only for organ development and tissue regeneration, but also for various pathologic processes, such as tumor development and vasculopathy. The maintenance of the postnatal vascular system requires constant remodeling, which occurs through angiogenesis, vasculogenesis, and arteriogenesis. Vasculogenesis is mediated by the de novo differentiation of mature endothelial cells from endothelial progenitor cells (EPCs). Early studies provided evidence that bone marrow-derived CD14+ monocytes can serve as a subset of EPCs because of their expression of endothelial markers and ability to promote neovascularization in vitro and in vivo. However, the current consensus is that monocytic cells do not give rise to endothelial cells in vivo, but function as support cells, by promoting vascular formation and repair through their immediate recruitment to the site of vascular injury, secretion of proangiogenic factors, and differentiation into mural cells. These monocytes that function in a supporting role in vascular repair are now termed monocytic pro-angiogenic hematopoietic cells (PHCs). Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by excessive fibrosis and microvasculopathy, along with poor vascular formation and repair. We recently showed that in patients with SSc, circulating monocytic PHCs increase dramatically and have enhanced angiogenic potency. These effects may be induced in response to defective vascular repair machinery. Since CD14+ monocytes can also differentiate into fibroblast-like cells that produce extracellular matrix proteins, here we propose a new hypothesis that aberrant monocytic PHCs, once mobilized into circulation, may also contribute to the fibrotic process of SSc.
- PublicationOpen AccessInhibition of muscle fibrosis and improvement of muscle histopathology in dysferlin knock-out mice treated with halofuginone(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Halevy, Orna; Genin, Olga; Barzilai-Tutsch, Hila; Pima, Yaniv; Levi, Oshrat; Moshe, Itai; Pines, MarkAbsence of, or loss-of-function mutations in the dysferlin gene (dysf) result in dysferlinopathy, characterized by increased muscle inflammation, collagen deposition and deterioration in muscle function. We evaluated halofuginone efficacy in improving muscle histopathology in mice with deleted dysf transmembrane domain. Quadriceps sublumbar and longissimus muscles of 9-month-old dysf-/- mice treated with halofuginone for 4 months exhibited a reduction in centrally-nucleated myofibers, inflammatory infiltrates and collagen content. Late onset of dysferlinopathy makes it ideal for evaluating the efficacy of early treatments on late outcome. The dysf-/- mice were treated with halofuginone for 3 to 4 months starting at 1, 5 or 9 months of age, and quadricep muscle histopathology was evaluated at 12 months. Collagen content and number of centrally nucleated myofibers decreased after early halofuginone treatment, administered when myofibers with central nuclei and inflammatory infiltrates are evident, but there was almost no fibrosis. When administered at the beginning of fibrosis it resulted in a further decrease in the number of centrally-nucleated myofibers with no additional decrease in collagen levels. Cardiac fibrosis was almost completely abolished following early halofuginone treatment. Halofuginone inhibited Smad3 phosphorylation and its translocation to the nucleus and increased the activity of matrix metalloproteinases 9 and 2 responsible for resolution of pre-existing collagen. Macrophage and myofibroblast invasion into the dystrophic muscle at the site of myofibers with central nuclei was inhibited by halofuginone. These results suggest that early halofuginone treatment can prevent the late outcome of dysferlinopathy and can cause resolution of the established fibrosis when administered at later stages.
- PublicationOpen AccessThyroid hormone receptor (TR)alpha and TRbeta expression in breast cancer(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Ditsch, Nina; Toth, Bettina; Himsl, Isabelle; Lenhard, Miriam; Ochsenkühn, Robert; Friese, Klaus; Mayr, Doris; Jeschke, UdoThere is evidence that breast cancer patients suffer from thyroid disorders. However, the relation between thyroid receptor (TR) expression and breast cancer remains unknown so far. Therefore, the aim of this study was an immunohistochemical analysis of TR expression in breast cancer patients. Materials and methods: The expression of the combined antibody TRalpha1 and 2 and TRalpha1 or 2 alone as well as the expression of combined TRbeta1 and 2 and TRbeta1 or 2 alone was investigated with specific monoclonal or polyclonal antibodies in 82 patients. All patients presented with a first diagnosis of sporadic breast cancer. The ABC method was used for staining and staining intensities were analyzed using the IRS score. Results: Both TRalpha and TRbeta were expressed in the nuclei of breast cancer cells. In 24% (28/78) of the slides TRalpha1 and 2 IRS was positive. Immunopositivity for TRalpha1 was found in 55/78 slides, for TRalpha 2 in 54/79 slides (71 and 68%, respectively). The expression of TRbeta1 and 2 showed a positive detection in 33/77 (43%) of the slides, for TRbeta1 it was 43/79 (54%), for TRbeta2 60/76 (79%). Significant correlations of the expression of TRs - especially TRalpha2 - were found with further prognostic histopathological parameters such as tumor size, axillary lymph node involvement, grading and hormone receptor status. Multivariate analysis showed a trend for TRalpha2 as an independent predictor of disease-free and overall survival. Discussion: Our results revealed specific alterations in the expression of TRs - especially of TRalpha2 - in breast cancer patients, suggesting it as a marker with possible prognostic validity.
- PublicationOpen AccessPulmonary toxicity of instilled cadmium-doped silica nanoparticles during acute and subacute stages in rats(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Coccini, Teresa; Barni, Sergio; Vaccarone, Rita; Mustarelli, Piercarlo; Manzo, Luigi; Roda, ElisaPotential risk associated with new nanomaterial exposure needs to be assessed. This in vivo study investigated pulmonary effects of engineered cadmium-containing silica nanoparticles Cd/SiNPs (1 mg/rat), silica SiNPs (600 µg/rat) and CdCl 2 (400 µg/rat) 1, 7 and 30 days after intratracheal instillation. Comprehensive histopathological and immunocytochemical characterization of lung damage in terms of apoptosis, cell proliferation, inflammation, fibrosis and metabolism were obtained. After exposure to all treatments, lung parenchyma showed injury patterns characterized by collapsed alveoli, inflammation, granuloma formation, thickened alveolar septa and bronchiolar epithelium exfoliation. Type II pneumocytes, containing scarcely surfactantlamellated bodies, were also observed. Apoptotic phenomena enhanced as following, Cd/SiNPs>CdCl 2> SiNPs. In parallel with these findings, a significant increase of PCNA-immunoreactive cells was detected together with high mitotic activity. Cellular localization and distribution of IL-6, IP-10 and TGF-ß1 revealed an increased expression of these cytokines as evidence of an enhanced cellular inflammatory response. CYP450- immunoreactivity was also enhanced, at bronchiolar (e.g. Clara cells) and alveolar (e.g. macrophages) level after both Cd/SiNPs and CdCl 2. These overall effects were observed acutely and lasted until the 30th day, with Cd/SiNPs producing the most marked effects. Collagen-immunolabelling changed particularly 7 and 30 days after Cd/SiNPs, when a strong stromal fibrogenic reaction occurred. The present findings suggest that Cd/SiNPs produce significantly greater pulmonary alterations than either SiNPs or CdCl2 under the present experimental conditions.
- PublicationOpen AccessStaining of MUC1 in ovarian cancer tissues with PankoMab-GEX™ detecting the tumour-associated epitope, TA-MUC1, as compared to antibodies HMFG-1 and 115D8(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2013) Dian, Darius; Lenhard, Miriam; Mayr, Doris; Heublein, Sabine; Karsten, Uwe; Goletz, Steffen; Kuhn, Christina; Wiest, Irmi; Friese, Klaus; Weissenbacher, Tobias; Jeschke, Udoy. PankoMab-GEX™ is a novel humanized and glycooptimized antibody, which recognizes a novel specific tumour epitope of MUC1 (TA-MUC1). The aim of this study was to evaluate PankoMab-GEX™ binding to a variety of ovarian cancer specimens (n=156) and to normal ovarian tissue. In addition, PankoMab-GEX™ staining was compared to that of the well-known antiMUC1 antibodies HMFG-1 and 115D8. PankoMabGEX™ showed positive reactivity in serous (100% of cases, mean IRS 8.23), endometrioid (95% of cases, mean IRS 6.40), mucinous (58% of cases, mean IRS 4.17), and clear cell (92% of cases, mean IRS 7.58) carcinomas. In contrast to HMFG-1, healthy ovarian tissue was not recognized by PankoMab-GEX™. Staining with antibody 115D8 was increased with staging. Cytoplasmic PankoMab-GEX™ staining increased with tumour grade, but no correlation was found with staging. Univariate Kaplan-Meier analysis revealed a tendency of reduced survival of patients with high expression of TA-MUC1. The findings are encouraging with respect to a potential use of PankoMab-GEX™ as a new therapeutic antibody for the treatment of ovarian cancer patients.