Heparanase and macrophage interplay in the onset of liver fibrosis

Abstract The heparan sulfate endoglycosidase heparanase (HPSE) is involved in tumor growth, chronic inflammation and fibrosis. Since a role for HPSE in chronic liver disease has not been demonstrated to date, the current study was aimed at investigating the involvement of HPSE in the pathogenesis of chronic liver injury. Herein, we revealed that HPSE expression increased in mouse livers after carbon tetrachloride (CCl4)-mediated chronic induction of fibrosis, but with a trend to decline during progression of the disease. In mouse fibrotic liver tissues HPSE immunostaining was restricted in necro-inflammatory areas, co-localizing with F4/80 macrophage marker and TNF-α. TNF-α treatment induced HPSE expression as well as HPSE secretion in U937 macrophages. Moreover, macrophage-secreted HPSE regulated the expression of α-SMA and fibronectin in hepatic stellate LX-2 cells. Finally, HPSE activity increased in the plasma of patients with liver fibrosis but it inversely correlated with liver stiffness. Our results suggest the involvement of HPSE in early phases of reaction to liver damage and inflammatory macrophages as an important source of HPSE. HPSE seems to play a key role in the macrophage-mediated activation of hepatic stellate cells (HSCs), thus suggesting that HPSE targeting could be a new therapeutic option in the treatment of liver fibrosis

The Story of SPATA2 (Spermatogenesis-Associated Protein 2): From Sertoli Cells to Pancreatic Beta-Cells

In an attempt to isolate new spermatogenesis-associated genes, pd1 was initially identified and cloned as a novel human cDNA sequence from testis cDNA library. The novel gene was submitted to GenBank under accession n° U28164 in 1996. PD1 expression was demonstrated at the Sertoli cell level with a production which appeared to be under the influence of neighbouring spermatogenic cells. The rat orthologue of human pd1 was further cloned and, according to the Gene Nomenclature Committee, was renamed spata2 (spermatogenesis-associated protein 2) gene on the basis of its FSH-dependent up-regulation and developmental expression. The analysis of the human and rat cDNA sequences disclosed an open reading frame for a protein of 520 and 511 amino acids respectively, with an overall identity of 85%. Subsequently, a zebrafish orthologue of the human spata2 gene was identified. The consensus open reading frame (1650 bp) encodes a polypeptide of 550 amino acids, which shares 37% identity with the human spata2. By means of whole-mount in situ hybridisation it has been shown that spata2 transcripts are maternally derived and become strongly localised in the central nervous system at early developmental stages. At the same time, RT-PCR analysis demonstrated that several adult zebrafish tissues expressed high level of spata2 mRNA providing evidence that this gene may have a broader function than previously described. More recently, novel findings have highlighted a potential role of spata2 during pancreatic development and β-cell proliferation. In this review we will discuss spata2 gene expression and regulation as well as focus on novel evidence, which suggests a role for this protein in pancreatic β-cell function

Heparanase: A Multitasking Protein Involved in Extracellular Matrix (ECM) Remodeling and Intracellular Events

Heparanase (HPSE) has been defined as a multitasking protein that exhibits a peculiar enzymatic activity towards HS chains but which simultaneously performs other non-enzymatic functions. Through its enzymatic activity, HPSE catalyzes the cutting of the side chains of heparan sulfate (HS) proteoglycans, thus contributing to the remodeling of the extracellular matrix and of the basal membranes. Furthermore, thanks to this activity, HPSE also promotes the release and diffusion of various HS-linked molecules like growth factors, cytokines and enzymes. In addition to being an enzyme, HPSE has been shown to possess the ability to trigger different signaling pathways by interacting with transmembrane proteins. In normal tissue and in physiological conditions, HPSE exhibits only low levels of expression restricted only to keratinocytes, trophoblast, platelets and mast cells and leukocytes. On the contrary, in pathological conditions, such as in tumor progression and metastasis, inflammation and fibrosis, it is overexpressed. With this brief review, we intend to provide an update on the current knowledge about the different role of HPSE protein exerted by its enzymatic and non-enzymatic activity

Spondylolisthesis in an Etruscan Woman from Spina (Ferrara, Italy): an Iron Age Case Report

Spondylolisthesis consists of the slippage of a vertebra in relation to the one beneath. It is caused by separation of the neural arch from the vertebral body (spondylolysis), and predominantly occurs at the isthmus (pars interarticularis). Originally thought to be a congenital anomaly, its strict correlation with certain activities that seem to exert stress on lower spine was later demonstrated. This paper describes a case of progression of spondylolysis to spondylolisthesis found on an adult female skeleton from the Etruscan necropolis of Spina (Ferrara, Italy). The case in question was identified among 209 skeletons exhumed at Spina. As spondylolisthesis is strictly connected with activities that exert stress on lower spine, the evidence suggests that this woman was engaged in stressful physical activity, perhaps related to the specific trade function of the site

Project and construction of a cw, single-frequency and tunable Ti : Sapphire laser, actively stabilized to the resonance of an external reference cavity

Orientadores: Flavio Caldas da Cruz, Evandro ConfortiObservação: Faltam as páginas 61-62, 85-86-87 e 96Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de ComputaçãoResumo: Este trabalho apresenta o projeto e construção de um laser de Tirsafira (safira dopada com titânio) sintonizável, operando em regime contínuo, de freqüência única e estabilizada à ressonância de uma cavidade externa de referência. Descrevemos os componentes do laser: cristal de Ti:Safira, cavidade óptica em anel, diodo óptico, filtro birrefringente e etalons. Apresentamos medidas de espectro do ruído de intensidade e do ruído de freqüência com o laser de Ti:safira operando sem controle ativo da freqüência. Estas medidas foram feitas para dois lasers de bombeio: um laser iônico de Argônio e um laser de estado sólido de Vanadato de Itrio dopado com Neodímio e duplicado em freqüência. A freqüência do laser de Ti:Safíra é duplicada em um cristal não linear de Niobato de Potássio colocado em uma cavidade ressonante para maior eficiência. São discutidos os sistemas de controle do etalon grosso, de estabilização da cavidade do laser e da cavidade de duplicação, incluindo os respectivos métodos para derivação dos sinais de erro. Este laser é uma ferramenta versátil e essencial para espectroscopia atômica e molecular de alta resolução e, uma vez duplicado, foi usado para desacelerai- e aprisionar átomos de cálcio.Abstract: In this work we discuss the project and construction of a cw, single-frequency and tunable Titanium:sapphire laser, actively stabilized to the resonance of an external reference cavity. We describe the laser components - Ti:Sapphire cristal, ring cavity, optical diode, birrefringent filter and etalons- and present measurements of the spectral distribution of amplitude and frequency noise for the free-running laser. These measurements were carried out for pumping with an Ar+ laser and with a frequency doubled NdiYVCU. The Tr.sapphire frequency has been doubled in a potassium niobate crystal (KNbOj) placed inside a power enhancement cavity for greater efficiency. We discuss the control systems, used for the thick etalon and laser and doubling cavities, and also the methods used to derive the error signals. This laser is a versatile tool for high resolution atomic and molecular spectroscopy and, when frequency doubled, has been used to decelerate and trap Calcium atoms.MestradoEletrônica, Microeletrônica e OptoeletrônicaMestre em Engenharia Elétric

Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling

The small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However, it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERb-positive MDA-MB-231, cells with the respective ERb-suppressed (shERbMDA-MB-231), and lowly invasive ERa-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell–cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of a1, a2, a3, aVb3, and aVb5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of a2 and b1 integrin subunits both in MDA-MB-231 and in shERbMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for integrins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer

Heparanase activity in alveolar and embryonal rhabdomyosarcoma: implications for tumor invasion

<p>Abstract</p> <p>Background</p> <p>Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-β-D-glucuronidase that cleaves heparan sulphate proteoglycans.</p> <p>Methods</p> <p>We determined HPSE expression by Western blot analysis in ARMS and ERMS cells lines and activity in supernatants by an ELISA assay. Stable <it>HPSE </it>silencing has been performed by shRNA technique in RH30 and RD cell lines and their invasiveness has been evaluated by Matrigel-invasion assay. HPSE activity and mRNA expression have also been quantified in plasma and biopsies from RMS patients.</p> <p>Results</p> <p>HPSE expression and activity have been detected in all RMS cell lines. Stable <it>HPSE </it>silencing by shRNA technique determined a significant knockdown of gene expression equal to 76% and 58% in RH30 and RD cell lines respectively and induced a less invasive behaviour compared to untreated cells. Finally, we observed that <it>HPSE </it>mRNA expression in biopsies was higher than in foetal skeletal muscle and that plasma from RMS patients displayed significantly more elevated HPSE levels than healthy subjects with a trend to higher levels in ARMS.</p> <p>Conclusion</p> <p>In conclusion, our data demonstrate for the first time HPSE expression and activity in RMS and highlight its involvement in tumor cell invasion as revealed by shRNA silencing. Moreover, HPSE expression in RMS patients is significantly higher with respect to healthy subjects. Further studies are warranted to assess possible relationships between HPSE and clinical behaviour in RMS.</p

Collagen Fiber Array of Peritumoral Stroma Influences Epithelial-to-Mesenchymal Transition and Invasive Potential of Mammary Cancer Cells

: Interactions of cancer cells with matrix macromolecules of the surrounding tumor stroma are critical to mediate invasion and metastasis. In this study, we reproduced the collagen mechanical barriers in vitro (i.e., basement membrane, lamina propria under basement membrane, and deeper bundled collagen fibers with different array). These were used in 3D cell cultures to define their effects on morphology and behavior of breast cancer cells with different metastatic potential (MCF-7 and MDA-MB-231) using scanning electron microscope (SEM). We demonstrated that breast cancer cells cultured in 2D and 3D cultures on different collagen substrates show different morphologies: i) a globular/spherical shape, ii) a flattened polygonal shape, and iii) elongated/fusiform and spindle-like shapes. The distribution of different cell shapes changed with the distinct collagen fiber/fibril physical array and size. Dense collagen fibers, parallel to the culture plane, do not allow the invasion of MCF-7 and MDA-MB-231 cells, which, however, show increases of microvilli and microvesicles, respectively. These novel data highlight the regulatory role of different fibrillar collagen arrays in modifying breast cancer cell shape, inducing epithelial-to-mesenchymal transition, changing matrix composition and modulating the production of extracellular vesicles. Further investigation utilizing this in vitro model will help to demonstrate the biological roles of matrix macromolecules in cancer cell invasion in vivo