Steroids and receptors in canine mammary cancer

The aims of this study were to investigate the serum and tissue content of androgens and estrogens in canine inflammatory mammary carcinomas (IMC) as well as in non-inflammatory malignant mammary tumors (MMT), and assessed the immunoexpression of estrogen and androgen receptors using immunohistochemistry. Profiles for the androgens dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T), and for the estrogens 17beta estradiol (E2) and estrone-sulphate (SO4E1) were measured both in tissue homogenates and in serum of MMT and IMC by EIA techniques in 42 non-inflammatory malignant mammary tumors (MMT) and in 14 inflammatory mammary carcinomas (IMC), prospectively collected from 56 female dogs. Androgen receptor (AR) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression was studied using immunohistochemistry (strepavidin-biotin-peroxidase method) in samples of 32 MMT and 14 IMC, and counted by a computer image analyzer. IMC serum and tissue levels of androgens were significantly higher than MMT levels. Tissue content of estrogens was also significantly higher in IMC than in MMT. Serum values of SO4E1 were significantly higher in IMC, but serum levels of E2 were significantly lower in IMC compared to MMT cases. Medium-high androgen receptor intensity was observed in 64.28% of IMC and 40.62% of MMT. No important differences were found between ERalpha expression in IMC (100% negative) and MMT (90% negative). ERbeta and AR were intensely expressed in highly malignant inflammatory mammary carcinoma cells. To our knowledge, this is the first report relative to AR immunohistochemistry in canine mammary cancer and to estrogens or androgens in serum of dogs with benign or malignant mammary tumors.     

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

Expression of Connexin 43 in normal canine testes and canine testicular tumors

In human testis, gap junctions containing connexin(Cx)43 are located within the seminiferous epithelium between Sertoli cells and between Sertoli and germ cells. Cx43 is known to play a role in the differentiation and proliferation of these cell types. It can further be associated with human seminoma development. The dog has been proposed as a model for studies of the male reproductive system, because of the frequent occurrence of testicular neoplasms. Thus, we investigated Cx43-mRNA and -protein expression in testes of normal prepubertal dogs, adult dogs, and in canine testicular tumors. Sertoli cells in prepubertal cords express Cx43 mRNA, but do synthesize only less Cx43 protein. Within the seminiferous tubules, Cx43 mRNA was detected in Sertoli cells, spermatogonia, and spermatocytes. Cx43 protein was mainly present in the basal compartment. In canine testicular tumors Cx43 mRNA was detectable in both seminoma and neoplastic Sertoli cells, whereas Cx43 protein was only found in neoplastic Sertoli cells. Our data indicate that Cx43 is regulated differentially in testicular tumors and that alterations of Cx43 expression may be involved in the pathogenesis of canine testicular malignancies. This study represents the first morphological work on the spatiotemporal expression pattern of Cx43 in normal and neoplastic canine testis.     

Enhanced assay of endothelial exocytosis using extracellular matrix components

Vascular inflammation plays a key role in the pathogenesis of atherosclerosis. The first step in vascular inflammation is endothelial exocytosis, in which endothelial granules fuse with the plasma membrane, releasing prothrombotic and proinflammatory messenger molecules. The development of cell culture models to study endothelial exocytosis has been challenging because the factors that modulate exocytosis in vitro are not well understood. Here we report a method for studying endothelial exocytosis that optimizes extracellular matrix components, cell density, and duration of culture. Human umbilical vein endothelial cells plated on collagen I-coated plates and cultured in the confluent state for 7–12 days in low-serum medium showed robust secretion of von Willebrand factor when stimulated with various agonists. This exocytosis assay is rapid and applicable to high-throughput screening.     

Changes in the cellular phenotype and extracellular matrix during progression of estrogen-induced mesenchymal kidney tumors in Syrian hamsters

The cellular origin of estrogen-induced kidney tumors in male Syrian hamsters has been repeatedly the subject of controversy. Several authors have proposed that the tumors arise from proximal tubules, from a combination of tubular and interstitial stromal cells, or solely from interstitial cells. Because of the model character of this tumor for hormone-associated cancer, it was further investigated in this study with respect to morphology, enzyme and intermediate filament pattern, the expression of α-smooth muscle actin and the extracellular matrix proteins fibronectin and tenascin. These analyses were carried out with early and late tumors as well as metastases to determine possible changes in expression of biochemical parameters during the development and progression of this neoplasm. The enzyme histochemical and intermediate filament patterns were usually the same as those described previously for proliferative foci and early tumors, i.e. highly elevated activities of glucose-6-phosphate dehydrogenase, adenylate cyclase and alkaline phosphatase, a lack of glucose-6-phosphatase and γ-glutamyltransferase and coexpression of vimentin and desmin. α-smooth muscle actin could not be detected in early lesions. In five of 24 advanced tumors inclusions of kidney tubules were found which showed various degrees of alteration in their morphology and enzyme histochemical pattern, but were often directly connected with tubular segments of normal appearance outside the tumor. Like the normal tubules, the enclosed tubular segments were strongly positive for cytokeratin but never expressed vimentin or desmin. Among the 24 tumors studied, two contained cysts which expressed cytokeratin and sometimes also vimentin but not desmin. The enzyme histochemistry of the cells lining the cysts was similar to that of the surrounding tumor mass, except adenylate cyclase was lacking and alkaline phosphatase was not uniformly distributed. In tumors containing cytokeratin-positive cysts, there often were cytokeratin-positive, vimentin-negative and desmin-negative tumor formations in close contact to these cysts. With the exception of cyst formation, the pattern of metastases were identical to that of the primary tumors. All large tumors and the main component of the metastases expressed vimentin, desmin and fibronectin. Mesothelia surrounding metastatic tumor complexes were positive for vimentin, desmin, α-smooth muscle actin, fibronectin, cytokeratin and tenascin. It was concluded from these and previous observations on early stages of tumor development that the estrogen-induced hamster kidney tumor originates from mesenchymal interstitial cells (probably pericytes) which may rarely acquire an epithelial phenotype by metaplastic transformation during tumor progression.     

Accelerated extracellular matrix turnover during exacerbations of COPD

Background

Exacerbations of chronic obstructive pulmonary disease (COPD) contribute significantly to disease progression. However, the effect on tissue structure and turnover is not well described. There is an urgent clinical need for biomarkers of disease activity associated with disease progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD.

Methods

69 patients with COPD hospitalised for an exacerbation were recruited at admission and returned for a 4 weeks follow-up. Competitive ELISAs measuring circulating protein fragments in serum or plasma assessed the formation and degradation of collagen types III (Pro-C3 and C3M, respectively), IV (P4NP 7S and C4M, respectively), and VI (Pro-C6 and C6M, respectively), and degradation of elastin (ELM7 and EL-NE) and versican (VCANM).

Results

Circulating levels of C3M, C4M, C6M, ELM7, and EL-NE were elevated during an exacerbation of COPD as compared to follow-up (all P <0.0001), while VCANM levels were decreased (P <0.0001). Pro-C6 levels were decreased and P4NP 7S levels were elevated during exacerbation (P <0.0001). Pro-C3 levels were unchanged. At time of exacerbation, degradation/formation ratios were increased for collagen types III and VI and decreased for collagen type IV.

Conclusions

Exacerbations of COPD resulted in elevated levels of circulating fragments of structural proteins, which may serve as markers of disease activity. This suggests that patients with COPD have accelerated ECM turnover during exacerbations which may be related to disease progression.     

Cartilage link protein 1 (Crtl1), an extracellular matrix component playing an important role in heart development

To expand our insight into cardiac development, a comparative DNA microarray analysis was performed using tissues from the atrioventricular junction (AVJ) and ventricular chambers of mouse hearts at embryonic day (ED) 10.5-11.0. This comparison revealed differential expression of approximately 200 genes, including cartilage link protein 1 (Crtl1). Crtl1 stabilizes the interaction between hyaluronan (HA) and versican, two extracellular matrix components essential for cardiac development. Immunohistochemical studies showed that, initially, Crtl1, versican, and HA are co-expressed in the endocardial lining of the heart, and in the endocardially derived mesenchyme of the AVJ and outflow tract (OFT). At later stages, this co-expression becomes restricted to discrete populations of endocardially derived mesenchyme. Histological analysis of the Crtl1-deficient mouse revealed a spectrum of cardiac malformations, including AV septal and myocardial defects, while expression studies showed a significant reduction in versican levels. Subsequent analysis of the hdf mouse, which carries an insertional mutation in the versican gene (CSPG2), demonstrated that haploinsufficient versican mice display septal defects resembling those seen in Crtl1(-/-) embryos, suggesting that reduced versican expression may contribute to a subset of the cardiac abnormalities observed in the Crtl1(-/-) mouse. Combined, these findings establish an important role for Crtl1 in heart development.     

Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells

Surface-associated protein expression by Streptococcus uberis was influenced by the presence of collagen, laminin and bovine mammary epithelial cells in the culture medium. After electrophoresis and silver staining, four proteins stained more intensively in samples from S. uberis cultivated with epithelial cells and extracellular matrix components than in samples from S. uberis cultivated alone. Induction of these proteins was more obvious after multiple bacterial passages. The correlation between the phenotype of S. uberis and its potential virulence status as illustrated by an immunoblotting study with sera obtained from infected cows revealed that these proteins are probably expressed in vivo during infection.     

Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells

Two strains of Streptococcus uberis (UT 888 and UT 366) isolated from cows with clinical mastitis were co-cultured with bovine mammary epithelial cells (MAC-T) with and without laminin, fibrinogen, fibronectin or collagen. Incubation of S. uberis with extracellular matrix proteins (ECMPs) increased adherence to and internalization into MAC-T cells. Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP. However, adherence was always higher when strains were co-cultured with ECMP than in medium alone. S. uberis UT 888 adhered better to MAC-T cells than S. uberis UT 366. The influence of ECMPs on bacterial internalization into MAC-T cells was similar to adherence, however, differences among ECMPs were less noticeable. S. uberis UT 888 had a higher internalization index than S. uberis UT 366. It is possible that ECMPs induce or up-regulate proteins that selectively adhere to ECMPs which could serve as a bridge between the eukaryotic cell and the bacterial pathogen that leads to internalization of the ECMP-bound pathogen into the mammary epithelial cell.     

Immunohistochemical evaluation of versican,in relation to chondroitin sulphate,in canine mammary tumours

The expression of increased amounts of versican, a chondroitin sulphate proteoglycan, in neoplastic tissues may play a role in promoting tumour cell proliferation and migration. This study investigated the immunolocalization of versican in normal and neoplastic canine mammary tissues, using antibodies 12C5 and 2B1, against different epitopes of the protein core of versican. Antibody CS56, recognising chondroitin sulphate (CS), was used to investigate the relation between versican and CS, which accumulates in canine mammary tumours. We found enhanced versican expression in both benign and malignant tumours, appearing in three main patterns: in periductal tissues, probably in association with basement membranes of ducts; in peripheral invasive areas of malignant tumours; and in spindle cell proliferations and myxoid areas of complex and mixed tumours. The 12C5 and 2B1 immunoreactivities co-localised in all types of tumours, and could be improved by chondroitinase digestion. The only exception was the abundant extracellular matrix (ECM) of spindle cell proliferations, particularly in myxoid areas of complex and mixed tumours, which displayed intense and diffuse 12C5 immunoreactivity and patchy or absent 2B1 and CS56 immunoreactivities; versican immunoreactivity could not be enhanced by chondroitinase digestion. The results indicate that versican is one of the extracellular matrix components characteristic of canine mammary tumours. It appears likely that in complex and mixed tumours versican exists in at least two forms, one of them lacking the CS attachment domain and the 2B1 epitope. Furthermore, the enhanced versican expression in the invasive areas of malignant tumours indicates the involvement of this proteoglycan in tumour cell invasion.     

Extracellular matrix alterations in brains lacking four of its components

The organization of the brain extracellular matrix appears to be based on aggregates of hyaluronan and proteoglycans, connected by oligomeric glycoproteins. Mild phenotypical consequences were reported from several mouse strains lacking components of this matrix such as neurocan, brevican, tenascin-R, and tenascin-C. To further challenge the flexibility of the extracellular matrix network of the brain, mice lacking all four brain extracellular matrix molecules were generated, which were found to be viable and fertile. Analysis of the brains of 1-month-old quadruple KO mice revealed increased protein levels of fibulin-1 and fibulin-2. Histochemical analysis showed an unusual parenchymal deposition of these fibulins. The quadruple KO mice also displayed obvious changes in the pattern of deposition of hyaluronan. Further, an almost quadruple knockout like extracellular environment was noticed in the brains of triple knockout mice lacking both tenascins and brevican, since these brains had strongly reduced levels of neurocan.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

Summary A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of ³H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of ³H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.     

Monocyte-to-macrophage differentiation: synthesis and secretion of a complex extracellular matrix

Although monocyte- and macrophage-derived molecules are known to promote extracellular matrix (ECM) disruption and destabilization, it is less appreciated that they also synthesize molecules contributing to ECM formation, stabilization, and function. We have identified and characterized the synthesis of proteoglycans and related proteins, some not previously known to be associated with macrophages. Proteoglycan extracts of [(35)S]sulfate- and (35)S-trans amino acid-radiolabeled culture media from THP-1 monocytes induced to differentiate by treatment with phorbol myristate acetate revealed three major proteins of ~25, 90, and 100 kDa following chondroitin ABC lyase digestion. The 25-kDa protein was predominant for monocytes, whereas the 90- and 100-kDa proteins were predominant for macrophages. Tandem mass spectrometry identified (i) the 25-kDa core protein as serglycin, (ii) the 90-kDa core protein as inter-α-inhibitor heavy chain 2 (IαIHC2), and (iii) the 100-kDa core as amyloid precursor-like protein 2 (APLP2). Differentiation was also associated with (i) a >500-fold increase in mRNA for TNF-stimulated gene-6, an essential cofactor for heavy chain-mediated matrix stabilization; (ii) a >800-fold increase in mRNA for HAS2, which is responsible for hyaluronan synthesis; and (iii) a 3-fold increase in mRNA for versican, which interacts with hyaluronan. Biochemical evidence is also presented for an IαIHC2-APLP2 complex, and immunohistochemical staining of human atherosclerotic lesions demonstrates similar staining patterns for APLP2 and IαIHC2 with macrophages, whereas serglycin localizes to the underlying glycosaminoglycan-rich region. These findings indicate that macrophages synthesize many of the molecules participating in ECM formation and function, suggesting a novel role for these molecules in the differentiation of macrophages in the development of atherosclerosis.     

Localization of chondroitin sulfate proteoglycan versican in adult brain with special reference to large projection neurons

Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versican has two glycosaminoglycan attachment regions, named the GAGα and GAGβ domains, which are both regulated by alternative splicing and yield four protein isoforms. We have investigated the expression and localization of versican in the developing and adult brain by using anti-versican GAGα and GAGβ antibodies. Western analysis revealed that GAGα-reactive isoform was dominant in the adult brain. Immunohistochemical study demonstrated that GAGα immunoreactivity was detectable from neonatal periods to adulthood, whereas GAGβ immunoreactivity completely disappeared within 3 weeks of birth. In the adult brain, GAGα immunoreactivity was seen in the white matter regions and was also localized in the gray matter including somata and dendrites of cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells. In contrast, GAGα immunoreactivity was not localized on parvalbumin-positive interneurons and cerebellar stellate cells. Furthermore, GAGα immunoreactivity was not co-localized with perineuronal net markers such as Wisteria floribunda agglutinin lectin and phosphacan. Thus, versican was localized on large projection neurons rather than small interneurons. To confirm the binding mechanism of versican to neurons, hyaluronan and chondroitin sulfates were enzymatically removed from brain sections before the immunolabeling of versican. These treatments had no effect on the labeling pattern of versican, suggesting that other versican-interactive molecules are involved in the binding of versican to neurons. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Advanced Brain Science Project” from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Steroid pathway and oestrone sulphate production in canine inflammatory mammary carcinoma

Spontaneous canine mammary inflammatory carcinoma (IMC) shares epidemiologic, histopathologic and clinical characteristics with the inflammatory breast carcinoma (IBC) disease in humans. We have analysed the steroids levels in serum and in tissue homogenates of IMC, the expression of two of their receptors (androgen and β-estrogen) and of three enzymes included in the steroidogenesis pathway (aromatase (CYP19A1), steroid sulphatase (STS) and estrogen sulfotransferase (EST)) trying to explain the specific accumulation of steroids in IMC tissues generating deposits in the form of lipid droplets whose presence can be attributed to steroids secreted by IMC cells. According to our working hypothesis, oestrone sulphate would be the main component of these lipid droplets. The presence of these steroid deposits would contribute to the intense proliferation and invasive behaviour of IMC and IBC, although their involvement in angiogenesis is yet to be demonstrated.     

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

Summary Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.