A Role for Versican in the Development of Leiomyosarcoma Keire, Paul A.; Bressler, Steven L.; Lemire, Joan M. ...
Journal of biological chemistry/The Journal of biological chemistry,
12/2014, Letnik:
289, Številka:
49
Journal Article
Recenzirano
Odprti dostop
Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an ...extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.
Background: The cause of leiomyosarcoma (LMS) is unknown.
Results: Experimental modulation of versican levels in LMS cells resulted in altered cell proliferation, adhesion, migration, and tumor growth.
Conclusion: Versican regulates the growth of LMS tumors in a mouse model.
Significance: Collectively, these results suggest targeting versican in the treatment of LMS.
Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in ...a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.
•Downregulation of versican in leiomyosarcoma cells leads to elastic fiber formation.•Microarray analyses identified an increase in ECM- and elastin-related genes.•Combining cDNA and TaqMan low density arrays was useful for discovery and validation.•Results highlight a role for versican in regulating elastin fiber assembly in cancer.
A promising method to fabricate tissue-engineered blood vessels is to have cells synthesize the supportive extracellular matrix scaffold of the tissue-engineered blood vessel; however, a shortcoming ...of this method has been limited elastogenesis. Previously, we found that arterial smooth muscle cells (ASMCs) produced significant quantities of elastin when transduced with splice variant 3 of the proteoglycan versican (V3). In this study, we assessed whether elastogenesis and the structural properties of entirely cell-derived engineered vascular constructs could be improved by the incorporation of V3-transduced rat ASMCs. After 18 weeks of culture, V3 constructs had more tropoelastin, more elastin crosslinks, higher burst strengths, greater elasticity, and thicker collagen fiber bundles compared with empty-vector controls. The expression of elastin and elastin-associated proteins was increased in V3 and control ASMC monolayer cultures when ascorbic acid, which promotes collagen synthesis and inhibits elastogenesis, was removed from the medium. Engineered vascular constructs with ascorbate withdrawn for 14 weeks, after an initial 4-week exposure to ascorbate, exhibited increased elastin, desmosine content, elasticity, and burst strength compared with constructs exposed continuously to ascorbate. Our results show that V3 coupled with limited exposure to ascorbate promotes elastogenesis and improves the structural and functional properties of engineered vascular constructs.
Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, ...are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing, and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.
There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, ...chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle-specific alpha-actin-positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individual's own cells.
A Contemporary Atlas of the Mouse Diaphragm Stuelsatz, Pascal; Keire, Paul; Almuly, Ricardo ...
The journal of histochemistry and cytochemistry,
09/2012, Letnik:
60, Številka:
9
Journal Article
Recenzirano
Odprti dostop
The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell ...composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this “contemporary atlas” can be further explored in the context of diaphragm pathology and genetic disorders.
The role of the mammalian phospholipase D (PLD) in the control of key cellular responses has been recognised for a long time, but only recently have there been the reagents to properly study this ...very important enzyme in the signalling pathways, linking cell agonists with intracellular targets. With the recent cloning of PLD isoenzymes, their association with low-molecular-weight G proteins, protein kinase C and tyrosine kinases, the availability of antibodies and an understanding of the role of PLD product, phosphatidic acid (PA), in cell physiology, the field is gaining momentum. In this review, we will explore the molecular properties of mammalian PLD and its gene(s), the complexity of this enzyme regulation and the myriad physiological roles for PLD and PA and related metabolic products, with particular emphasis on a role in the activation of NADPH oxidase, or respiratory burst, leading to the generation of oxygen radicals.
Proteoglycans are extracellular matrix (ECM) macromolecules that interact with other ECM components to influence tissue organization and function. For example, the proteoglycan versican influences ...the expression and organization of elastin. Small interfering RNAs (siRNAs) were designed and tested for the ability to downregulate the expression of versican in a uterine leiomyosarcoma smooth muscle cell line (SK-LMS-1) that normally expresses abundant versican. Downregulation of versican expression resulted in increased levels of elastin expression, as shown by mRNA, protein, and immunohistochemistry. Moreover, downregulation of versican resulted in slower rates of cell proliferation and migration, increased cell adhesion, decreased hyaluronan (HA) accumulation, and changes in expression of genes associated with elastin synthesis and degradation. Addition of purified versican to siRNA-transduced SK-LMS-1 cell cultures restored cell proliferation to the level of control SK-LMS-1 cells and decreased cell adhesion in a dose-dependent manner. Versican siRNA-transduced SK-LMS-1 cells generated tumors in nude mice that had lower volumes and reduced levels of mitosis compared to control SK-LMS-1 tumors. By immunohistochemistry, the versican siRNA-transduced tumors expressed significantly less versican and HA, but more elastin than did control tumors. The V3 isoform of versican has been shown to induce elastogenesis in vitro and in vivo and, therefore, could be used to improve the limited elastogenesis that is typical of tissue-engineered blood vessels (TEBVs). Accordingly, smooth muscle cells were transduced to overexpress V3 and specific clonal lines were selected for elevated elastin synthesis. Subsequently, these lines were used to create TEBVs, which were evaluated for levels of elastin mRNA and protein, for deposition/organization of elastin and associated ECM components, and for collagen fiber thickness, burst strength, compliance, stress/strain response, and viability. V3-derived TEBVs had thicker collagen fibers, and greater elastin staining, desmosine content, and compliance than did control TEBVs. In conjunction with V3 overexpression, culture of TEBVs in the absence of ascorbate increased the expression of elastin and elastin-associated proteins. Collectively, the findings of this dissertation indicate that downregulation of versican using siRNA results in significant increases in elastogenesis that are associated with decreased tumor growth. Furthermore, the overexpression of V3 results in increased elastogenesis and improved performance of TEBVs.
There is a critical need for alternates to native vein or artery for vascular surgery. The clinical efficacy of synthetic or allogeneic/xenogeneic vessels has been limited by thrombosis, rejection, ...chronic inflammation, or poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from patients with advanced cardiovascular disease, we have constructed Tissue Engineered Blood Vessels (TEBVs) that served as arterial bypass grafts in long-term animal models. These TEBVs demonstrated mechanical properties similar to human blood vessels without relying upon synthetic or exogenous scaffolding. The TEBVs were antithrombogenic and mechanically stable through 8 months
in vivo
. Histology demonstrated complete tissue integration and vasa vasorum formation. The endothelium was confluent and von Willebrand factor positive. A smooth muscle specific α-actin positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy revealed an endothelial basement membrane, elastogenesis and a complex collagen network. These results demonstrate that a completely biological and clinically relevant TEBV can be assembled exclusively from a patient’s own cells.
PDF
