Shuji Toda

Evaluation of acoustic radiation force impulse elastography for fibrosis staging of chronic liver disease: a pilot study

Background: Acoustic radiation force impulse (ARFI) is a new technology integrated into conventional B‐mode ultrasonography. ARFI is used to evaluate tissue stiffness in several organs, but this method has not been applied for liver fibrosis.

Mature adipocytes, but not preadipocytes, promote the growth of breast carcinoma cells in collagen gel matrix culture through cancer-stromal cell interactions.

Interaction between cancer and stromal cells is critical for tumour cell growth and invasion. It has recently been suggested that mature adipocytes, a specific type of stromal cell that is abundant in the breast, affect the biological behaviour of some epithelial cell types. However, the nature of the interaction between mature adipocytes and breast cancer cells remains unclear. The present study has examined the effects of mature rat adipocytes, as the main cell type in adipose tissue, on the growth, apoptosis, and cell adhesion of breast carcinoma cell lines [human oestrogen receptor (ER)‐positive cell lines MCF‐7, ZR75‐1, and T47‐D; and the ER‐negative mouse cell line MMT 060562] by analysing bromodeoxyuridine (BrdU) uptake, apoptotic indices, and immunohistochemical expression of E‐cadherin. As a reference, the effects of rat preadipocytes (immature adipocytes) on the behaviour of the cell lines were examined. The cell lines were cultured in a three‐dimensional collagen gel matrix with mature adipocytes or preadipocytes. Mature adipocytes significantly promoted BrdU uptake by all cell lines other than MMT 060562 cells. Preadipocytes decreased the uptake of T47‐D cells and had no effect on that of MCF‐7, ZR75‐1 or MMT 060562 cells. Mature adipocytes or preadipocytes did not affect the apoptotic indices of any of the cell lines. Mature adipocytes did not influence E‐cadherin expression in any of the cell lines, but preadipocytes clearly promoted E‐cadherin expression by MCF‐7 and T47‐D cells, but not by R75‐1 and MMT 060562 cells. These data suggest that mature adipocytes may be involved in the mechanisms regulating the growth of breast cancer mass through their growth‐promoting effect on ER‐positive tumour cells. Copyright

Prostate cancer cell growth is modulated by adipocyte‐cancer cell interaction

To assess whether adipocytes (mesenchymal stromal cells thought to affect the proliferation and differentiation of epithelial cells) affect the behaviour of prostate cancer cells in vitro, as prostate cancer metastasizes to the bone, which is an adipocyte‐rich environment.

Periostin, a matricellular protein, accelerates cutaneous wound repair by activating dermal fibroblasts

Abstract:  Cutaneous wound repair is a highly ordered and well‐coordinated process involving various cell lineages and many molecular effectors. Cell–matrix interactions through integrin molecules provide key signals important for wound repair. Periostin is a matricellular protein that may provide signals important during tissue development and remodelling by interacting with several integrin molecules, via the phosphatidylinositol 3‐kinase/Akt and MAP kinase pathways. In this study, we examined the role of periostin in the process of cutaneous wound repair using periostin‐deficient mice and by analysing the effects of periostin on dermal fibroblasts. We first determined the expression profile and localization of periostin in a well‐characterized wound repair model mice. Periostin was robustly deposited in the granulation tissues beneath the extended epidermal wound edges and at the dermal–epidermal junctions in wounded mice. Moreover, periostin‐deficient mice exhibited delayed in vivo wound repair, which could be improved by direct administration of exogenous periostin. In vitro analyses revealed that loss of periostin impaired proliferation and migration of dermal fibroblasts, but exogenous supplementation or enforced periostin expression enhanced their proliferation. Combined, these results demonstrate that periostin accelerates the process of cutaneous wound repair by activating fibroblasts.

Effects of fat cells on keratinocytes and fibroblasts in a reconstructed rat skin model using collagen gel matrix culture

Background Fat cells (stromal tissue cells), not only have the function of lipid metabolism, but produce various cytokines that exert an influence on other cell types through paracrine or endocrine mechanisms.

Transforming Growth Factor-β1 Induces a Mesenchyme-Like Cell Shape without Epithelial Polarization in Thyrocytes and Inhibits Thyroid Folliculogenesis in Collagen Gel Culture

Transforming growth factor-beta1 (TGFbeta1) induces a mesenchyme-like cell shape in some epithelial cell types. To clarify the role of TGFbeta1 in the morphological regulation of thyrocytes, we performed collagen gel culture of porcine thyrocytes with serum-free medium. TGFbeta1-nontreated cells organized follicles. In contrast, the cells treated with 10 ng/ml TGFbeta1 became spindle shaped, i.e. they resembled mesenchymal fibroblasts, and did not form follicles. To characterize the spindle-shaped cells, we examined the fine structures and expression of thyroglobulin (Tg) and cytoskeletal proteins using electron microscopy, immunohistochemistry, and immunoblotting. TGFbeta1-nontreated cells had microvilli at the apical side facing follicle lumen and had basal lamina at the basal side in contact with collagen gel. TGFbeta1-treated cells showed both microvilli and basal lamina at the basal side. TGFbeta1-nontreated cells expressed Tg, whereas TGFbeta1-treated cells showed no expression. TGFbeta1-nontreated cells barely expressed vimentin, but they expressed enough cytokeratin. TGFbeta1-treated cells extensively displayed vimentin along with the change in shape to become spindle-like and retained a decreased expression of cytokeratin. TSH (10 mU/ml) did not essentially influence any TGFbeta1 effects on the cells. These results indicate that TGFbeta1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular change and the loss of both epithelial polarization and a function in thyrocytes, and that it results in inhibiting thyroid folliculogenesis with or without TSH.

Reconstruction of the skin in three-dimensional collagen gel matrix culture.

SummaryThe skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that is composed of all three layers. The topmost layer, epidermis, was exposed to air by a new method. The exposure induced an extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells. Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases of the skin.

A new organotypic culture of thyroid tissue maintains three-dimensional follicles with C cells for a long term

Thyroid follicles embedded in extracellular matrix (ECM) seem to be supplied enough oxygen by a dense network of capillaries in vivo. Air exposure (AE) causes cells to increase oxygen availability in vitro. We speculated that three-dimensional (3D) environment of ECM together with AE may be applied to a thyroid tissue-organotypic culture, simply simulating such a microenvironment of follicles. To address the issue, we performed 3D collagen gel culture of minced thyroid tissues with or without AE. Most follicles in the tissues without AE died within 7 days. In culture with AE, most of the follicles with calcitonin-positive C cells were kept for over one month. Immunohistochemistry showed that thyrocytes displayed thyroglobulin, thyrotropin receptor, thyroid transcription factor-1 (TTF-1), and pendrin, which are all crucial for thyroid function. C cells expressed calcitonin gene-related peptide and TTF-1. Our study is the first demonstration that 3D collagen gel culture with AE retains 3D thyroid follicles with C cells for a long term. This suggests that ECM and oxygen supply together may be crucial for maintenance of 3D follicle structure and function. Our method will possibly open a new path to the study of thyrocyte-C cell interaction and thyroid biology.

Dietary corn oil promotes colon cancer by inhibiting mitochondria-dependent apoptosis in azoxymethane-treated rats.

How dietary corn oil is involved in colon carcinogenesis and cancer development is poorly understood. The aim of this study was to investigate whether long-term dietary corn oil promotes colon cancer by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis in azoxymethane (AOM)-treated rats. Male Sprague-Dawley rats were injected with AOM or with saline and fed on a basal diet or basal diet supplemented with 10% corn oil for 48 weeks. Colonic aberrant crypt foci (ACF) and tumors, including adenomas and carcinomas, were examined. Colonic apoptosis and cell proliferation were evaluated. Wild type (wt) p53 was analyzed using reverse transcription–polymerase chain reaction (RT-PCR) and Western blotting. In addition, Bcl-2, Bcl-xL, Bax, and Bak localized in the mitochondria were detected. Long-term dietary corn oil increased ACF in AOM-treated rats at 12 weeks and promoted colon cancer invasion at 48 weeks. Cancer invasion was not observed in the AOM-treated rats without dietary corn oil, although colon adenomas and cancers were detected. Apoptosis was decreased and cell proliferation was increased in the AOM-treated rats with dietary corn oil, compared with the AOM-treated rats with dietary basal diet. In these rats, mitochondrial wt p53 was significantly inhibited through decreased mitochondrial localization of wt p53 and increased cytosolic p53, resulting in the upregulation of Bcl-2 and Bcl-xL and the downregulation of Bak in the mitochondria. Results suggest that long-term dietary corn oil promotes AOM-induced colon cancer development partly by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis.

Immunohistochemical expression of growth factors in subacute thyroiditis and their effects on thyroid folliculogenesis and angiogenesis in collagen gel matrix culture

The inflammatory‐mechanistic basis of subacute thyroiditis remains unclear. To elucidate the roles of vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet‐derived growth factor‐BB (PDGF), transforming growth factor‐β1 (TGF‐β1) and epidermal growth factor (EGF) in the inflammatory process, their immunoexpression was examined in biopsy specimens of ten cases. At the granulomatous stage, all cases expressed VEGF, bFGF, PDGF, and TGF‐β1 in monocytes/macrophages infiltrating into follicle lumina, and in both epithelioid histiocytes and multinucleated giant cells of the granulomas. In fibroblasts and endothelial cells around the granulomas, all cases displayed VEGF, bFGF, and PDGF, but TGF‐β1 was detected only in fibroblasts in two cases. No cases expressed EGF in any of the above cell types. At the regenerative stage, all cases expressed VEGF, bFGF, and EGF in regenerating thyrocytes, whereas three and no cases displayed PDGF and TGF‐β1, respectively. Ten, seven and six cases expressed PDGF in fibroblasts, endothelial cells, and monocytes, respectively. In these cell types, all cases expressed VEGF and bFGF, whereas no cases displayed TGF‐β1 and EGF. To estimate the roles of these growth factors in thyroid tissue regeneration, their effects on thyroid folliculogenesis and angiogenesis were examined using collagen gel culture of thyrocytes and endothelial cells, respectively. Cell proliferation was also studied by bromodeoxyuridine (BrdU) uptake. EGF decreased follicle formation and TGF‐β1 drastically inhibited it, but the others had no effect. VEGF showed the greatest effect on vessel formation, although all of the others promoted it. EGF and VEGF or bFGF caused the highest BrdU uptake in thyrocytes and endothelial cells, respectively. The data suggest firstly, that at the granulomatous stage of subacute thyroiditis, growth factor‐rich monocytes/macrophages infiltrating into follicle lumina trigger the granulomatous reaction, and VEGF, bFGF, PDGF, and TGF‐β1 produced by the stromal cell types tested mediate the reaction; secondly, that at the regenerative stage, EGF serves follicle regeneration through its mitogenic effect on thyrocytes, although some cofactors with EGF are involved in folliculogenesis and the decreased expression of TGF‐β1, a fibrogenic factor, contributes to thyroid tissue repair; and thirdly, that VEGF and bFGF are more responsible for the angiogenesis at both stages than the other factors studied. Copyright