Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice.

Tenascin-C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune-mediated hepatitis in wild-type (WT) and TNC-null (TNKO) mice. Eight-week-old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd-6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon-gamma, tumour necrosis factor-alpha, and interleukin-4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)-beta1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF-beta expression during progression of hepatitis to fibrosis.

Expression of tenascin in bile duct cancer of hamster liver by combined treatment of dimethylnitrosamine with Opisthorchis viverrini infections.

Tenascin is an extracellular matrix glycoprotein known to be an essential factor for the modulation of reciprocal interactions between the epithelium and mesenchyme during embryogenesis and tumourigenesis. The interactions between the expression of tenascin in the liver of Syrian golden hamster and the development of bile duct cancer in an Opisthorchis viverrini-associated cholangiocarcinoma model were investigated. The tenascin was expressed in connective tissues surrounding the dilated ducts, ductal rims and the stroma of cancers, and strongly in the stroma flame of necrotic cancer nodules. The mRNA signal for tenascin was also recognized in the stroma cells. The potential roles of tenascin as prognostic tumour markers are discussed.

The expression of tenascin mRNA in human temporomandibular joint specimens.

The expression of mRNA of tenascin in the temporomandibular joint (TMJ) disc and synovial membrane was examined in 20 human TMJ samples from patients with internal derangement of the TMJ and 10 control specimens by in situ hybridization technique using paraffine-embedded tissue, and antisense and sense cRNA probes. In control specimens, tenascin mRNA was not expressed. However, we were able to find tenascin mRNA expression in the surgical specimens. In 15 of 20 samples, ranging numbers of synovial cells expressed tenascin mRNA in the hypertrophic synovial membranes. Also, in 6 of 20 samples, tenascin mRNA was identified in fibroblasts. In four specimens, vascular endothelial cells were positive for the mRNA. In internal derangement cases, histopathological findings are often found such as synovitis, new capillary growth and fibrosis. The present study demonstrates that tenascin is produced specifically in synovial cells, vascular endothelial cells and fibroblasts affected in the portion of TMJ with internal derangement.

Serial extracellular matrix changes in neointimal lesions of human coronary artery after percutaneous transluminal coronary angioplasty: clinical significance of early tenascin-C expression.

It has become clear that deposition of extracellular matrix(ECM) proteins is a major cause of human restenosis after percutaneous coronary angioplasty (PTCA). To define the composition and organization of the involved ECM in human restenotic tissue, we morphologically and semiquantitatively analyzed specimens obtained by means of directional coronary atherectomy at various stages after PTCA with anti-fibronectin, tenascin-C, collagens I and III, and PG-M/versican antibodies. Tenascin-C deposition transiently increased within 1 month after PTCA, when smooth muscle cell migration and proliferation was active. Following the disappearance of tenascin-C, PG-M/versican accumulation increased and peaked between 1 month and 3 months when clinical restenosis was most actively progressing. At later stages, the PG-M/versican was replaced by a more mature ECM consisting of collagens I and III. The volume ratio of elastin remained at a low level throughout. Our results demonstrate that the matrix proteins of human restenotic lesions sequentially change after angioplasty and that tenascin-C could be a key molecule in the early stages.

[Complete remission of brain metastases from prostate cancer by gamma knife radiosurgery: a case report].

A 59-year-old male visited us with a chief complaint of dysuria. The serum prostate specific antigen (PSA) level was within normal limits, and intravenous pyelography and urethrocystography showed no abnormal findings. Because of his urinary retention, transurethral resection of prostate was performed under a clinical diagnosis of benign prostatic hyperplasia. The pathological diagnosis was poorly differentiated adenocarcinoma of the prostate. Not only combination hormone therapy with goserelin acetate and flutamide, but also intermittent arterial infusion chemotherapy with cisplatin (CDDP) and pirarubicin (THP) using a reservoir system was administered. Additionally total pelvic irradiation was delivered. Magnetic resonance imaging (MRI) demonstrated that his prostate was reduced to less than 50% in size and he had no difficulty in voiding. He suddenly developed dysarthria and hemiplegia 3 months later. MRI and computed tomography (CT) revealed multiple brain metastases. After the gamma knife radiosurgery, neurological findings disappeared and MRI showed dramatic shrinkage of metastatic brain tumors. Metastasis to the pancreas was recognized on CT and he died of multiple organ failure 30 months after his first visit.

Tenascin-C modulates adhesion of cardiomyocytes to extracellular matrix during tissue remodeling after myocardial infarction.

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for alpha-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization.

Halogen-free process for the conversion of carbon dioxide to urethanes by homogeneous catalysis.

Carbon dioxide is converted to urethanes (carbamic acid derivatives) through reaction with amine and alcohol catalyzed by tin complexes; th addition of acetals as a dehydrating agent under high CO2 pressure is the key to achieve high yields.

Expression of tenascin-C in stromal cells of the murine uterus during early pregnancy: induction by interleukin-1 alpha, prostaglandin E(2), and prostaglandin F(2 alpha).

Tenascin-C (TN-C), an extracellular matrix glycoprotein, is known to be expressed in uterine stroma in the peri-implantation period. Examination of the spatiotemporal pattern during early pregnancy using immunohistochemistry and in situ hybridization revealed TN-C expression in the stroma beneath the luminal epithelia of the murine endometrium on Days 0 and 1 of pregnancy, subsequent disappearance, and reappearance on Day 4. After decidualization, tissue around the deciduoma was positive. In situ hybridization demonstrated TN-C production by the stromal cells adjacent to the epithelia. To investigate the regulation of TN-C expression in vitro, murine uterine stromal and epithelial cells were isolated and cultured. Addition of interleukin-1 alpha (IL-1 alpha) and prostaglandin E(2) (PGE(2)) and F(2 alpha) (PGF(2 alpha)) induced TN-C expression in the stromal cells at both protein and mRNA levels, while the sex steroid hormones, progesterone and ss-estradiol, exerted little effect. Immunohistochemistry using anti-IL-1 alpha antibody showed epithelial cells to be positive on Days 2-4 of pregnancy, and addition of progesterone but not ss-estradiol enhanced IL-1 alpha expression in epithelial cells in vitro. In a culture insert system, TN-C expression by stromal cells cocultured with epithelial cells was induced by addition of progesterone alone that was blocked by additions of anti-IL-1 alpha antibody. Collectively, these findings indicate that TN-C expression in the preimplantation period is under the control of progesterone, but not directly, possibly by the paracrine and autocrine intervention of IL-1 alpha secreted by epithelial cells and PGE(2) and PGF(2 alpha) secreted by stromal cells.

Clinical significance of tenascin-C expression in osteosarcoma: tenascin-C promotes distant metastases of osteosarcoma.

Tenascin-C (TN-C) is one of extracellular matrix glycoproteins. We have immunohistochemically examined the TN-C expression of 33 primary osteosarcoma paraffin-embedded samples. The TN-C expression of the patient group with metastases was higher than that of the group without metastases at significant difference, and the survival curves show a tendency for poor outcome in the high grade staining group. Moreover, the supplemental TN-C had an effect of easier migration of a human osteosarcoma cell line (HOS) in vitro. The results may suggest that TN-C help osteosarcoma cells to migrate and to metastasize.

Ultrastructural and immunohistochemical analysis of cholangiocarcinoma in immunized Syrian golden hamsters infected with Opisthorchis viverrini and administered with dimethylnitrosamine.

Utilizing the experimental model in Syrian golden hamsters, we explored the role of immunization in carcinogenesis. The animals, which were infected with liver flukes (Opisthorchis viverrini), and administered a subcarcinogenic dose of dimethylnitrosamine, developed cancer. Pre-immunizing with a crude somatic antigen did not reduce cancer development, but accelerated carcinogenesis. Histopathological analysis of the cancer tissues was done once at week 30 and again at week 39 using H and E staining, immunostaining for the p53 tumor suppressor phosphoprotein, and electron microscopy. Thirty weeks after immunization, the immunized hamsters developed tubular adenocarcinoma at a higher rate (71.43%) than the non-immunized group (20.00%). This rate (20.00%) increased to 63.64% by week 39. The small foci cancer in the non-immunized group decreased in frequency from 80.00% (at week 30) to 36.36% (by week 39), suggesting the small foci cancer progressed to tubular adenocarcinoma during the 9-week interval. Most of the observed tubular adenocarcinoma was well differentiated. Nearly all hamsters that tested positive for cancer also tested positive for p53 immunostaining in the epithelia of the small bile ducts. The positive reaction for p53-immunostaining was localized in the rough endoplasmic reticulum, Golgi apparatus and perinuclear membranes. The electron micrographs of these positive p53-immunostained cells showed characteristics of early cancer. The detection of p53 in early cancer development makes it a candidate as a tumor marker.

Involvement of tenascin-C in proliferation and migration of laryngeal carcinoma cells.

Tenascin-C (TN-C) is an extracellular matrix glycoprotein upregulated in various pathological processes. In this study, we investigated its distribution in dysplasia and carcinoma of the human larynx using immunohistochemistry and in situ hybridization (ISH) techniques. In all cancer tissues, TN-C immunostaining was markedly increased in the stroma, especially around the cancer cell nests. In addition, cytoplasmic staining of cancer cells was also observed in 62.5% of the invasive cases, the cells being distributed in the periphery of the nests adjacent to the stroma. TN-C mRNA signals in cancer cells were detected in all six cases examined by ISH. Furthermore, in vitro evaluation of the roles of TN-C demonstrated an increase in the proliferating cell fraction in a dose-dependent manner. In a wound closure assay, the addition of TN-C promoted migration. We conclude that TN-C secreted by cancer cells may be involved in their proliferation and migration in an autocrine fashion.

The expression of transforming growth factor beta (TGF-beta) in the synovial membrane of human temporomandibular joint with internal derangement: a comparison with tenascin expression.

We examined the expression of the transforming growth factor beta (TGF-beta) in 28 human temporomandibular joint (TMJ) samples (internal derangement of TMJ and control specimens) by an immunohistological method using paraffin-embedded tissues and a polyclonal antibody specific to human TGF-beta. The resulting reaction of TGF-beta expression divided into three types as follows. The first type, around the fibrocyte and in the lacunae of chondrocytes in the disc. The second type, at the stroma of the mildly hypertrophic synovial membrane and severely hypertrophic synovial membrane. The first type was observed in all the cases including the control cases. The second type showed only in the internal derangement of TMJ, and its expression pattern resembled that of tenascin (TN) within the stroma of hypertrophic synovial membranes. In conclusion, TGF-beta and TN were distributed in the affected synovial membrane of TMJ with internal derangement. These findings suggested that TGF-beta and TN might have a close relationship with synovitis, followed by tissue repair.

Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone.

We recently reported that osteopontin (OPN) and calprotectin (CPT) are present in the matrix of urinary calcium stones, and that OPN mRNA is expressed in the renal distal tubular cells. In the present study, we examined the immunohistochemical distributions of OPN and CPT in urinary stones. The stones used in this study were passed spontaneously from the upper urinary tract. One half of each of the stones was analyzed with an infrared spectrophotometer, and were shown to be comprised of calcium oxalate, calcium phosphate, uric acid and cystine. The other half of each stone was immersed in tetrasodium ethylenediamine-tetraacetate (EDTA) solution. The half-stones were embedded in paraffin and cut into 5-microm sections. The avidin-biotin-peroxidase complex technique was employed. A monoclonal antibody to human milk-derived OPN and a monoclonal antibody to human granulocyte-derived CPT were used as primary antibodies. The immunochemical study using the OPN and CPT antibodies showed positive staining of the matrix of the urinary calcium stones. The stones showed staining in two distinct zones: a core area was stained with randomly aggregated OPN and CPT, and peripheral layers were stained in concentric circles. On the basis of our observations, it is reasonable to presume that OPN and CPT play roles as the matrix in the structure of urinary calcium stones.

Calcium phosphate stones produced by Madin-Darby canine kidney (MDCK) cells inoculated in nude mice.

The canine renal distal tubular cell line Madin-Darby canine kidney (MDCK) forms calcium phosphate microliths during a long-term culture in vitro. We identified osteopontin (OPN) and calprotectin (CPT) from a urinary stone matrix. We recently also detected the expression of OPN and CPT in MDCK cells. The relationship between the mechanism of the stone formation and these stone matrix proteins is not yet known. Here, MDCK cells were cultured and inoculated in the subcutis of nude mice. After 4, 8 and 12 weeks, the inoculated tissues were resected, fixed and immunostained with polyclonal anti-human OPN and polyclonal anti-human CPT antibodies. Some serial specimens were stained with von Kossa's procedure. MDCK cells formed some follicular formations in the subcutis of nude mice at least at 12 weeks after transplantation. At 8 weeks after the inoculation, we detected small calcium phosphate stones with MDCK cells trapped in the follicles. The cells forming the stones also expressed both OPN and CPT. The CPT expression sites coincided with the stone formation sites. We confirmed that MDCK cells inoculated in nude mice had stone-forming potential, and we speculate that OPN and CPT play important roles in stone formation by MDCK cells.

Expression of fibronectin isoforms in human breast tissue: production of extra domain A+/extra domain B+ by cancer cells and extra domain A+ by stromal cells.

The expression of fibronectin (FN) isoforms including extra domain A (EDA) and extra domain B (EDB) segments, was investigated in 36 invasive ductal carcinomas and 13 benign tumors of human breast tissues by in situ hybridization using probes specific to alternative splicing sites. Signals for the constant region of FN mRNA in cancer cells were found in 53% of the invasive ductal carcinomas. The EDA+ and EDB+ mRNA signals were found in 47% and 33%, respectively. Stromal cells expressing FN, EDA+ and EDB+ mRNA signals were present in 100%, 69% and 14% of cases, respectively. Expression of FN mRNAs by cancer cells was most frequent in intraductal lesions or large cancer nests, and that by stromal cells was associated with desmoplastic areas. In representative cases, proportions of FN mRNA-positive cancer cells expressing EDA and EDB segments were 45% and 39%, respectively, signals for both being frequently found in the same cells. EDA+ and EDB+ mRNA were labeled in 25% and 6% of the FN mRNA-positive stromal cells, a large proportion thus being EDA-/EDB- FN. In conclusion, the splicing pattern of FN pre-mRNA is dependent on the cell type and histology of breast cancer tissues. The observed lack of expression in fibroadenomas and other benign conditions suggests a link with tumor progression.

Low cytoplasmic pH causes fragmentation and dispersal of the Golgi apparatus in human hepatoma cells.

The centrosomal localization of the Golgi apparatus in interphase cells is thought to be maintained by retrograde microtubule-based motility. It is well established that, when intracellular pH is lowered, lysosomes and endosomes, also showing pericentrosomal localization, translocate towards the plus ends of microtubules within 15 min. In this study, we found that prolonged incubation in low pH medium (pH 6.6) with 20 mM Na acetate induced the fragmentation and dispersal of the Golgi apparatus in the human hepatoma cell line PLC/PRF/5. The fraction of Golgi-dispersed cells increased in a time-dependent manner, and reached over 60% after the 16-h incubation. The cytoplasmic pH was dropped to approximately 7.10. Replacement with normal pH medium restored the structure and localization of the apparatus within 30 min. In the low pH condition, the microtubular network and endoplasmic reticulum appeared normal, and cytoplasmic dynein was still bound to the fragmented Golgi membranes. These findings suggest that low cytoplasmic pH suppresses the retrograde movement of the Golgi apparatus as well as that of lysosomes and endosomes.

Expression of tenascin-C and the integrin alpha 9 subunit in regeneration of rat nasal mucosa after chemical injury: involvement in migration and proliferation of epithelial cells.

Nasal mucosa covered by pseudostratified ciliated epithelia can be injured by microbial infection and physical and chemical agents. To elucidate mechanisms of regeneration, erosion of rat nasal mucosa was produced by intranasal instillation of trichloroacetic acid, and tissue specimens were then sequentially obtained after 1-14 days. Since tenascin-C (TN-C) and its receptor, alpha 9 beta 1 integrin, are assumed to play important roles in regeneration of stratified squamous epithelia, their expression was evaluated by immunohistochemistry and in situ hybridization. Three to five days after the injury, TN-C mRNA was found in epithelial cells of migrating fronts and in epithelial sheets recovering ulcerated surfaces between the fronts and normal regions. TN-C deposition was increased under such sheets. Enhanced alpha 9 staining was also evident in the involved epithelium. 5-Bromo-2'-deoxyuridine incorporation assays revealed significant increase in proliferating cells in cell sheets over TN-C deposits at 3-7 days. Therefore, we conclude that regenerating epithelial cells produce and secrete TN-C, associated with an increase in alpha 9 expression, and that interactions between these molecules could regulate migration and proliferation of the epithelial cells in an autocrine manner.

The localization of matrix metalloproteinase-3 and tenascin in synovial membrane of the temporomandibular joint with internal derangement.

OBJECTIVES: The aim of this investigation was to study the immunohistochemical localization of MMP-3 and tenascin in the temporomandibular joint and to compare it with control specimens. MATERIALS AND METHODS: Localizations of matrix metalloproteinase-3 (MMP-3) and tenascin in the temporomandibular joint disc and the synovial membrane in 26 human temporomandibular joint samples (internal derangement of TMJ; n = 16, and control; n = 10) by an immunohistological method with monoclonal antibodies specific to human MMP-3 and tenascin. RESULTS: MMP-3 was not distributed in control specimens while it was observed in the internal derangement cases. MMP-3 showed two staining profiles: (1) diffuse staining was observed within the stroma of severely deformed disc with osteophyte and/or disc displacement (three of 16 specimens); and (2) the localization was specifically detected on the surface of severely hypertrophic synovial membrane (six of 16 specimens). The latter localization pattern resembled that of the tenascin on the surface of the severely hypertrophic synovial membrane. CONCLUSION: Comparative localization of MMP-3 and tenascin revealed intense staining for both in the synovial membrane presenting inflammation, proliferation and hypertrophy. These findings suggest that tissue repair and remodeling in the temporomandibular joint disc and the inflammatory hypertrophic reaction in the synovial membrane might proceed at the same time.