OBJECTIVETo investigate the expression of Tenascin-C mRNA in keloids and hypertrophic Total RNA was isolated from normal adult skin. A cDNA fragment (base 5941-6481bp) of the scars.

METHODSfull-length human Tenascin-C cDNA was synthesized by polymerase chain reaction and subcloned in pGEM-T-easy. Dioxygen-labeled anti-sense and sense probes were synthesized by using a Sp6/T7 RNA synthesis kit in the present of Dig-UTP in vitro. The samples were taken from keloids in 10, hypertrophic scars in 10 and normal adult skin in 5. The hybridization was performed with 4% paraformaldehyde-fixed and wax-embedded sections to detect the Tenascin-C mRNA.

RESULTSThe Tenascin-C mRNA was negative in the normal adult epidermis and weakly located in the fibroblasts of the papillary dermis and the epidermal adnexa. In all of the 10 keloid specimens, the Tenascin-C mRNA was positive throughout the epidermis and widely distributed in the dermis included in the fibroblasts, endothelial cells and epidermal adnexa. In the specimens of the 3 hypertrophic scars,the Tenascin-C mRNA was also positive in the epidermis, but in the other 7 cases, it became negative. In the dermis of the hypertrophic scar,the Tenascin-C mRNA was weaker than that in the keloid, but stronger than that in the normal skin.

CONCLUSIONSThe expression of Tenascin-C mRNA is markedly enhanced in the keloids.

Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Female ; Humans ; Keloid ; genetics ; metabolism ; pathology ; Male ; RNA, Messenger ; metabolism ; Tenascin ; genetics ; metabolism

OBJECTIVETo investigate the abnormalities of human epiderm in keloids and hypertrophic scars.

METHODSBiopsies from ten untreated keloids (duration of disease 3 - 30 years) and ten hypertrophic scars (duration of disease 6 - 10 months) and five normal adult skin specimens. Total RNA was isolated from normal adult skin. A cDNA fragment (base 5941 - 6481 bp) of the full-length human Tenascin-C cDNA was synthesized by polymerase chain reaction and subcloned in pGEM-T-easy. Dioxigen-labeled anti-sense and sense probes were synthesized by using a Sp6/T7 in vitro RNA synthesis kit in the present of Dig-UTP. In situ hybridization was performed on 4% paraformaldehyde-fixed and wax-embedded sections of keloids and hypertrophic scars. NBT-NCIP was used in color detection. Immunohistochemical procedure. The sections were incubated with antibodies (anti-Tenascin-C, anti-CK-16 and anti-Ki-67). Ultrasensitive Streptavidin Peroxidase staining was performed following established procedures.

RESULTSThe study show that the proliferation of epidermal keratinocytes in keloids and hypertrophic scars is very clear. The expressions of Tenascin-C mRNA in keloids epidermal keratinocytes markedly increased in contrast with epidermal keratinocytes of hypertrophic scars and adult skin. The CK-16 and Ki-67 stainings significantly enhanced in the epidermal keratinocytes of keloids and hypertrophic scars.

CONCLUSIONSThe different expressions of Tenascin-C, CK-16 and Ki-67 among normal adult skin, keloids and hypertrophic scars show the abnormalities of epidermal keratinocytes proliferation and differentiation in keloids and hypertrophic scars.

Cicatrix, Hypertrophic ; metabolism ; pathology ; Epidermis ; metabolism ; pathology ; Female ; Humans ; Hyperplasia ; Immunohistochemistry ; In Situ Hybridization ; Keloid ; metabolism ; pathology ; Keratins ; genetics ; metabolism ; Ki-67 Antigen ; genetics ; metabolism ; Male ; Tenascin ; genetics ; metabolism

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer's disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.
Aged, 80 and over ; Alzheimer Disease/*metabolism ; Brain/*metabolism ; Female ; Gelsolin/genetics/metabolism ; Humans ; Male ; Membrane Proteins/genetics/metabolism ; Neoplasm Proteins/genetics/metabolism ; Organ Specificity ; Proteome/genetics/*metabolism ; Tenascin/genetics/metabolism ; Ubiquitin C/genetics/metabolism

OBJECTIVETo understand the mechanism by which tenascin-C regulates osteoblast differentiation and the role of tenascin-C in osteoporosis.

METHODSTenascin-C protein expression in femoral spongy bone of mice with or without osteoporosis was analyzed using Western blotting. In MC3T3-E1 osteoblasts with or without tenascin-C depletion by a specific siRNA targeting tenascin-C, alkaline phosphatase activity and Dickkopf-1 (DKK-1) expression were determined using quantitative RT-PCR and Western blotting, and the transcriptional activity of Wnt signaling pathway was analyzed using a luciferase reporter assay. The possible interaction of tenascin-C with DKK-1 predicted by STRING software was verified by immunoprecipitation.

RESULTSs Tenascin-C was markedly down-regulated in hemoral spongy bone of mice with osteoporosis as compared with the control mice. Osteoblastic differentiation was markedly suppressed in MC3T3-E1 osteoblast after tenascin-C depletion, and was significantly reversed by simultaneous β-catenin over-expression. siRNA-mediated knockdown of tenascin-C, which bound DKK-1, up-regulated the expression of DKK-1 and consequently lowered the transcriptional activity of Wnt pathway.

CONCLUSIONTenascin-C knockdown attenuates its negative control on DKK-1 to suppress the transcriptional activity of Wnt pathway, which in turn suppresses osteoblastic differentiation and promotes osteoporosis.

Animals ; Cell Differentiation ; Gene Knockdown Techniques ; Mice ; Osteoblasts ; cytology ; Osteogenesis ; Osteoporosis ; physiopathology ; RNA, Small Interfering ; genetics ; Tenascin ; genetics ; metabolism ; Up-Regulation ; Wnt Signaling Pathway ; beta Catenin ; metabolism

BACKGROUNDTreating metastatic osteosarcoma has been challenged in past decades. Extracelluar matrix (ECM) proteins play an important role in the progression of osteosarcoma as they are pivotal components of the tumor microenvironment. Here, we identified potential genes belonging to the ECM and characterized the roles of these genes in the progression of osteosarcoma and their association with outcomes.

METHODSOsteosarcoma parental cell line MG63 and its derivative MG63-A1 with a high metastatic potential underwent oligonucleotide microarray analysis. Gene ontology analysis was used to screen deregulated genes between the 2 cell lines which were either upregulated or downregulated by more than 4 fold, particularly focusing on mRNAs encoding extracellular matrix proteins. The expression of resulting candidate genes was then validated by reverse transcription-PCR for mRNA expression as well as Western blotting for protein expression. Immunohistochemistry was performed on 37 osteosarcoma specimens to examine the potential role of the candidate genes in a clinical context.

RESULTSMicroarray data and gene ontology analysis showed that Tenascin-C, a critical component of the ECM, is significantly down-regulated in the highly metastatic cell line MG63-A1 compared with the parental osteosarcoma cell line MG63-wt. This finding was validated at mRNA and protein levels. Immunohistochemical analysis found that Tenascin-C is located in the intercellular space in osteosarcoma specimens. Furthermore, low-grade Tenascin-C expression (less than 20%) in osteosarcoma specimens was associated with poor survival.

CONCLUSIONSTenascin-C expression level correlates with the survival of osteosarcoma patients. Its biological functional role and underlying molecular mechanisms in the progression of osteosarcoma needs further investigation.

Adolescent ; Adult ; Bone Neoplasms ; metabolism ; mortality ; Cell Line, Tumor ; Child ; Female ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; Osteosarcoma ; metabolism ; mortality ; Prognosis ; RNA, Messenger ; analysis ; Tenascin ; analysis ; genetics

OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*

OBJECTIVETo investigate the role of GATA4 gene in the endocardial cushions development.

METHODSTarget gene eukaryote expression vectors were constructed by pcDNA3.1(-) vector plasmid, and were identified by DNA sequence analysis. Recombinant plasmids were transfected into Hela cells with lipofectamine 2000, meanwhile Hela cells transfected with empty vector or those without transfection served as transfection control group and blank control group, respectively. Real-time PCR and Western blot were performed to detect the relative expression of mRNA and protein of transcription factors GATA4, Sox9, Scleraxis and ECM proteins Aggrecan, Tenascin in each group.

RESULTSThe relative mRNA expression of GATA4 in experimental group was significantly higher than in transfection control group and blank control group. GATA4 mRNA expression in Hela(GATA4), Hela(H436Y), Hela(Null) and Hela group was 310.83 ± 2.39, 146.35 ± 1.74, 0.94 ± 0.32, 1.00 ± 0.28, respectively (F = 72.508, P < 0.05). Western blot results were consistent with the results obtained by qRT-PCR. The relative mRNA and protein expressions of Sox9, Scleraxis, Aggrecan and Tenascin in both experimental groups were significantly higher than that in transfection control group and blank control group (P < 0.05), and above gene expressions were significantly downregulated in GATA4(H436Y) group, while they were similar between transfection control group and blank control group (all P > 0.05).

CONCLUSIONSGATA4 H436Y mutation reduces it's transcriptional activation, which might serve as a theoretical framework to demonstrate the roles of GATA4 gene in endocardial cushion development.

Aggrecans ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Down-Regulation ; Endocardial Cushions ; embryology ; GATA4 Transcription Factor ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; HeLa Cells ; Humans ; RNA, Messenger ; SOX9 Transcription Factor ; metabolism ; Tenascin ; metabolism ; Transfection

OBJECTIVETo study the expression of tenascin in normal and fibrotic rat liver.

METHODSLiver fibrosis induced in rat with CCl(4) were divided into three stages: the stage of hepatic injury (4 weeks), early stage of hepatic fibrosis (8 weeks) and later stage of hepatic fibrosis (12 weeks). Tenascin expression in liver tissue was observed by immunohistochemical method and in situ hybridization using digoxigenin-labelled DNA probe.

RESULTSIn normal rat liver there was a weak staining for tenascin. In both liver injury stage and early stage of hepatic fibrosis, both mRNA signal and immunostaining for tenascin were significantly increased as compared to that in normal liver. In later stage of hepatic fibrosis, mRNA signal and immunostaining for tenascin were decreased compared with that in early stage of hepatic fibrosis. The cellular source of tenascin in liver mainly restricted in mesenchymal cells.

CONCLUSIONSTenascin is a component of the extracellular matrix of liver tissue. Plays a role in early matrix organization during liver fibrogenesis.

Animals ; Carbon Tetrachloride ; Disease Models, Animal ; Extracellular Matrix Proteins ; biosynthesis ; genetics ; Image Processing, Computer-Assisted ; Immunohistochemistry ; In Situ Hybridization ; Liver Cirrhosis ; chemically induced ; metabolism ; Male ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Tenascin ; biosynthesis ; genetics

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the alpha v beta 3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of alpha v mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the alpha v beta 3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the alpha v beta 3 protein. No staining was evident in control aortas for the alpha v beta 3 antigen, tenascin or alpha v mRNA. The upregulation of av mRNA and the alpha v beta 3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.
Adult ; Aorta, Abdominal/immunology/pathology ; Aortic Aneurysm, Abdominal/*pathology ; Biological Markers/analysis/metabolism ; Female ; Humans ; Integrin alphaVbeta3/analysis/genetics/metabolism ; Male ; Neovascularization, Pathologic/genetics/*metabolism ; Nitric-Oxide Synthase/analysis/metabolism ; RNA, Messenger/analysis/metabolism ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, P.H.S. ; Tenascin/analysis/metabolism