CircSLC7A2 protects against osteoarthritis through inhibition of the miR-4498/TIMP3 axis.

OBJECTIVES: Circular RNAs (circRNAs) are noncoding RNAs that compete against other endogenous RNA species, such as microRNAs, and have been implicated in many diseases. In this study, we investigated the role of a new circRNA (circSLC7A2) in osteoarthritis (OA). MATERIALS AND METHODS: The relative expression of circSLC7A2 was significantly lower in OA tissues than it was in matched controls, as shown by real-time quantitative polymerase chain reaction (RT-qPCR). Western blotting, RT-qPCR and immunofluorescence experiments were employed to evaluate the roles of circSLC7A2, miR-4498 and TIMP3. The in vivo role and mechanism of circSLC7A2 were also conformed in a mouse model. RESULTS: circSLC7A2 was decreased in OA model and the circularization of circSLC7A2 was regulated by FUS. Loss of circSLC7A2 reduced the sponge of miR-4498 and further inhibited the expression of TIMP3, subsequently leading to an inflammatory response. We further determined that miR-4498 inhibitor reversed circSLC7A2-knockdown-induced OA phenotypes. Intra-articular injection of circSLC7A2 alleviated in vivo OA progression in a mouse model of anterior cruciate ligament transection (ACLT). CONCLUSIONS: The circSLC7A2/miR-4498/TIMP3 axis of chondrocytes catabolism and anabolism plays a critical role in OA development. Our results suggest that circSLC7A2 may serve as a new therapeutic target for osteoarthritis.

MiR-23b Promotes the Migration of Keratinocytes Through Downregulating TIMP3.

BACKGROUND: Wound healing is a complex process aiming at repairing the damaged skin. MiR-23b has been reported to be upregulated during wound healing. In this study, we intended to explore the working mechanism of miR-23b during wound healing. METHODS: Quantitative real-time polymerase chain reaction was performed to detect the enrichment of miR-23b and tissue inhibitor of metalloproteinase-3 (TIMP3) in HaCaT cells. Scratch wound assay was carried out to measure the migration of HaCaT cells. The target of miR-23b was predicted by microT-CDS software, and the combination was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. The abundance of TIMP3 protein was detected by Western blot assay. RESULTS: The abundance of miR-23b was positively related to the concentration and time of transforming growth factor ß1 treatment in HaCaT cells. MiR-23b promoted the migration of keratinocytes. TIMP3 was a direct target of miR-23b and was negatively regulated by miR-23b. TIMP3 inhibited the migration of keratinocytes. MiR-23b accelerated the migration of keratinocytes by downregulating the abundance of TIMP3. CONCLUSIONS: MiR-23b promoted the migration of keratinocytes partly through reducing the enrichment of TIMP3. MiR-23b might be a promising target for the treatment of wound healing-associated diseases.

Comparative analysis of the expression of metalloproteases (MMP-2, MMP-9, MMP-11 and MMP-13) and the tissue inhibitor of metalloprotease 3 (TIMP-3) between previous negative biopsies and radical prostatectomies. / Análisis comparativo de la expresión de metaloproteasas (MMP-2, MMP-9, MMP-11 y MMP-13) y del inhibidor tisular de la metaloproteasa 3 (TIMP-3) entre las biopsias previas negativas y las prostatectomías radicales.

Metalloproteases (MMPs) and tissue inhibitor of metalloprotease-3 (TIMP-3) have been associated to the risk of having cancer and tumor aggressiveness. When facing the difficulties of prostate cancer diagnosis, the expression of MMPs and TIMP-3 in negative biopsies could be helpful to evaluate a diagnostic suspicion. Our objective is to carry out a comparative study of the expression of MMPs and TIMP-3 in previous negative biopsies and radical prostatectomies (RP). MATERIAL AND METHODS: Retrospective analysis of a hospital-based cohort including 21 patients with suspicion of prostate carcinoma, whose expressions of MMP-2, 9, 11 and 13 and TIMP-3 were evaluated by immunohistochemistry in the tumor area from previous negative biopsies and RP. RESULTS: Immunohistochemical staining values (Score) for MMPs (-11 and -13) and TIMP-3 showed no significant differences when comparing the areas of negative biopsies where tumors subsequently developed with those of the RP. However, we did observe a significant difference in the increased expression of MMP-2 (P=.002) and MMP-9 (P=.001) in the tumor area of the RP with respect to the corresponding area of the previous negative biopsy. CONCLUSIONS: Our data indicate a higher overall expression of MMP-2 and MMP-9 in the tumor area of the RP compared to the corresponding areas of the negative previous biopsy, which seems to be associated to the process of malignant transformation.

Immunohistochemical markers of advanced basal cell carcinoma: CD56 is associated with a lack of response to vismodegib.

Vismodegib is an effective treatment for advanced basal cell carcinoma (BCC), but primary resistance to vismodegib remains to be elucidated. Alternative approaches are warranted to help selecting patients most likely to be responsive to treatment. The identification of immunohistochemical markers may support this perspective, as well as better understanding of resistance mechanisms. To determine the level of expression of CD56, PDGF-R, CD117, MMP9, TIMP3, and CXCR4 in advanced BCC, and explore whether expression levels are associated with non-response to vismodegib. A cross-sectional study was conducted. Immunohistochemical markers were selected based on their roles in tumour proliferation and/or migration in skin tumours. Tissue samples included pretreatment advanced BCC samples from patients treated with vismodegib, with an available response after six months of treatment. Regression optimised models were used to build hypotheses regarding a possible association between expression levels and non-response to vismodegib, which was then tested by logistic regression. Twenty-three patients were included. The percentage of samples expressing markers ranged from 43.5% (CD117) to 91.3% (CXCR4). CD56 expression was significantly associated with an increased risk of non-response to vismodegib (OR = 5.5; CI 95%: 3.4-29.8; p = 0.0488); a similar association was suggested for CXCR4 (p = 0.066), but not identified for other markers. These results provide a better understanding of the expression of immunohistochemical markers in advanced BCC. Further detailed analysis of CD56 expression may provide insights into guiding further investigation of the correlation between this marker and non-response to vismodegib.

Effect of psychological stress on the structure of the temporomandibular joint and the expression of MMP-3 and TIMP-3 in the cartilage in rats.

Our aim was to observe the effects of psychological stress on the structure of the temporomandibular joint (TMJ), and to evaluate the expression of matrix metallopeptidase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in condylar chondrocytes in rats. The rats were divided into 3 groups of 12 according to the duration of psychological stress: 3 weeks or 6 weeks, and 6 weeks of recovery. A fourth group of 12 rats was used as controls. Each rat was evaluated by the open-field test and the weight measured. The results confirmed psychological stress in 24 of the 36 rats (67%). The tissues of the TMJ were stained with haematoxylin and eosin and pathological changes were studied under a light microscope. MMP-3 and TIMP-3 expression was investigated using the SP kit. The experimental groups showed thinning of articular cartilage, shedding of collagen fibres, cracks in the articular discs, and other structural changes that were aggravated with time, from three weeks to six weeks. The 6-week recovery group showed an improvement in these changes, which indicated the initiation of joint repair. The MMP-3 expression rate correlated with the degree of joint lesion, while the TIMP-3 rate showed an opposite trend and was highest in the 6-week recovery group. Our findings clearly indicate that psychological stress may play an important part in the development of TMJ diseases in rats; further studies should be made to extrapolate the results to other models before clinical use.

Effects of chemically induced hypoxia on in vitro expression of hypoxia inducible factor-lα, vascular endothelial growth factor, aggrecanase-1, and tissue inhibitor of metalloproteinase-3 in rat mandibular condylar chondrocytes.

AIMS: To examine the expression of hypoxia inducible factor-lα (HIF-1α), vascular endothelial growth factor (VEGF), aggrecanase-1 (ADAMTS-4), and tissue inhibitor of metalloproteinase-3 (TIMP-3) in rat mandibular condylar chondrocytes under hypoxic conditions and examine the relationship of these expressed factors in early condylar cartilage growth. METHODS: CoCl2 was used to mimic a hypoxic microenvironment by chemically inducing hypoxia. Chondrocytes, which were obtained from the mandibular condyles of 3-week-old female Sprague-Dawley rats, were treated with 125 µmol/L CoCl2 for 48 hours. The HIF-1α, VEGF, ADAMTS-4, and TIMP-3 mRNA levels in chondrocytes were detected using a semiquantitativepolymerase chain reaction (PCR) at 12, 24, and 48 hours after the initiation of hypoxia and normoxia conditions. A univariate variance analysis (pairwise least significant difference t test) using a SPSS 13.0 software package was performed to test for differences between different groups. RESULTS: PCR analysis of the chondrocytes showed that the expression of HIF-1α and VEGF mRNA increased at 12, 24, and 48 hours after induction of hypoxia. HIF-1α expression gradually increased throughout the study duration, while VEGF expression peaked at 12 hours. Compared to normoxia conditions, hypoxia resulted in constantly elevated expression levels of both. On the other hand, there was no significant difference in the ADAMTS-4 and TIMP-3 mRNA expression between hypoxic and normoxic conditions throughout the study (P > .05). CONCLUSION: An upregulated HIF-1α and VEGF mRNA expression indicates their important roles in cartilage vascularization and development in newly hypoxic microenvironments. Additionally, chemically induced hypoxia has little effect on the expressions of ADAMTS-4 and TIMP-3.

Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN).

BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003). CONCLUSIONS: We concluded that these proteins/enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour.

Age-related alterations in gene expression of gingival fibroblasts stimulated with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Elderly people exhibit increased susceptibility to a number of autoimmune and infectious diseases, such as periodontitis. Although aging is reportedly associated with a decline in immune function, age-related alterations in periodontal tissue have remained elusive. In the present study, we comprehensively analyzed the effect of aging on the expression of selected genes using mouse gingival fibroblasts. MATERIAL AND METHODS: Gingival fibroblasts derived from young (8 wk of age) and old (≥ 24 mo of age) C57BL/6 mice were stimulated with Porphyromonas gingivalis lipopolysaccharide or live P. gingivalis strain W83. Expression of cytokines/chemokines, innate immune receptors, growth factors, matrix metalloproteinases, tissue inhibitors of metalloproteinases and osteoclastogenesis-related molecules were evaluated using real-time polymerase chain reaction and ELISA for interleukin-6 and transforming growth factor-ß1. RESULTS: Gingival fibroblasts derived from old mice exhibited decreased gene expression of Il-6, Cxcl1, Tlr2, Tlr4, Irak3 (IRAK-M), Kgf, Timp1, Timp3 and Rankl under resting conditions, whereas the expression levels of Tgfß1, Mmp3, Mmp13 and Opg were increased. Age-related differences were also detected at the protein level. Although P. gingivalis W83 upregulated Vegf, Fgf-2 and Bmp2 expression in both young and old gingival fibroblasts, the stimulatory effect on these genes was significantly lower in old gingival fibroblasts. CONCLUSION: Our findings demonstrated that aging altered the expression of a number of genes in gingival fibroblasts. Thus, alterations in the balance of these molecules could play a critical role in periodontitis progression in the elderly.

Immunohistochemical analysis of TIMP-3 and MMP-9 in actinic keratosis, squamous cell carcinoma of the skin, and basal cell carcinoma.

The expression of metalloproteinases and their inhibitors has been related to different invasive and metastatic potentials in cancer. This study aims to investigate the immunohistochemical expression of TIMP-3 and MMP-9 in samples of basal cell carcinoma (BCC), squamous cell carcinoma of the skin (SCC), and actinic keratosis (AK). Immunohistochemistry was performed to evaluate the expression of TIMP-3 and MMP-9 in samples of BCC (n=22), SCC (n=10), and AK (n=15). Ten fields of both tumor parenchyma and tumor stroma were photographed and counted in image software. The ratio of positive cells to total cells was used to quantify the staining. A higher expression of MMP-9 was found in tumor stroma of SCC compared to BCC and AK. No significant differences in TIMP-3 expression were observed among the groups. Considering the well-described differences between these neoplasms, these results provide additional evidence of the role of MMP-9 in tumor invasiveness of keratinocyte-derived tumors.

Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile.

BACKGROUND: Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. METHODS: Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). RESULTS: Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. CONCLUSIONS: We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer.

Promoter methylation and expression of TIMP3 gene in gastric cancer.

BACKGROUND: Gastric carcinoma development is a multi-stage process that involves more than one gene. Aberrant changes in DNA methylation are considered as the third mechanism that leads to anti-oncogene inactivation, which plays an essential role in tumor development. In this study, we assessed the relationship among the aberrant methylation of the promoter CpG islands of tissue inhibitor of metalloproteinase 3 (TIMP3) gene, its protein expression, and the clinicopathological features of gastric adenocarcinoma. METHODS: The methylation status of the promoter CpG islands and the protein expression of TIMP3 gene in tumors and adjacent normal mucosal tissues of 78 patients with gastric adenocarcinoma were detected by methylation-specific PCR (MSP) and immunohistochemistry. RESULTS: The CpG island methylation of TIMP3 was detected in tumor tissues, cancer-adjacent tissues, and lymph nodes with metastasis. In increasing order, the hypermethylation frequency of these tissues were 35.9% (28 of 78 non-neoplastic tissues), 85% (17 of 20 early-stage cases), 89.7% (52 of 58 progressive-stage cases), and 100% (78 of 78 metastatic lymph node). A marked difference was found between tumors and non-neoplastic tissues (P<0.05), but no difference existed among the subgroups of tumors (P>0.05). Immunohistochemistry analysis confirmed TIMP3 down-regulation in tumor tissues. The rate of TIMP3 gene expression was 100% in non-neoplastic tissues but apparently decreased to various extents at different stages, i.e., decreased to 30% (6/20) at the early stage, to 3.4% (2/58) at the progressive stage, and to 0% (0/78) in metastatic lymph nodes. Among the 70 tumor tissues with negative TIMP3 expression, 64 (91.4%) were hypermethylated and 6 were unmethylated (8.6%), indicating a significant association between hypermethylation and reduced or negative TIMP3 expression (P<0.01). CONCLUSION: The hypermethylation of the promoter region in CpG islands is the main mechanism of TIMP3 gene expression and may provide evidence for the molecular diagnosis and stage evaluation of gastric cancer. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1756134016954958.

Gene and protein localisation of tumour necrosis factor (TNF)-α converting enzyme in gingival tissues from periodontitis patients with drug-induced gingival overgrowth.

OBJECTIVE: It is known that tumour necrosis factor (TNF)-α converting enzyme (TACE) plays a crucial role in fibrotic inflammatory diseases, and is specifically inhibited by tissue inhibitor of metalloproteinase (TIMP)-3. Fibrotic drug-induced gingival overgrowth (GO) is often combined with periodontitis. However, neither TACE nor TIMP-3 has been thoroughly examined in periodontal tissues to date. The aim of the present study was to analyse mRNA expression of TACE and TIMP-3, and protein localisation of TACE in gingival tissues removed from drug-(calcium-channel blocker) induced GO and periodontitis. METHODS: A total of 30 gingival tissue samples were taken from 15 GO and 15 periodontitis patients. The mRNA expression levels were analysed by quantitative reverse transcription polymerase chain-reaction (qRT-PCR) and the protein localisation was investigated by immunohistochemistry. Statistical analysis was performed using the Mann-Whitney U-test. RESULTS: TACE and TIMP-3 mRNA levels were significantly higher in GO compared to the periodontitis groups, as revealed by qRT-PCR (p<0.05). TACE-producing cells were immunohistochemically detected among monocytes/macrophages, plasma cells and some epithelial cells. TACE immunoreactivity was shown to be more intense in GO than in periodontitis-gingival tissue. CONCLUSIONS: We have demonstrated TACE expression in cells such as macrophages, plasma cells and epithelial cells, and its predominant expression in GO tissues. This data suggests that TACE expression in GO-gingiva could be involved in the pathogenesis of disease.

Altered relationship between MMP-8 and TIMP-2 in gingival crevicular fluid in adolescents with Down's syndrome.

BACKGROUND AND OBJECTIVE: Periodontitis is more frequently found in subjects with Down's syndrome. The aim was to investigate whether the relationship between MMPs and TIMPs) in the gingival crevicular fluid of subjects with Down's syndrome is altered compared with controls. MATERIAL AND METHODS: Twenty-one adolescents with Down's syndrome and gingivitis (DS-G), 12 subjects with Down's syndrome and periodontitis (DS-P), 26 controls with gingivitis (HC-G) and eight controls with periodontitis (HC-P) were clinically examined. All patients were between 11 and 20 years of age. Gingival crevicular fluid was collected from each subject and the concentrations of MMPs (2, 3, 8, 9 and 13) and TIMPs (1, 2 and 3) (expressed as pg/µL adjusted for volume of gingival crevicular fluid) were determined using multianalyte kits from R&D Systems. RESULTS: The concentrations of MMP-2, MMP-3, MMP-8, MMP-9 and TIMP-2 in gingival crevicular fluid were significantly higher (p < 0.005) in the DS-G group compared with the HC-G group. The correlation coefficient between MMP-8 and TIMP-2 differed significantly (p = 0.006) between the DS-G group and the HC-G group. On the contrary, the correlation coefficients between MMPs and TIMPs did not differ significantly between the DS-P group and the HC-P group. However, the DS-P group exhibited a significantly lower concentration of TIMP-2 in the gingival crevicular fluid compared with the HC-P group. CONCLUSION: Down's syndrome subjects with gingivitis exhibit higher concentrations of MMPs in gingival crevicular fluid with an altered relationship between MMP-8 and TIMP-2, which might impair the periodontal tissue turnover.

Effects of electrical stimulation on periodontal tissue remodeling in rats.

BACKGROUND AND OBJECTIVE: Electric current is used to promote wound healing. However, it is unclear whether electrical stimulation contributes to gingival tissue remodeling. This study examined the effects of electrical stimulation on gingival tissue remodeling in a rat periodontitis model. MATERIAL AND METHODS: Male Wistar rats (n = 28, 8 wks of age) were divided into four groups of seven rats each. The control group did not receive any treatment for 6 wks. In the other groups, periodontitis was ligature-induced for 4 wks. After 4 wks, the rats with periodontitis were given daily electrical stimulation of 0, 50 or 100 µA for 2 wks. RESULTS: The periodontitis group stimulated with 0 µA showed a higher density of polymorphonuclear leukocytes and a lower density of collagen in gingival tissue compared with the control group (p < 0.05). The two remaining groups treated with 50 or 100 µA of electrical stimulation exhibited a lower density of polymorphonuclear leukocytes (p < 0.05) and a higher density of collagen than the group stimulated with 0 µA (p < 0.05). They also showed higher expression of fibroblast growth factor-2 than the group treated with 0 µA of electrical stimulation (p < 0.05). CONCLUSION: Electric stimulation may offer a novel approach to promote gingival tissue remodeling in periodontal lesions.

Expression of a disintegrin and metalloprotease in human abdominal aortic aneurysms.

OBJECTIVES: The a disintegrin and metalloprotease (ADAM) family of metalloproteases possesses a proteolytic function and activates various inflammatory factors. Their expression pattern in an abdominal aortic aneurysm (AAA) is as yet unknown. The aim of this study was to make a detailed analysis of the expression of ADAMs 8, 9, 10, 12, 15 and 17, and their tissue inhibitors of metalloprotease (TIMP)-1 and TIMP-3 in patients with AAA. DESIGN: The aortic vessel walls of AAA patients (n=20) and non-aneurysmal aortic specimens (n=10) were obtained by conventional surgical repair and autopsy. SYBR green-based real-time PCR, histology and immunohistochemistry were performed on all samples. MAIN OUTCOME MEASURES: Quantitative expression analysis and the localisation of various ADAMs in AAA. RESULTS: ADAMs tested in our study were expressed in both AAA and control aorta without any significant differences between the groups. In contrast, expression of TIMP-1 was significantly reduced in AAA compared to control vessels. Smooth muscle cells (SMCs), neovessels and macrophages were positive for all ADAMs and TIMPs tested. Infiltrates were negative for TIMP-3, and luminal endothelial cells were positive for ADAMs 15 and 17. A significant positive correlation was observed between ADAMs 10, 12, 15, 17, TIMP-3 and SMCs. CONCLUSION: ADAMs are constitutively expressed in normal aortic vessel walls and AAA, particularly in SMCs.

Dysregulated gene expression of extracellular matrix and adhesion molecules in saphenous vein conduits of hemodialysis patients.

OBJECTIVE: The incidence of cardiovascular disease was approximately 10 times higher in hemodialysis patients with end-stage renal disease than in the general population. The saphenous vein is the most commonly used conduit for coronary artery bypass grafting. However, the extracellular matrix and adhesion molecule characteristics of saphenous vein in hemodialysis patients remain unclear. The aim of the present study was to survey the extracellular matrix gene expression profile of the saphenous vein in hemodialysis patients undergoing coronary artery bypass grafting. METHODS: A total of 34 patients undergoing elective coronary artery bypass grafting were enrolled. Of the 34 patients, 15 with end-stage renal disease required maintenance hemodialysis. The control group consisted of the other 19 patients without preoperative renal disease. Samples of the saphenous vein were obtained at coronary artery bypass grafting. The expression profile of the extracellular matrix genes was analyzed by microarray. The tissue matrix metallopeptidase/tissue inhibitor of metallopeptidase protein activities in the saphenous vein were evaluated by immunocytochemistry and Western blotting. RESULTS: Nineteen extracellular matrix and adhesion molecule-focused genes demonstrated at least a threefold difference in expression between the 2 groups. Upregulation was observed in 16 genes, and 3 genes appeared to be downregulated. Notable imbalanced matrix metallopeptidase/tissue inhibitor of metallopeptidase protein activities of saphenous vein exposed to end-stage renal disease conditions was found. CONCLUSIONS: The results from present study suggest that the native extracellular matrix gene expression profile of the saphenous vein conduits in hemodialysis patients show signs of the vein graft disease process before coronary surgery. Furthermore, some preoperative profiles of hemodialysis patients undergoing coronary artery bypass grafting might provide some useful clues regarding vein graft quality and prompt adjustment in surgical strategy.

Association of microRNA-21 expression with its targets, PDCD4 and TIMP3, in pancreatic ductal adenocarcinoma.

Since the discovery of small non-coding RNAs, the analyses of microRNA (miRNA) expression patterns in human cancer have provided new insights into cancer biology. miRNA-21 has been suggested to be one of the miRNAs that have an important role in the development or biological behavior of a variety of malignancies, including pancreatic cancer. This study was conducted to evaluate the relationship between the expression of miRNA-21 and that of its molecular targets, programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinase (TIMP3), in pancreatic ductal adenocarcinoma. The study included 65 pancreatic ductal adenocarcinomas and 5 normal pancreatic tissue specimens for comparison. The miRNA expression profiling of five selected pancreatic ductal adenocarcinomas and five normal pancreatic specimens was performed using a microarray platform, and was evaluated by a hierarchical clustering analysis. The miRNA most highly expressed in pancreatic ductal adenocarcinomas (ie, miRNA-21) was further assessed by quantitative real-time reverse transcription PCR (RT-PCR) assays in the 65 pancreatic ductal adenocarcinoma cases. The expression pattern of its molecular targets (eg, PDCD4 and TIMP3) in pancreatic ductal adenocarcinoma was examined immunohistochemically. In the microarray analyses, 28 miRNAs were upregulated in pancreatic ductal adenocarcinoma compared with normal pancreatic tissue, whereas 48 miRNAs were downregulated. miRNA-21 was the most significantly overexpressed miRNA in the pancreatic ductal adenocarcinomas analyzed, and was also highly expressed in 75% of the 65 pancreatic ductal adenocarcinomas examined by real-time RT-PCR. High miRNA-21 expression was correlated with a worse prognosis in the pancreatic ductal adenocarcinoma patients (P=0.045). The immunohistochemical expression patterns of PDCD4 (reduced nuclear staining pattern) and TIMP3 (downregulated expression) were significantly associated with both the upregulated miR-21 expression (P<0.05) and the poor survival of the patients (P<0.001 and P=0.001, respectively). Our data suggest that an overexpression of miRNA-21 is, therefore, associated with the biological behavior of pancreatic ductal adenocarcinoma via the downregulation of the expression of tumor suppressors, PDCD4 and TIMP3, thus resulting in tumor progression and the adverse clinical course of pancreatic ductal adenocarcinoma.

Altered matrix metalloproteinases and tissue inhibitors of metalloproteinases in embryos from diabetic rats during early organogenesis.

Maternal diabetes increases the risks for embryo malformations. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are two relevant MMPs for embryo development. Here, we addressed whether changes in these MMPs and in tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-2 are altered in embryos and decidua from type 1 diabetic rats during early organogenesis. Our results demonstrate MMP-2 and MMP-9 overactivities and overexpression, together with increases in lipid peroxidation and nitric oxide production in embryos and decidua from diabetic animals. There is a concomitant increase in the inhibitory activity of TIMP-1 and TIMP-2 in embryos and decidua, and an increase in protein expression of embryonic TIMP-1 and TIMP-2. In situ zymography demonstrated MMPs overactivities despite increased TIMPs in embryos and decidua in maternal diabetes during early organogenesis. This study reveals that maternal diabetes leads to profound alterations in MMPs/TIMPs balance during embryo organogenesis, the gestational period during which most malformations are induced.

Expression of matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-1 (TIMP-1, TIMP-2 and TIMP-3) in women with uterine prolapse but without urinary incontinence.

OBJECTIVE: To investigate the activities of matrix metalloproteinase-2 (MMP-2) and its inhibitors, tissue inhibitor of metalloproteinase-1, -2 and -3 (TIMP-1, TIMP-2 and TIMP-3), in the pelvic support and nonsupport tissue of women with uterine prolapse but without urinary incontinence. STUDY DESIGN: Paired samples of uterosacral ligament and cervical tissue were obtained from 11 postmenopausal and 8 premenopausal women with severe uterine prolapse. Nine premenopausal women without prolapse were selected as normal controls. Immunoreactivity of MMP-2 and TIMPs was demonstrated by immunohistochemistry. Steady state of MMP-2 as well as TIMPs messenger RNA (mRNA) expression was analyzed by polymerase chain reaction (PCR) with quantitative expression determined by multiplex PCR. RESULTS: A significantly higher expression of MMP-2 mRNA and lower expression of TIMP-2 mRNA were found in uterosacral ligament in uterine prolapse women compared to controls. In the cervical tissue, however, the MMP-2 and TIMPs mRNA expression was comparable between prolapse and control groups. With regard to menopausal status, there was no significant difference in MMP-2 and TIMPs mRNA expression between premenopausal and postmenopausal women with uterine prolapse. CONCLUSIONS: An increase in MMP-2 mRNA and a decrease in TIMP-2 mRNA expression in uterosacral ligament are related to uterine prolapse in women without urinary incontinence.

Role of tumor necrosis factor-alpha in wound repair in human vocal fold fibroblasts.

OBJECTIVES/HYPOTHESIS: Tumor necrosis factor-alpha (TNF-alpha) is an inflammatory cytokine and apoptotic molecule that appears to be a mediator in inflammation and fibrosis. The objective of this investigation was to examine the effects of TNF-alpha on 3D Carbylan-GSX in vitro cultured human vocal fold fibroblasts (hVFFs), to provide insight into the mechanism responsible for the improved vocal fold wound healing that has been previous reported with Carbylan-GSX treatment. STUDY DESIGN: In vitro cell culture. METHODS: hVFF were cultured in 3D Carbylan-GSX and on polystyrene with different dosages of TNF-alpha (0, 0.1, 1, 10, and 100 ng/mL) with and without 10% fetal bovine serum (FBS). hVFF response to TNF-alpha was characterized by morphology, proliferation rates, and gene transcript levels for matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 3 (TIMP3), collagen I, collagen III, fibronectin, and TNF-alpha receptor. RESULTS: In 3D Carbylan-GSX, TNF-alpha inhibited hVFF proliferation in a dose-dependent manner. TNF-alpha (0.1-100 ng/mL) was shown to significantly downregulate TIMP3 and extracellular matrix-related mRNA transcript levels for collagen III and fibronectin and to upregulate MMP1 and MMP2 expression, resulting in increased MMP/TIMP3 ratios. TNF-alpha receptor expression was significantly upregulated in Carbylan-GSX compared to control polystyrene. Responses were more marked in 10% FBS culture. CONCLUSIONS: After vocal fold injury, locally injected Carbylan-GSX can enhance the role of TNF-alpha in remodeling the lamina propria layer of the vocal fold, accelerating wound healing. Carbylan-GSX has potential as a new therapeutic approach that may lead to better treatment of vocal fold wound healing.