Gene Expression Levels Of MMP-2 Research Articles

Tendon Healing in a Mouse Model of Loeys-Dietz Syndrome: Controlled Study Using a Patellar Tendon Transection Model.

Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by genetic mutations in the transforming growth factor-β (TGFβ) signaling pathway. In addition to vascular malformations, patients with LDS commonly present with bone and tendon abnormalities, including joint laxity. While TGFβ signaling dysregulation has been implicated in many of these clinical manifestations, the degree to which it influences the tendinopathy and tendon healing issues in LDS has not been determined. Wound healing after patellar tendon transection was compared between wild-type (WT) and Tgfbr2-mutant (LDS) mice (7 mice per group). In all mice, the right patellar tendon was transected at midsubstance, while the left was untouched to serve as a control. Mice were euthanized 6 weeks after surgery. Tendon specimens were harvested for histopathologic grading according to a previously validated scoring metric, and gene expression levels of Mmp2, Tgfb2, and other TGFβ-signaling genes were assayed. Between-group comparisons were made using 1-way analysis of variance with post hoc Tukey honestly significant difference testing. Expression levels of assayed genes were similar between LDS and WT tendons at baseline; however, at 6 weeks after patellar tendon transection, LDS tendons showed sustained elevations in Mmp2 and Tgfb2 compared with baseline values; these elevations were not seen in normal tendons undergoing the same treatments. Histologically, untreated LDS tendons had significantly greater cellularity and cell rounding compared with untreated WT tendons, and both WT and LDS tendons had significantly worse histologic scores after surgery. We present the first mechanistic insight into the effect of LDS on tendons and tendon healing. The morphologic differences between LDS and WT tendons at baseline may help explain the increased risk of tendon/ligament dysfunction in patients with LDS, and the differential healing response to injury in LDS may account for the delayed healing and weaker repair tissue. Level V.

Neonatal Maternal Separation Alters Gelatinase Activity in Mouse Ovarian Preantral Follicles.

ObjectiveThe early life environment is critical for normal growth and development for future reproductive function. This study aims to investigate the effect of neonatal maternal separation (MS) on gelatinase activity of mouse ovarian follicles.Materials and MethodsIn this experimental study, infants from female NMRI mice were randomly allocated into two groups immediately after birth: i. MS isolated from their mothers for 6 hours per day, from postpartum days 2 to 16) and ii. Control (undisturbed during the 16 days). Ovarian tissues were dissected to perform differential counts of the ovarian follicle type by haematoxylin and eosin staining. The isolated follicles were cultured for 12 days. Gelatinase activity and the gene expressions of matrix metalloproteinases, MMP2 and MMP9, and their tissue inhibitors, TIMP1 and TIMP2, were evaluated by zymography and real-time polymerase chain reaction (PCR), respectively. Results Follicle counts at the different developmental stages were significantly different between the control and MS groups. There was a significant decrease in gelatinase activity in the MS group compared to the control group. The MS group showed significantly decreased gene expression levels of MMP2 and MMP9 compared to the control group. In contrast, the gene expression levels of TIMP1 and TIMP2 significantly increased in the MS group compared to the control group. ConclusionMS is a stressor agent that compromises ovarian follicle development, at least via disruption of gelatinase activity and its related gene expressions.

Signaling pathways associated with structural changes in varicose veins: a case-control study.

In varicose veins, blood pressure increases in the veins of the lower extremities due to mechanical stimulation and function remodeling. The aim of this study is assessment of Signaling pathways associated with structural changes in varicose veins. This pilot study was performed on patients with varicose veins, which had undergone surgery. The healthy tissues from trauma patients or vascular bypass without underlying diseases were used for control samples. Hematoxylin-eosin, trichrome, and elastin staining were used for histopathological examination. The levels of MDA (malondialdehyde), total thiol, SOD (Superoxide dismutase) and NO (nitric oxide) level were measured using Elisa kits to evaluate the oxidative stress level. Gene expression levels of MMP2, MMP9, FOXO3a, APOE and p53 genes were determined using Real-time PCR. This study showed, the vascular Vein wall changes are visible in vascular collagen staining. Although these changes are observed in the structure of vascular wall collagen fibers, the accumulation of collagen and elastin was increased in the walls of varicose veins compared to the control group. The amount of nitric oxide and thiol were increased in the varicose group (P < 0.0001). The expression of metalloproteinase2 gene associated with extracellular matrix change was increased in varicose veins. However, the amount of metalloproteinase 9 was decreased in this group compared to control group. FOXO3a, APOE Genes were down-regulated in the varicose veins compared to control group, while p53 gene expression was significantly increased in the varicose group (P < 0.0001). This study demonstrated changes in oxidative stress, morphological structure, and aging pathways in varicose when compared to non-varicose veins. The changes in oxidative stress may be associated with the variations in morphological structure and aging pathways which contribute to the pathogenesis of varicose veins.

Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models.

Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity of these extracts. Methanol, ethyl-acetate and hexane were used as a solvents for extraction from both branch-body part of the plant (extracts 1, 2 and 3) and from plant flowers (extracts 4, 5 and 6). The cytotoxic effects of the extracts were determined against 2D and 3D cancer cell models. Cell cycle changes of treated HeLa cells were analyzed by flow cytometry. Measurements of gene and microRNA expression levels in treated HeLa cells weredone by quantitative real time PCR. Five examined extracts (2-6) exerted selective concentration-dependent cytotoxic effects on HeLa, K562, and A549 cancer cells, while the extract 1 exhibited very weak cytotoxicity. The extract 6 showed the highest intensity of cytotoxic activity. All tested extracts (2-6) demonstrated the ability to induce apoptosis in HeLa cells through activation of caspase-3. These extracts remarkably decreased gene expression levels of MMP2, MMP9, TIMP3, and VEGFA in HeLa cells. Flower extracts might have stronger effects on miR128/193a-5p/335 level changes than branch-body extracts. Hypericum perforatum extracts exerted weaker cytotoxic effects on 3D HeLa spheroids when compared with their effects on 2D monolayer HeLa cells. Taken together, results of our research may suggest the promising anticancer properties of the Hypericum perforatum extracts.

Effect of Lapachol on the Inhibition of Matrix Metalloproteinase Related to the Invasion of Human Fibrosarcoma Cells.

Anti-cancer effect of lapachol contained in Tabebuia avellandae has been poorly understood until now. The aim of this study was to investigate the inhibitory effect of lapachol on MMPs related to cell invasion. Its action mechanism was elucidated by analyzing the activity and the expression of MMPs and the proteins involved in the signaling pathway of cell invasion. The cytotoxicity of lapachol was evaluated by MTT assay in HT1080 cells. The effects of lapachol on the expression and the activation of MMPs were analyzed by western blot, immunofluorescence staining, and gelatin zymography assays. Their gene expression was analyzed by RT-PCR, and metastasis was evaluated by cell invasion assay. Lapachol below 2 μM showed no cytotoxicity. It was observed that lapachol above 0.5 μM inhibited the activation of MMP-2 and MMP-9 stimulated by PMA. In particular, the protein and gene expression levels of MMP-2 stimulated by PMA were remarkably decreased in the presence of lapachol at 1 μM compared with the PMA treatment group. In addition, lapachol increased the expression level of TIMP-1 compared with the PMA treatment group. Moreover, lapachol decreased the expression level of p-p38 among MAPKs compared with the PMA treatment group. It was also found that the expression level of p65, a part of NF-kB, in nuclei was reduced in the presence of lapachol above 0.5 μM compared with the PMA treatment group. In addition, lapachol inhibited the invasion of human fibrosarcoma cells stimulated with VEGF. Above results suggest that lapachol could play an important role in the modulation of MMPs related to cell invasion via the increase in TIMP-1 expression as well as the inactivation of p38 through NF-kB transcription factor.

A Co-Culture Model of Fibroblasts and Adipose Tissue-Derived Stem Cells Reveals New Insights into Impaired Wound Healing After Radiotherapy.

External radiation seems to be associated with increased amounts of cytokines and other cellular modulators. Impaired microcirculation and fibrosis are examples of typical long term damage caused by radiotherapy. Adipose tissue-derived stem cells (ASC) are discussed to enhance wound healing, but their role in wounds due to radiotherapy is poorly understood. Normal human fibroblasts (NHF) and ASCs were co-cultured and external radiation with doses from 2–12 Gray (Gy) was delivered. Cell proliferation and mRNA levels of matrix metalloproteinases (MMP1, MMP2 and MMP13) were determined 48 h after irradiation of the co-cultures by qPCR. Additionally, tissue inhibitors of matrix metalloproteinases (TIMP1, TIMP2) were determined by enzyme-linked immunosorbent assay (ELISA). There was a reduction of cell proliferation after external radiation in mono-cultures of NHFs and ASCs compared to controls without irradiation. The co-culture of ASCs and NHFs showed reduced impairment of cell proliferation after external radiation. Gene expression of MMP1 and MMP13 was reduced after external irradiation in NHF. MMP2 expression of irradiated NHFs was increased. In the co-culture setting, MMP1 and MMP2 gene expression levels were upregulated. TIMP1 and TIMP2 protein expression was increased after irradiation in NHFs and their co-cultures with ASCs. ASCs seem to stimulate cell proliferation of NHFs and modulate relevant soluble mediators as well as proteinases after external radiation.

Role of Matrix Metalloproteinases 2 and 9 in Lacrimal Gland Disease in Animal Models of Sjögren's Syndrome.

Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. There was a 2.5- and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands.

Expression profile of matrix metalloproteinase-2 and -9 and their endogenous tissue inhibitors in osteonecrotic femoral heads

The pathogenetic mechanisms of osteonecrosis of the femoral head are unresolved to date. This study analyzed the matrix metalloprotease (MMP) and tissue inhibitor of matrix metalloprotease (TIMP) expression and activity which might add to the impaired bone matrix repair capacity affecting the balance between bone resorption and de novo bone formation in osteonecrosis of the femoral head (ONFH). Cancellous bone biopsies were taken at the femoral head and neck of patients with advanced ONFH and patients with primary osteoarthritis (OA) who were undergoing total hip arthroplasty. We assessed the gene expression levels of MMP-2 and -9, TIMP-1 and -2, Il-1beta, Il-6 and TNF-alpha in both biopsies. These data were corroborated by MMP activity screening, collagen profiling and ELISAs for determination of MMP, TIMP, Il-1beta, Il-6 and TNF-alpha concentrations. Gene expression rates of MMP-2 and TIMP-1 were higher in ONFH biopsies whereas TIMP-2 and MMP-9 gene expression was not significantly altered. MMP protein profile shifted towards increased biosynthesis of both, active and pro-MMP-2 in ONFH bone lysates and decreased pro-MMP-9 production. Expression profiles of pro-inflammatory cytokines and collagens in OA and ONFH bone lysates were highly similar. Increased biosynthesis and activation of MMP-2 in ONFH samples may add to shifting the bone matrix turnover balance towards resorption of bone macromolecules thereby counter-acting new bone matrix production. OA- and ONFH-affected bone exhibits a similar pro-inflammatory cytokine and collagen expression profile suggesting a relationship on the molecular level of inflammation and collagenous matrix composition between both diseases.

Vascular Endothelial Growth Factor C Gene Expression Is Closely Related to Invasion Phenotype in Gynecological Tumor Cells

Objective. The correlation between the gene expression of various angiogenic factors and in vitro invasive activity in 16 human gynecological cancer cell lines was investigated.Methods. Semiquantitative reverse transcription polymerase chain reaction analysis was performed to investigate the mRNA expression levels of vascular endothelial growth factors (VEGF-A, -B, -C, and -D), basic fibroblast growth factor (bFGF), and matrix metalloproteinase (MMP)-2 with β-actin coamplified as an internal standard. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were evaluated by haptotactic migration and invasion assay.Results. Expression of VEGF-A mRNA was detected in all 16 cell lines, whereas the relative expression levels of other VEGF family members and bFGF, differed markedly among the cell lines. There was a statistical correlation between VEGF-C gene expression and the number of cells that migrated and invaded (P < 0.01). However, expression of mRNAs of other angiogenic factors did not correlate with motility and invasive activity of the cells. Moreover, there was a close correlation between VEGF-C and MMP-2 gene expression levels (P < 0.05).Conclusion. Tumor cells that produce VEGF-C may have a higher invasive and metastatic potential because of their capacity to pass through tissue barriers.