Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As the field of dental implants continues to grow at a rapid rate so does our quest to find new techniques to enhance bone grafting. Tissue engineering is an exciting new technique in bone grafting. Therefore, the purposes of this study were to develop a simple, reproducible method to isolate human osteoblast-like cells (HOBs) and to evaluate in vitro cell proliferation within 2 different 3-dimensional (3-D) constructs targeted for tissue engineering applications. Ultimately, HOBs that have been amplified within 3-D constructs may be employed for bone regeneration techniques, such as onlay and sinus grafting prior to implant placement. Our cell isolation protocol employed human fetal calvaria tissue sequentially digested with trypsin and collagenase. The HOB cells from only the third and fourth digests were obtained, cultured and evaluated within the constructs. An osteoblast-like phenotype was in part verified for these HOB cells by demonstrating a significantly higher alkaline phosphatase activity than for human gingival fibroblasts, and a comparable level to the osteoblast cell line MG-63. The HOB cells were cultured within either poly (D,L-lactide) (PLA) or a fused fiber ceramic and evaluated for the ability to support in vitro HOB amplification. HOB proliferation was validated by scanning electron microscopy, identifying cells throughout the 3-D constructs. Continuous cell viability was demonstrated for the duration of the 33-day evaluation period and the extent of cell amplification reached approximately 20 times the seeding density. The in vitro amplification results further indicate that tissue engineering strategies with either the PLA or fused fiber construct may be suitable for bone regeneration therapy for dental implants.
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PMID:Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering. 984 35

Clinical trials with monoclonal antibodies directed against TNF alpha (anti-TNF mAbs) and soluble TNF receptor fusion proteins (sTNFR-IgGs) have demonstrated that systemic and synovial trapping of TNF alpha results in long lasting anti-inflammatory and anti-nociceptive effects in patients with rheumatoid arthritis. Clinical indices of inflammatory synovitis and laboratory parameters (CRP and ESR) respond to single and repeated administrations of anit-TNF alpha therapies in a dose-dependent fashion. Studies on the immuno-pharmacological profile in patients suggest evidence that TNF alpha trapping down-regulates the effector mechanisms involved in the immuno-inflammatory response in rheumatoid arthritis. Inhibition of PLA 2- and COX-2-derived pathways of mediators of inflammation (prostanoids and leukotrienes) decreases signs and symptoms of inflammatory synovitis such as joint swelling, tenderness and pain. Down-regulating of the cytokine-inducible adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in endothelial cells and synoviocytes results in a marked inhibition of transendothelial migration of inflammatory and immune cells. A decrease of cytokine-regulated metalloproteinase expression results in normalization of circulating MMP-1 and MMP-3 levels. The effect of TNF alpha neutralization on mechanisms of rheumatoid joint destruction has the long-term potential for preventing or decreasing the rate of erosive changes of cartilage and bone.
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PMID:[Immunopharmacologic profile and therapeutic prospects of anti-TNF-alpha therapy]. 986 33

Neutrophil (PMN) accumulation frequently occurs at the site of snakebite as part of the inflammatory response to envenoming. We demonstrate here that the venoms of the cobras, Naja naja and N. mossambica, and two purified venom phospholipase A(2)s (PLA(2)s) isolated from the latter venom, stimulate CD11b translocation from the PMN granule store to the plasma membrane and enhance neutrophil motility on collagen-coated surfaces. These effects were partially attenuated by the PLA(2) inhibitor, aristolochic acid, and almost completely abolished by the specific cytosolic PLA(2) inhibitor, methylarachidonylfluorophosphonate (MAFP). Annexin V and inhibitors of collagenase, cyclo-oxygenase and lipo-oxygenase, all inhibited PMN motility to a variable extent. FACS analysis and confocal microscopy showed that Annexin V interfered with binding and rapid endocytosis of the venom PLA(2). These results indicate that venom and venom PLA(2) preparations first caused a non-enzymatic stimulation of PMN leading to the activation of cytosolic PMN PLA(2) and production of arachidonate metabolites involved in stimulation of PMN degranulation and motility. The evidence suggests that venom PLA(2) then interacts with anionic phospholipids exposed on stimulated PMN, becomes endocytosed, and then contributes itself to the production of chemoattractants responsible for PMN accumulation at the site of the snakebite.
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PMID:The role of phospholipases A2 in the stimulation of neutrophil motility by cobra venoms. 1265 15

Thrombin stimulation of isolated rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent PLA(2) (iPLA(2)) that selectively hydrolyzes plasmalogen phospholipids and results in increased production of arachidonic acid and lysoplasmenylcholine. To determine whether MAPK regulates myocardial iPLA(2) activity, we isolated ventricular myocytes from rabbit heart by collagenase digestion and pretreated them with MAPK inhibitors before stimulating them with thrombin. Pretreatment with PD-98059 to inhibit p42/44 MAPK or SB-203580 to inhibit p38 MAPK had no significant effect on thrombin-stimulated, membrane-associated iPLA(2) activity. Thrombin stimulation resulted in significant increases in both p42/44 and p38 MAPK activity after 2 min. Pretreatment with the iPLA(2)-selective inhibitor bromoenol lactone completely inhibited thrombin-stimulated MAPK activity, suggesting that activation of MAPKs was dependent on iPLA(2) activation. Ventricular myocyte MAPK activity was increased by incubation of the myocytes with lysoplasmenylcholine, a metabolite produced by iPLA(2)-catalyzed membrane plasmalogen phospholipid hydrolysis. Altogether, these data suggest that activation of MAPKs occurs downstream of and is dependent on iPLA(2) activation in thrombin-stimulated rabbit ventricular myocytes.
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PMID:Activation of MAPKs in thrombin-stimulated ventricular myocytes is dependent on Ca2+-independent PLA2. 1633 69

Human processed lipoaspirate is a source of multipotent adult stem cells that are able to differentiate between mesenchymal and neurogenic lineage. We characterized PLA cells by cytometry and then they were cultured to induce differentiation into myogenic and neurogenic lineage. Lipoaspirates were digested with collagenase to obtain the pellet, which was labelled with anti-CD44, anti-CD45, and anti-CD90. We used BD FACS Calibur flow cytometer to acquire cellular events. Some cells were cultured at 37 degrees C and 5% CO(2) in neurogenic or myogenic medium and analysed by immunocytochemistry, using Neuron specific enolase, Vimentin, Glial fibrillary acidic protein, Tau, MAP2 to confirm neurogenic differentiation, MyoD1, Myosin heavy chain, Actin smooth muscle, vimentin to confirm myogenic differentiation. The cytometry results suggest that a part of the cells are of a mesenchymal origin, among which there are progenitor endothelial cells and leucocytes. Microscopy observation already reveals neuronal morphology and longitudinal multinucleated cells compared to control cells. Neurogenic cells only express NSE (early neuronal progenitor marker), but not GFAP, Tau, MAP2 (mature neuron and glial markers); myogenic cells are positive for MyoD1 and Myosin heavy chain. This demonstrates that lipoaspirate cells are capable of differentiating in vitro over a short period of time, and could be employed in biological and clinical research, like mesenchymal adult stem cells.
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PMID:Characterization and induction of human pre-adipocytes. 1711 45

Cyclooxygenases (COX) are rate-limiting enzymes involved in the conversion of PLA(2)-mobilized arachidonic acid into prostaglandins and thromboxanes. COX-2 is a key mediator of inflammation during both physiologic and pathologic responses to endogenous stimuli and infectious agents. Its overexpression has been detected in different cancers, including that of the breast. Using RNA interference, we have reduced the expression of COX-2 in the highly malignant breast cancer cell line MDA-MB-231 below detectable levels in response to interleukin-1 beta or 12-O-tetradecanoylphorbol-13-acetate treatment. Microarray analysis showed that COX-2 silencing resulted in the loss of mRNA expression of several oncogenic markers, such as matrix metalloproteinase-1, chemokine (C-X-C motif) receptor 4, and interleukin-11, which have been correlated with poor disease outcome, and in the up-regulation of antimetastatic transcripts, such as thrombospondin-1 and Epstein-Barr-Induced 3. Cells lacking COX-2 were less able to invade reconstituted extracellular matrix than parental cells in vitro. Consistent with these changes, loss of COX-2 resulted in the abolition or the significant delay of tumor onset when the cells were injected in the mammary fat pad of severe combined immunodeficient mice. Finally, silencing of COX-2 resulted in the inhibition of metastasis to the lungs of severe combined immunodeficient mice after intravenous injection. These data show that silencing of COX-2 abolishes the metastatic potential of MDA-MB-231 cells in vivo.
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PMID:Silencing of cyclooxygenase-2 inhibits metastasis and delays tumor onset of poorly differentiated metastatic breast cancer cells. 1751 Mar 10

The creation of tissue-engineered constructs with autologous cells is a central goal in regenerative medicine. With respect to ligament replacement, we have evaluated the influences of matrix and growth factors on hMSCs (human mesenchymal stromal cells). hMSCs were seeded in two different 3D (three-dimensional) systems consisting of either a collagen type I gel or a synthetic PLA [poly-(L-lactic acid)] scaffold. After cultivation for 14 days with rhTGFbeta1 (recombinant human transforming growth factor beta1), rhPDGF-BB (recombinant human platelet-derived growth factor homodimer of B-chain) or rhBMP13 (recombinant human bone morphogenetic protein 13), we assessed the proliferation potential, mRNA expression and protein expression of various matrix-interacting and matrix-degrading molecules by quantitative real-time RT (reverse transcriptase)-PCR, immunohistochemistry and gelatin zymography in comparison with unstimulated cells. Cellular reactions to the type of scaffold or soluble factors could be found in the expression of tenascin-C as well as integrin subunits alpha1, alpha3 and beta1. Collagen type X expression was induced by 3D culture and stimulated by rhTGFbeta1 on PLA. The expression of MMP-1 (matrix metalloproteinase 1) tended to increase, and MMP-13 was induced in the collagen culture system. The activation of MMP-2 was stimulated by the cultivation of MSCs within the collagenous matrix. These results demonstrated that various interactive effects of growth factors and scaffolds influence the cell-biological behaviour of MSCs. It is important to take these complex interactions, which partly differ from differentiated cells, into account in further tissue-engineering approaches.
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PMID:Interactive effects of growth factors and three-dimensional scaffolds on multipotent mesenchymal stromal cells. 1764 Jan 72

Stem cell therapy seems promising in reducing deficits after focal cerebral ischemia. As stroke may result from intracerebral hemorrhage (ICH) in up to 20% we investigated whether human processed lipoaspirate mesenchymal stem cells (PLA-MSC) influence the functional outcome, migration behavior and the activation of endogenous progenitor cells. Experimental ICH was induced by stereotactic administration of collagenase in rats randomly assigned to the control or treatment group. The latter received 3 x 10(6) PLA-MSC by intravenous (i.v.) injection 24h after ICH induction. The outcome was continuously monitored using the RotaRod test over a period of 4 weeks. Morphometric analysis of ICH was performed consecutively by magnetic resonance imaging (MRI) studies and immunohistochemical analysis. The RotaRod test revealed a significant 1.5-fold improvement (p<0.005) in functional outcome for the PLA-MSC treated group after 4 weeks compared to controls. Histological and MRI assessment of lesion size showed no difference between the two groups. Although i.v. injected human cells could not be detected in the post mortem brain, evaluation of the number of endogenous progenitor cells revealed a twofold increase in the treated animals compared to controls. Treatment with PLA-MSC improved the functional outcome significantly in an experimental ICH model. This effect was achieved by stimulation of endogenous progenitor cells rather than integration and differentiation of the infused PLA-MSC.
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PMID:Lipoaspirate-derived adult mesenchymal stem cells improve functional outcome during intracerebral hemorrhage by proliferation of endogenous progenitor cells stem cells in intracerebral hemorrhages. 1869 31

The purpose of this study was to prove the effect of cyclic uniaxial intermittent strain on the mRNA expression of ligament-specific marker genes in human mesenchymal stem cells (MSC) and anterior cruciate ligament-derived fibroblasts (ACL-fibroblasts) seeded onto a novel textured poly(L-lactide) scaffold (PLA scaffold). Cell-seeded scaffolds were mechanically stimulated by cyclic uniaxial stretching. The expression of ligament matrix gene markers: collagen types I and III, fibronectin, tenascin C and decorin, as well as the proteolytic enzymes matrix metalloproteinase MMP-1 and MMP-2 and their tissue specific inhibitors TIMP-1 and TIMP-2 was investigated by analysing the mRNA expression using reverse transcriptase polymerase chain reaction and related to the static control. In ACL-fibroblasts seeded on PLA, mechanical load induced up-regulation of collagen types I and III, fibronectin and tenascin C. No effect of mechanical stimulation on the expression of ligament marker genes was found in undifferentiated MSC seeded on PLA. The results indicated that the new textured PLA scaffold could transfer the mechanical load to the ACL-fibroblasts and improved their ligament phenotype. This scaffold might be suitable as a cell-carrying component of ACL prostheses.
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PMID:Effects of mechanical strain on human mesenchymal stem cells and ligament fibroblasts in a textured poly(L-lactide) scaffold for ligament tissue engineering. 2272 94

We used a murine model to assess the evolving biomechanical properties of tissue engineered vascular grafts (TEVGs) implanted in the arterial circulation. The initial polymeric tubular scaffold was fabricated from poly(lactic acid)(PLA) and coated with a 50:50 copolymer of poly(caprolactone) and poly(lactic acid)(P[PC/LA]). Following seeding with syngeneic bone marrow derived mononuclear cells, TEVGs (n=50) were implanted as aortic interposition grafts in wild-type mice and monitored serially using ultrasound. A custom biaxial mechanical testing device was used to quantify the in vitro circumferential and axial mechanical properties of grafts explanted at 3 or 7 months. At both times, TEVGs were much stiffer than native tissue in both directions. Repeated mechanical testing of some TEVGs treated with elastase or collagenase suggested that elastin did not contribute significantly to the overall stiffness whereas collagen did contribute. Traditional histology and immunostaining revealed smooth muscle cell layers, significant collagen deposition, and increasing elastin production in addition to considerable scaffold at both 3 and 7 months, which likely dominated the high stiffness seen in mechanical testing. These results suggest that PLA has inadequate in vivo degradation, which impairs cell-mediated development of vascular neotissue having properties closer to native arteries. Assessing contributions of individual components, such as elastin and collagen, to the developing neovessel is needed to guide computational modeling that may help to optimize the design of the TEVG.
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PMID:Characterization of evolving biomechanical properties of tissue engineered vascular grafts in the arterial circulation. 2470 63


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