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PubMed2002

Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways.

A provisional matrix consisting of fibrin and fibronectin (FN) is deposited at sites of tissue damage and repair. This matrix serves as a scaffold for fibroblast migration into the wound where these cells deposit new matrix to replace lost or damaged tissue and eventually contract the matrix to bring the margins of the wound together. Tenascin-C is expressed transiently during wound repair in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. Using a synthetic model of the provisional matrix, we have found that tenascin-C regulates cell responses to a fibrin-FN matrix through modulation of focal adhesion kinase (FAK) and RhoA activation. Cells on fibrin-FN+tenascin-C redistribute their actin to the cell cortex, downregulate focal adhesion formation, and do not assemble a FN matrix. Cells surrounded by a fibrin-FN+tenascin-C matrix are unable to induce matrix contraction. The inhibitory effect of tenascin-C is circumvented by downstream activation of RhoA. FAK is also required for matrix contraction and the absence of FAK cannot be overcome by activation of RhoA. These observations show dual requirements for both FAK and RhoA activities during contraction of a fibrin-FN matrix. The effects of tenascin-C combined with its location around the wound bed suggest that this protein regulates fundamental processes of tissue repair by limiting the extent of matrix deposition and contraction to fibrin-FN-rich matrix in the primary wound area.

3T3 Cells↗
PubMed1998

Use of a collagen-hydroxyapatite matrix in spinal fusion. A rabbit model.

STUDY DESIGN: The efficacy of a specially designed mineralized bovine collagen matrix as a carrier for bone marrow stem cells was studied in a rabbit posterolateral spinal fusion model. OBJECTIVES: To determine if bone marrow cells added to Healos matrix will lead to fusion rates, biomechanical properties, and histologic properties comparable with those of fusions using autologous iliac crest bone graft; and to determine if the addition of preservative-free heparin to anticoagulate the bone marrow during harvest will adversely affect the fusion rate. SUMMARY OF BACKGROUND DATA: Although the development of new preparations of osteoinductive agents has advanced rapidly in recent years, the carrier systems that have been used in their application have received less attention. The composition and structure of the matrix used are key components affecting the ability of the matrix to function as a scaffold on which cells can migrate, adhere, proliferate, and form bone. The composition and design of matrix components also determine the ability of osteoinductive agents to influence local and hematogenously derived osteogenic precursor cells, which migrate to or are brought into the fusion site. Thus, the properties of the carrier can affect the behavior and efficacy of the osteoinductive agent that is used. The authors studied the properties of a new mineralized collagen matrix called Healos, which has been engineered specifically for spinal fusion application. METHODS: Forty-four adult female New Zealand white rabbits were divided into five groups. Groups 1-4 underwent bilateral intertransverse fusion between L5 and L6. The fusions were augmented with either autologous iliac crest bone graft, Healos matrix alone, Healos matrix mixed with autologous bone marrow, or Healos matrix combined with heparinized autologous bone marrow. At 8 weeks after surgery, the fusions were characterized radiographically, histologically, and biomechanically. The rate of fusion was determined by radiographic analysis. The fifth group consisted of two animals whose bone marrow was aspirated from their tibias and femurs and then sent for determination of total nucleated cell count. RESULTS: At 8 weeks, the radiographically determined fusion rate for autologous bone graft was 75% (9/12 animals), compared with 100% (10/10 and 9/9 animals) for groups in which fusions were done by using Healos matrix augmented with bone marrow (P < or = 0.1). Matrix used alone yielded a fusion rate of 18% (2/11 animals, P < or = 0.006). Histologically, the most mature bone was seen in the group augmented with autologous iliac crest graft, followed in decreasing order by the groups augmented with Healos with heparinized bone marrow, Healos with unheparinized bone marrow, and Healos alone. Biomechanically, the group augmented with autologous graft had the highest mean stiffness, followed by the groups augmented with Healos with heparinized bone marrow, Healos with untreated bone marrow, and finally Healos matrix alone. However, the differences in stiffness between groups were not statistically significant with the number of spines tested. CONCLUSIONS: These results show that Healos is an osteoconductive matrix that can be a useful carrier in the biologic and mechanical environment of a posterolateral intertransverse fusion site. In combination with bone marrow, it produces fusion rates that are comparable with those of autologous bone graft. However, it must be combined with an osteoinductive or osteogenic agent to ensure reliable fusion rates and alone cannot produce reliable osteogenesis. The Healos matrix was not compared with other commercially available matrices currently in use. Therefore, the efficacy of Healos relative to these other materials could not be determined.

Animals↗
PubMed2002

Matrix metalloproteinases and angiogenesis.

Angiogenesis is a prominent feature of numerous diseases, including cancer and arthritis, and appears to play an important role in kidney disease and hypertension. The matrix metalloproteinases, especially matrix metalloproteinase-2, play a vital role during angiogenesis by degrading the surrounding extracellular matrix and allowing endothelial cell invasion. Membrane type 1 matrix metalloproteinase directly degrades matrix components as well as activating matrix metalloproteinase-2 on the cell surface. The integrin receptors, particularly alpha(v)beta(3), can recruit and possibly activate matrix metalloproteinases to localized microdomains on the cell membrane. This restricts matrix metalloproteinase activity to the pericellular region, preventing excessive matrix degradation which would otherwise impede endothelial invasion. Inhibitors of matrix metalloproteinase activity may actually promote cell invasion by preventing uncontrolled matrix degradation. In addition to degrading the matrix, matrix metalloproteinases produce protein fragments that impede their angiogenic action. These multiple regulatory pathways permit fine control over cell invasion during angiogenesis and provide new, precise strategies for targeting abnormal angiogenesis, through control of matrix metalloproteinase activity.

Humans↗
PubMed1997

Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells.

Polymerization of soluble fibronectin into extracellular matrix fibers occurs through the interaction between the amino terminus of fibronectin contained within a 70 kDa fragment and 'matrix assembly sites' on the cell surface. The present studies were performed to localize the 'matrix assembly sites' (defined by 70 kDa binding sites) on newly adherent cells and on cells containing preformed fibronectin matrix. Matrix nucleation sites on newly spread cells were visualized using Texas Red conjugated 70 kDa fragment and were found to colocalize with vinculin and substrate fibronectin fibrils. Cells plated onto vitronectin coated coverslips did not exhibit any 70 kDa binding sites although these cells were well-spread with fully developed focal adhesions. Time course studies indicated that 70 kDa binding sites could be detected on newly adherent cells within 30-40 minutes following cell plating onto fibronectin coated coverslips, prior to the reorganization of substrate fibronectin into fibrils. Similarly, exogenous fibronectin conjugated with Texas Red was also colocalized with vinculin when added to newly adherent cells. The disruption of actin filaments with cytochalasin D both prevented the expression of 70 kDa binding sites and also resulted in the loss of established 70 kDa binding sites on newly spread cells. After 3 days in culture, cells organized an extensive fibronectin matrix and 70 kDa was colocalized with two distinct types of matrix fibronectin fibers: fine linear cell-associated fibers which co-stained with the beta1 integrin and coarse extracellular fibers which did not stain for the beta1 integrin. There was also a third type of fibronectin fiber which was organized into a meshwork structure. There was no localization of either beta1 or 70 kDa to these structures. Treatment of 3-day cells with cytochalasin D resulted in the disruption of cell-matrix fibers and cell-associated 70 kDa binding sites. In contrast, the coarse extracellular matrix fibers as well as the meshwork fibers were unaffected by cytochalasin. In the presence of cytochalasin D, 70 kDa bound to sites which colocalized with the coarse extracellular matrix fibers. These data suggest that de novo assembly of fibronectin matrix occurs at sites of focal adhesion and as fibronectin polymerization proceeds, matrix nucleation sites colocalize along cell associated fibronectin fibers. At later times 70 kDa is localized to a subset of more mature fibronectin-containing fibers. These results suggest that there are at least three morphologically distinct 70 kDa binding sites on adherent cells: one which colocalizes with beta1 to focal adhesions, a second which colocalizes with beta1 and fibronectin in matrix contacts, and a third which localizes to extracellular matrix fibers.

Actins↗
PubMed1992

Lanthanum tracer and freeze-fracture studies suggest that compartmentalisation of early bone matrix may be related to initial mineralisation.

In adult bone the calcified matrix and enclosed osteocytes are separated from the extracellular space by a continuous layer of bone lining cells. It thus appears that bone matrix is compartmentalised and, as such, may constitute a 'milieu intérieur' which is different from the general extracellular space. Since adult bone matrix is compartmentalised and matrix vesicles also form a microcompartment, it is conceivable that compartmentalisation, in early osteogenesis, may be a requirement for the initial events of the mineralisation process. We have therefore conducted an ultrastructural, tracer, and freeze-fracture study to determine the stage in which bone matrix becomes compartmentalised and also to find out whether there are tight junctions between osteoblasts. The results show that in early nonmineralised stages and in incipient mineralisation, lanthanum penetrates all intercellular spaces and the newly forming bone matrix which is rich in matrix vesicles and collagen. With the progression of mineralisation, when all matrix vesicles appear mineralised and calcification is 'spreading' to the surrounding matrix, lanthanum is restricted to intercellular spaces and conspicuous macular tight junctions are present between osteoblasts. We suggest that matrix vesicles act as microcompartments for calcification when the early bone matrix is in continuity with the surrounding extracellular space. In later stages, when lanthanum fails to penetrate the matrix, matrix vesicles may no longer be necessary because the bone matrix itself is compartmentalised, thus allowing for localised changes in composition that might favour mineral deposition.

Animals↗
PubMed1998

Association between ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 and local recurrence, metastasis, and survival in human chondrosarcoma.

Chondrosarcoma, a malignant cartilage-forming mesenchymal tumor, displays a wide range of clinical behavior that can be difficult to predict with histological analysis. Matrix metalloproteinases contribute to the processes of local invasion and metastasis by controlling the ability of a tumor to transverse tissue boundaries. The specificity of matrix metalloproteinase-1 (interstitial collagenase) for fibrillar collagen may be central to those processes. Matrix metalloproteinase-2 facilitates invasion by degradation of such basement-membrane structures as type-IV collagen. The balance between the activity of tissue inhibitors of metalloproteinase and the activity of matrix metalloproteinase determines the proteolytic activity and may, in part, determine the overall invasiveness and potential for metastasis. The measurement of the ratio of matrix metalloproteinase to tissue inhibitor of metalloproteinase may have prognostic value for determining whether individual chondrosarcomas are locally invasive or will metastasize. Furthermore, there may be a specific pattern of expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in chondrosarcomas that is related to local invasion and probability of metastasis. Sixteen paraffin-embedded archival specimens of tumors were examined. Six twenty-micrometer-thick sections were cut from each tumor, and the amounts of cDNA formed from the mRNA were determined with reverse transcription-polymerase chain reaction with use of novel primers for matrix metalloproteinase-1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2. The amounts of cDNA for the matrix metalloproteinases and their inhibitors were determined by chemiluminescence and band densitometry. The ratio of the amount of cDNA for matrix metalloproteinase-1 to that for its tissue inhibitor and the ratio of the amount of cDNA for matrix metalloproteinase-2 to that for its tissue inhibitor were calculated, and the results were compared with use of the Student t test, enabling log-rank analysis of Kaplan-Meier survival curves. These ratios as well as the age and gender of the patient; the grade, size, and location of the tumor; the type of adjuvant therapy; and the operative margins were examined for significance with use of stepwise logistic-regression analysis. The patients who had recurrent disease had a significantly higher (p < 0.003) ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 (mean, 0.939; range, 0.647 to 1.101) than the patients who were free of disease (mean, 0.703; range, 0.629 to 0.772). Moreover, there was a striking difference between the Kaplan-Meier survival curve associated with a high ratio (more than 0.8) and that associated with a low ratio (p = 0.0015). The mean ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 was 1.814 (range, 1.206 to 3.77) in the patients who had recurrent disease compared with 1.473 (range, 1.073 to 2.390) in those who were free of disease; this difference was not found to be significant, with the numbers available. Analysis of the survival curves indicated that a worse prognosis was associated with a high ratio, but again this relationship was not found to be significant. Regression analysis revealed that a high ratio of matrix metalloproteinase-1 to its tissue inhibitor was a moderately significant independent predictor of a poor outcome (alpha = 0.07).

Adult↗
PubMed2004

Decreased collagen-degrading activity could be a marker of prolonged mesangial matrix expansion.

BACKGROUND: Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion. METHODS: We prepared two rat models, showing reversible or prolonged mesangial matrix expansion, induced by a single injection or two consecutive injections of anti-Thy-1.1 monoclonal antibody 1-22-3, respectively. We analyzed the glomerular expression of type I and type IV collagens; MMP-2, -9, and -13; membrane type 1-MMP (MT1-MMP); TIMP-1; and urinary type I collagen-degrading activity in both models. RESULTS: There were no differences in glomerular mRNA levels of type I and type IV collagens between the reversible and the prolonged models. MMP-9 mRNA expression and protein level was lower in the prolonged model than in the reversible one, whereas there were no differences in mRNA levels of MMP-2, -13, MT1-MMP, or TIMP-1 between the two models. Urinary type I collagen-degrading activity in the prolonged model was lower than that in the reversible one. Furthermore, there was a significant correlation between the mesangial matrix expansion and urinary type I collagen-degrading activity. CONCLUSIONS: Impaired expression of MMP-9 may contribute to the development of prolonged mesangial matrix expansion. Analysis of urinary type I collagen-degrading activity may provide additional diagnostic information in mesangial proliferative glomerulonephritis. Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion.

Animals↗
PubMed2006

Tackling matrix effects during development of a liquid chromatographic-electrospray ionisation tandem mass spectrometric analysis of nine basic pharmaceuticals in aqueous environmental samples.

When developing an LC-MS/MS-method matrix effects are a major issue. The effect of co-eluting compounds arising from the matrix can result in signal enhancement or suppression. During method development much attention should be paid to diminish matrix effects as much as possible. The present work evaluates matrix effects from aqueous environmental samples in the simultaneous analysis of a group of nine specific pharmaceuticals with LC-ESI/MS/MS: flubendazole, propiconazole, pipamperone, cinnarizine, ketoconazole, miconazole, rabeprazole, itraconazole and domperidone. Solutions to diminish signal suppression were examined: optimisation of the sample preparation, decrease of the flow rate, and the use of appropriate internal standards. Several SPE-stationary phases were tested in view of retention of the analytes: Oasis HLB, C8, Phenyl, Strata X-polymer RP sorbent and Strata X-polymeric SCX/RP sorbent. Oasis HLB showed the best retention for all analytes. The Oasis HLB SPE-method was optimised, but analyses showed high matrix suppression. Therefore, a second SPE-method, on a phenyl stationary phase (the second best option), was also optimised. A comparison of the matrix effect was made between the two procedures: the phenyl-method was less subject to matrix effects, however, the average matrix effect (ME%) of 46% indicated that matrix effects where still present. Several optimisation options for the phenyl-method were evaluated: addition of a ferric nitrate solution before extraction, application of an alkaline wash step, and use of a second SPE-cartridge, either a NH2-column or a florisil column. A more efficient sample clean-up was achieved by applying the extract after extraction on the phenyl column and after dilution with chloroform, onto a NH2-column (average ME%: 53%). In addition, applying a post-column split (1:5), further reduced matrix effects (average ME%: 65%). Labelled internal standards are the best way to tackle matrix effects, but no such internal standards were commercially available for the analytes of interest. The thorough search and application of four internal standards (structural analogues) was beneficial and compensates the matrix effect partially (average ME%: 83%). In an attempt to reduce the analysis time Speedisk phenyl columns were applied. Under these conditions matrix effects decreased even more while recoveries were between 91 and 109%. Different kinds of surface water samples were analyzed, and different matrix effects were observed. For this reason, standard addition will be used to perform quantitative analysis.

Chromatography, Liquid↗
PubMed2003

The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria.

Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extramitochondrial free calcium, and finally activation of the permeability transition. Relationships between these phases, free and total matrix calcium concentration, and phosphate concentration are investigated in rat liver and brain mitochondria. Slow, continuous calcium infusion is employed to avoid transient bioenergetic consequences of bolus additions. Liver and brain mitochondria undergo permeability transitions at precise matrix calcium loads that are independent of infusion rate. Cytochrome c release precedes the permeability transition. Cyclosporin A enhances the loading capacity in the presence or absence of acetoacetate. A remarkably constant free matrix calcium concentration, in the range 1-5 microM as monitored by matrix-loaded fura2-FF, was observed when total matrix calcium was increased from 10 to at least 500 nmol of calcium/mg of protein. Increasing phosphate decreased both the free matrix calcium and the matrix calcium-loading capacity. Thus the permeability transition is not triggered by a critical matrix free calcium concentration. The rate of hydrogen peroxide detection by Amplex Red decreased during calcium infusion arguing against a role for oxidative stress in permeability pore activation in this model. A transition between a variable and buffered matrix free calcium concentration occurred at 10 nmol of total matrix calcium/mg protein. The solubility product of amorphous Ca3(PO4)2 is consistent with the observed matrix free calcium concentration, and the matrix pH is proposed to play the major role in maintaining the low matrix free calcium concentration.

Acetoacetates↗
PubMed1992

Cell maturation-specific autocrine/paracrine regulation of matrix vesicles.

Matrix vesicles are extracellular organelles produced with distinctive phospholipid composition and enzyme activity. They are produced by cells which typically calcify their extracellular matrix and their characteristics are cell-maturation dependent. Regulation of matrix vesicle structure and function occurs at the genomic and non-genomic levels. By following alkaline phosphatase gene transcription, protein concentration, and enzyme specific activity, we have shown that steroid hormones and growth factors exhibit a regulatory influence over gene transcription, protein synthesis, and matrix vesicle activity. Matrix vesicles respond to peptide hormones, other matrix proteins, like alpha 2-HS-glycoprotein, and autocoid mediators as well. Matrix vesicle metabolism can be directly affected by vitamin D metabolites, even in the absence of cells. The results indicate that 1,25-(OH)2D3(1,25D) or 24,25-(OH)2D3(24,25D) produced by the cells in culture can modulate matrix vesicle activity, and suggest that calcifying cells can modulate events in the matrix via autocrine/paracrine stimulation or inhibition of the matrix vesicles. 1,25D and 24,25D regulate matrix vesicle phospholipase A2 activity, fatty acid turnover, arachidonic acid release, PGE2 production and membrane fluidity, which act on the matrix vesicle to alter enzyme activity. Since vitamin D metabolite production is sensitive to both hormones and growth factors, there is potential for fine tuning matrix vesicle behavior.

Animals↗
PubMed2000

Increased concentration of pro-matrix metalloproteinase 9 in term fetal membranes overlying the cervix before labor: implications for membrane remodeling and rupture.

OBJECTIVES: Regional structural alterations that develop before labor are important in the mechanisms of both physiologic and pathologic membrane rupture, because they are also detected in preterm prelabor rupture of the fetal membranes, the most common cause of preterm birth (as great as 60%). Matrix metalloproteinases are located in the fetal membranes and are the main mediators of extracellular matrix degradation. The objective of this study was to examine whether gelatinases (matrix metalloproteinases 2 and 9) could be involved in the development of these regional structural changes seen at term before labor. STUDY DESIGN: Fetal membranes from patients undergoing elective cesarean delivery were regionally sampled from over the cervix (cervical membranes) and midway between this area and the placental edge (midzone). Fetal membranes obtained after spontaneous labor and delivery at term were also regionally sampled. Matrix metalloproteinase 2 and 9 activities were assessed by gelatin zymography, whereas total matrix metalloproteinase 9 protein was determined by enzyme-linked immunosorbent assay. RESULTS: Zymography only detected activity corresponding to the pro-matrix metalloproteinase 2 (72 kd) and 9 (92 kd) forms in prelabor fetal membranes. Although the levels of pro-matrix metalloproteinase 2 showed no regional differences, the pro-matrix metalloproteinase 9 level was higher in the cervical area than in the midzone (2.5 +/- 0.98 vs 0.76 +/- 0.28 optical density units/20 microg protein; P <.01). The concentration of pro-matrix metalloproteinase 9 protein in the cervical area was also significantly higher than that in the midzone (6.69 +/- 4.8 vs 1.58 +/- 1.14 ng/mg protein; P <.01). After delivery both pro-matrix metalloproteinase 2 and 9 activities were elevated, whereas pro-matrix metalloproteinase 9 protein activity showed no regional difference between the rupture site and midzone (23.47 +/- 4.5 vs 25. 3 +/- 6.2 ng/mg protein). Active bands of matrix metalloproteinases 2 (66 kd) and 9 (83 kd) were also detected after delivery. CONCLUSION: This study suggests that a specific regional induction of pro-matrix metalloproteinase 9 occurs in the cervical area before labor and may play a role in "programming" this area for subsequent rupture after activation during labor.

Amnion↗
PubMed2003

Angiogenesis: vascular remodeling of the extracellular matrix involves metalloproteinases.

Endothelial cell invasion is an essential event during angiogenesis (the formation of new blood vessels). This process involves the degradation of the extracellular matrix, the basement membrane, and interstitial stroma, and is governed by the activation of matrix metalloproteinases. However, the contribution of matrix metalloproteinases in angiogenesis is much more complicated. Tumor growth above a certain size is dependent on new vessels. A number of studies have demonstrated that treating tumors with matrix metalloproteinase inhibitors results in tumor reduction and a decrease in tumor angiogenesis. Matrix metalloproteinases as sole matrix eaters or degraders is a matter of the past. Not only tumor cells but more importantly bystander cells such as stromal cells produce matrix metalloproteinases. Matrix metalloproteinases therefore are also part of the pathologic microenvironment in different diseases. This enzymatic microenvironment dictates the endothelial cell fate, the angiogenic switch, and finally angiogenesis. During recent years, the role of matrix metalloproteinases has expanded, and their function as modulators of biologically active signaling molecules has drawn much attention. Depending on their substrate (growth factors or their receptors, extracellular matrix components, and angiogenic factors), matrix metalloproteinase activation results in the generation of proangiogenic or antiangiogenic factors. These data challenge the old concept that matrix metalloproteinases are simply proangiogenic. The knowledge of the local enzymatic profile and what, where, and how matrix metalloproteinases are involved in angiogenesis of tumors or other diseases will help design future therapeutic strategies better reflecting the complexity of the underlying biologic process of angiogenesis.

Animals↗
PubMed1985

Glycosaminoglycan production in cultures of early and late passage human endothelial cells: the influence of an anionic endothelial cell growth factor and the extracellular matrix.

An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL less than or equal to 20) and late (CPDL greater than 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on a matrix from early passage endothelial cells than when grown on matrix from late passage cells. We conclude that EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; early passage cells contain more GAG but less heparan sulfate than late passage cells, extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Adhesion↗
PubMed1991

Immunologically-related nucleic acid-binding proteins associated with the nuclear matrix of Physarum polycephalum.

The nuclear matrix of Physarum polycephalum is composed of two abundant polypeptides of 27 and 38 kDa as well as numerous minor polypeptides of various molecular weight. By contrast, the nuclear matrix of vertebrates consists of three major (the lamins) and many minor polypeptides mainly in the 60-70 kDa molecular weight range. In order to better characterize the major nuclear matrix proteins of P. polycephalum and, perhaps, define their relationship with the major nuclear matrix proteins of vertebrates, we have purified the abundant nuclear matrix proteins of P. polycephalum. In Western blot analyses, polyclonal antibodies raised against the purified 27 kDa polypeptide recognised polypeptides of 50 kDa, 45 kDa and several low molecular weight species (14-21 kDa) in the P. polycephalum nuclear matrix. The polyclonal antibodies did not react with the other abundant nuclear matrix protein of 38 kDa from P. polycephalum nor with polypeptides of the mouse nuclear matrix. Two-dimensional gel electrophoresis showed that the major nuclear matrix proteins of P. polycephalum were more basic than the major nuclear matrix proteins of vertebrates, the lamins. Moreover, both the 27 and 38 kDa polypeptides are post-translationally modified by either D-mannosyl or D-glycosyl moieties, and not by phosphorylation as has been demonstrated for the vertebrate lamins. DNA-binding assays further revealed that the immunologically related polypeptides of 50 kDa, 45 kDa, 27 kDa and low molecular weight species of 14-21 kDa preferentially bound single-stranded DNA, but the 38 kDa polypeptide of Physarum matrix did not. Based on these findings, we conclude that the abundant nuclear matrix protein of 27 kDa belongs to a group of immunologically-related nucleic acid-binding proteins, and is immunologically and functionally distinct from the other major nuclear matrix protein of 38 kDa from P. polycephalum and the vertebrate lamins.

Autoradiography↗
PubMed1994

Deposition and selective degradation of structurally-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro.

Collagen matrix deposition and turnover were studied in skin fibroblasts from a control and from a patient with lethal perinatal osteogenesis imperfecta (OI) identified as a Gly667 to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in the control fibroblast cell layer. In contrast, total collagen deposition by the OI fibroblasts was poor, with the quantity of collagen deposited only about a quarter of that of the control cells. Detailed analysis of the OI fibroblast matrix revealed that the mutant collagen chains were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts reproduced the abnormal matrix deposition pattern of OI tissues in vivo. The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cells was only slightly less than that of the control cells. The incorporation of the newly-synthesized OI collagen into the matrix was less efficient than in control cells, reflecting the cooperative nature of matrix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experiment and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proportion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indicated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composition fibers, or was unable to form copolymers efficiently and polymerized into mutant-only fibrillar assemblies more prone to proteolytic attack.

Collagen↗
PubMed2005

Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium.

Neovascularization may improve cardiac function and prevent further scar tissue formation in infarcted myocardium. A number of studies have demonstrated that bone marrow-derived cells have the potential to induce neovascularization in ischemic tissues. In this study, we hypothesized that implantation of bone marrow mononuclear cells (BMMNCs) using injectable fibrin matrix further enhances neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. To test this hypothesis, infarction was induced in rat myocardium by cryoinjury. Three weeks later, rat BMMNCs were mixed with fibrin matrix and injected into the infarcted myocardium. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Eight weeks after the treatments, histological analyses indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium compared to BMMNC implantation without matrix. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye prior to implantation survived in the infarcted myocardium at 8 weeks of implantation. Importantly, implantation of BMMNCs using fibrin matrix resulted in much more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. The microvessel density in infarcted myocardium was significantly higher (p < 0.05) when BMMNCs were implanted using fibrin matrix (350 +/- 22 microvessels/mm2) compared to BMMNC implantation without matrix (262 +/- 13 microvessels/mm2) and medium injection (76 +/- 9 microvessels/mm2). In addition, average internal diameter of microvessels was significantly larger (p < 0.05) in BMMNC implantation with fibrin matrix group (14.6 +/- 1.2 microm) than BMMNC implantation without matrix group (10.2 +/- 0.7 microm) and medium injection group (7.3 +/- 0.5 microm). These results suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy for myocardial infarction treatment.

Animals↗
PubMed2006

Temporal disparity in the induction of matrix metalloproteinases and tissue inhibitors of metalloproteinases after thoracic aortic aneurysm formation.

BACKGROUND: An important component of matrix remodeling during thoracic aortic aneurysm progression is the balance between matrix metalloproteinases and their endogenous inhibitors (tissue inhibitors of metalloproteinases). However, whether and to what degree matrix metalloproteinase/tissue inhibitor of metalloproteinases profiles change over time with an evolving thoracic aortic aneurysm remains unclear. METHODS: Descending thoracic aortic aneurysms were induced in mice (FVB strain, 15 minutes of 0.5 mol/L CaCl2 exposure) and followed for 24 hours, 72 hours, 1 week, 2 weeks, 4 weeks, or 8 weeks (each group, n = 13). Thoracic aortic aneurysm size was determined by means of video micrometry, and immunoblotting was used to measure aortic matrix metalloproteinase 2, 8, 9, and 12 and tissue inhibitor of metalloproteinases 1 and 4 levels (expressed as a percentage of control values, n = 13). RESULTS: Increased aortic diameter was detected by 72 hours and reached a maximal size at 4 weeks (135% +/- 4% increase from baseline, P < .05), which is consistent with thoracic aortic aneurysm progression. Active matrix metalloproteinase 8 (collagenase) levels increased at 72 hours (178% +/- 49%, P < .05 from control), and active matrix metalloproteinase 12 (elastase) levels increased by 24 hours (138% +/- 11%, P < .05), whereas active matrix metalloproteinase 2 levels increased at 72 hours and 1 week after thoracic aortic aneurysm induction (72 hours: 158% +/- 12%, 1 week: 162% +/- 19%; P < .05). At 1 week after thoracic aortic aneurysm induction, active matrix metalloproteinase 9 and 12 levels decrease (matrix metalloproteinase 9: 55% +/- 5%; matrix metalloproteinase 12: 63% +/- 5%; P < .05); however, matrix metalloproteinase 9 and 12 levels were increased from these values at 4 and 8 weeks (P < .05). Tissue inhibitor of metalloproteinases 1 levels were decreased at 1 week (52% +/- 15%, P < .05) and later returned to control values, whereas tissue inhibitor of metalloproteinases 4 levels increased at the late thoracic aortic aneurysm time points (4 weeks: 278% +/- 46%; 8 weeks: 213% +/- 40%; P < .05). CONCLUSIONS: These findings show 2 phases of matrix metalloproteinase abundance during murine thoracic aortic aneurysm formation. The late tissue inhibitor of metalloproteinases 4 increase might explain prevention of further aortic dilation past 4 weeks. Unique matrix metalloproteinase/tissue inhibitor of metalloproteinases temporal relationships occurred during the natural history of thoracic aortic aneurysm progression that might hold both diagnostic and therapeutic relevance.

Animals↗
PubMed1993

A mouse 3T6 fibroblast cell culture model for the study of normal and protein-engineered collagen synthesis and deposition into the extracellular matrix.

Mouse 3T6 fibroblasts deposited an organized collagenous extracellular matrix during long-term culture in the presence of ascorbic acid. The matrix produced by the cells had a similar distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III and V collagens. By day 8 of culture more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB3H4 into reducible crosslinks and aldehydes directly demonstrated the involvement of the alpha 1 (I)CB6 and alpha 2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesised collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable matrix present, 29% of the collagen synthesised was deposited, while by day 15, 88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix.

Aldehydes↗