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Beta1-integrins in the cartilage matrix.

Integrins are cell-surface receptors that mediate cell attachment to extracellular matrix components. The pericellular matrix in cartilage not only is a mechanical framework, but is also important for chondrocyte differentiation and stabilization of the phenotype. The interaction between chondrocytes and pericellular matrix is mediated, in part, by integrin receptors. We have previously demonstrated the presence of beta1-integrins in the cartilage matrix of organoid culture of limb buds from 12-day-old mouse embryos by immunohistological methods. In order to corroborate these findings, we have further investigated the distribution of integrins in the cartilage matrix by immunoelectron microscopy and by immunoprecipitation methods. Cartilage tissue of limb buds of 17-day-old mouse embryos was treated with collagenase and the cell-free and cellular protein-free supernatant was removed and used for immunoprecipitation experiments. Immunoprecipitation with antibodies against beta1-, alpha1-, alpha3-, and alpha5beta1-integrins and collagen type II, followed by immunoblotting with the same antibodies, demonstrated the presence of these integrins and collagen type II in the supernatant. The integrins found in the cartilage matrix could have been either secreted or shed by the cells. The question as to whether they have a function in the cartilage matrix, such as interlinking, in the matrix organization or in the stabilization of matrix components remains to be elucidated.

Animals↗

Experimental colonic obstruction increases collagen degradation by matrix metalloproteinases in the bowel wall.

PURPOSE: Emergency resections for colonic obstruction are accompanied with increased risk of anastomotic dehiscence. Elevated local degradation of submucosal collagens by matrix metalloproteinases may predispose to anastomotic leakage. This study was designed to study the effect of colon obstruction and surgical trauma on matrix metalloproteinase activities and correlate these results to collagen concentration in the colon wall. METHODS: Colonic obstruction was induced in male, Sprague-Dawley rats (n = 58) by applying a constricting silicone ring around the left colon 3 cm above the peritoneal reflection. After four days of obstruction, 2-mm wide colonic segments were resected approximately 3 mm proximal and 3 mm distal to the stenosis for biochemical analyses. Colonic segments at corresponding locations were obtained from sham-operated rats (n = 5) without obstruction but with silicone ring placed adjacent to colon and from normal, nontraumatized rats (n = 10). Matrix metalloproteinase activity was determined by liberation of fragmented collagens from homogenized colonic tissue incubated ex vivo. Matrix metalloproteinase-2 specifically was analyzed by gelatin zymography. RESULTS: Endogenous collagenolysis by matrix metalloproteinases increased (P < 0.001) in colon as a consequence of obstruction (4.1-fold) and trauma (1.7-fold) compared with normal colon. In the proximity of the colon stenosis, total matrix metalloproteinase activity and matrix metalloproteinase-2 were significantly (P < 0.05) higher above than below the obstruction. Total activity was 22.9 (13.1-32.9) units/mg collagen proximal and 16.6 (12.7-18.4) units/mg collagen distal to the stenosis. Collagen concentration correlated inversely (r = -0.76; P < 0.001) with total matrix metalloproteinase activity. CONCLUSION: Colonic obstruction and trauma up-regulated matrix metalloproteinases and decreased collagen concentration in colonic wall.

Animals↗

The local matrix distribution and the functional development of tissue engineered cartilage, a finite element study.

Assessment of the functionality of tissue engineered cartilage constructs is hampered by the lack of correlation between global measurements of extra cellular matrix constituents and the global mechanical properties. Based on patterns of matrix deposition around individual cells, it has been hypothesized previously, that mechanical functionality arises when contact occurs between zones of matrix associated with individual cells. The objective of this study is to determine whether the local distribution of newly synthesized extracellular matrix components contributes to the evolution of the mechanical properties of tissue engineered cartilage constructs. A computational homogenization approach was adopted, based on the concept of a periodic representative volume element. Local transport and immobilization of newly synthesized matrix components were described. Mechanical properties were taken dependent on the local matrix concentration and subsequently the global aggregate modulus and hydraulic permeability were derived. The transport parameters were varied to assess the effect of the evolving matrix distribution during culture. The results indicate that the overall stiffness and permeability are to a large extent insensitive to differences in local matrix distribution. This emphasizes the need for caution in the visual interpretation of tissue functionality from histology and underlines the importance of complementary measurements of the matrix's intrinsic molecular organization.

Biomechanical Phenomena↗

Fluoride binding by matrix proteins in rat mineralizing tissue.

Chronic fluoride exposure in vivo results in alterations in the formation of mineralizing tissues. One possible mechanism for the formation of fluorosed tooth enamel and bone is a binding of fluoride to matrix proteins, resulting in an alteration in their structure and function. Studies were designed to investigate fluoride binding to matrix proteins in vivo and their possible role in fluorosis. Rats were given either 0 or 100 parts/10(6) fluoride in drinking water for 6 weeks to allow the formation of fluorotic mineralizing tissues. The animals were killed by CO2 inhalation, and the enamel and bone were analysed for fluoride and calcium. Matrix binding by fluoride in enamel was determined after extraction of proteins from undemineralized matrix. In bone, the matrix was demineralized and F, Ca and P were determined in both ashed and unashed samples. The studies showed ionic binding of fluoride to the matrix in both enamel and bone, possibly associated with calcium binding by the matrix. There was no difference in the amount of matrix-bound fluoride in control as compared to fluorosed bone or maturation-stage enamel. This indicates that although matrix proteins can bind fluoride, it is not likely that this mechanism is important in the formation of fluorosed mineralizing tissues.

Animals↗

Interaction of lipoprotein lipase with subendothelial extracellular matrix.

We have analyzed the binding of lipoprotein lipase (LPL) to the subendothelial extracellular matrix produced by cultured endothelial cells. Binding was linear up to a concentration of 0.5 microgram/ml (10 nM) enzyme used in this study, and equilibrium was achieved after 2 h of incubation with bovine 125I-LPL at 4 degrees C. Heparin and heparan sulfate effectively inhibited the binding of LPL to extracellular-matrix-coated plates; chondroitin sulfate had no effect, while high concentrations of dermatan sulfate or keratan sulfate inhibited binding of LPL to extracellular matrix by only 40%. Basic fibroblast growth factor (bFGF) did not affect LPL binding, while antithrombin-III (AT-III) caused up to a 50% inhibition of enzyme binding to extracellular matrix. alpha-Thrombin. 5.10(-6) M, and its esterolytically inactive derivative, DIP-alpha-thrombin, effectively inhibited binding of LPL to extracellular-matrix-coated plates. alpha-Thrombin was also able to release the extracellular-matrix-bound LPL in an active form. Extracellular-matrix-bound LPL detached into medium containing triolein emulsion and/or serum, and was catalytically active after being released. Extracellular-matrix-bound LPL lost 30% of its activity following incubation at 37 degrees C for 4 h. in contrast to soluble LPL which lost 75% of its activity. It is plausible to conclude from these data that in vivo the subendothelial basement membrane, similarly to extracellular matrix, sequesters and stabilizers LPL secreted into the subendothelial space by non-endothelial cells, and thus may play an important role in determining the route of LPL from its site of synthesis to its site of action.

Animals↗

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices.

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Electroelution for purification of influenza A matrix protein for use in immunoassay.

A new preparative method for isolation of matrix protein from type A influenza virus was developed. Commercially available whole virus or split virus vaccines were lysed, and the soluble proteins separated by electrophoresis on polyacrylamide gel. The matrix protein was located on the gel by precipitation with KCl, and recovered by electroelution. The method was technically simple and required little direct supervision during the two-step recovery process. Yields of A matrix were consistently high, averaging 68.1% in five trials with A/Brazil/X-71. The method was also successful with other A viruses, although not with influenza B virus. Isolated A matrix had less than 0.5% contamination by hemagglutinin or nucleoprotein, as determined by immunoblotting and ELISA. Matrix protein was immunoreactive in Western blots and was detectable in concentrations as low as 1 ng/ml with ELISA. The isolated matrix provided a suitable standard for detection of matrix protein in nasal washes from patients with influenza A virus infection, and could also be used to detect anti-matrix antibodies, including monoclonal antibodies in tissue culture supernatants. The advantages of electroelution for separation of matrix protein compared to other methods were its technical simplicity, applicability to formalin-fixed influenza virus in commercially available vaccines, its consistently high yield, and its very high level of purification.

Antibodies, Monoclonal↗

Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta).

After demonstrating the presence of matrix vesicles in three osteosarcoma cell lines, MG-63, ROS 17/2.8 and MC-3T3-E1, we sought to determine whether two major enzymes localized to matrix vesicles, alkaline phosphatase and phospholipase A2, could be regulated by 1,25(OH)2D3 and/or TGF beta. Intravesicular calcification is probably dependent on these two enzymes. Alkaline phosphatase is essential for hydrolysis of phosphate-containing substrates and phospholipase A2 hydrolyzes diacylphosphatides in a calcium-mediated manner at lipid-aqueous interfaces leading to changes in membrane fluidity and possibly breakdown of the matrix vesicle. The 1,25(OH)2D3 induced increase of alkaline phosphatase in bone cells is localized to the matrix vesicle. TGF beta also increased alkaline phosphatase activity in two of the cell lines, MG-63 and ROS 17/2.8 but to a greater degree than 1,25(OH)2D3. Matrix vesicle alkaline phosphatase activity exhibited a greater response than that in the plasma membrane. TGF beta increased phospholipase A2 activity in both matrix vesicles and plasma membranes, therefore, no targeting was observed with respect to this enzyme. When TGF beta was combined with 1,25(OH)2D3, 1,25(OH)2D3 had no effect on phospholipase A2 and did not interfere with TGF beta stimulation of phospholipase A2 activity. When 1,25(OH)2D3 and TGF beta were combined, a tremendous synergy was observed in alkaline phosphatase specific activity in both plasma membranes and matrix vesicles with targeting to matrix vesicles. Therefore, TGF beta not only plays an important role in matrix formation and differentiation, but works in conjunction with 1,25(OH)2D3 to greatly potentiate the effects seen with 1,25(OH)2D3 alone.

Alkaline Phosphatase↗

The effects of various kinase and phosphatase inhibitors on the transmission of the prolactin and extracellular matrix signals to rabbit alpha S1-casein and transferrin genes.

In all species, milk protein genes are specifically expressed in the mammary gland under the control of lactogenic hormones and extracellular matrix. In rabbit, casein gene expression is induced by prolactin alone and this induction is amplified by extracellular matrix. Transferrin gene expression is induced by extracellular matrix in the absence of hormones. The transduction mechanisms of prolactin and extracellular matrix to milk protein genes is only partly known. The present study has been undertaken to determine if protein kinases and phosphatases are involved in these mechanisms. Rabbit primary mammary cells were cultured in three different conditions (i) directly on floating collagen I, (ii) on plastic after a trypsinization to remove endogenous extracellular matrix, and (iii) on floating collagen I after a trypsinization to restore a functional extracellular matrix. In these culture conditions, prolactin and several protein kinase and phosphatase inhibitors were added to the medium. The expression of alpha S1-casein and transferrin genes was evaluated using Northern blotting analysis. In cells cultured directly on collagen I, staurosporine, quercetin and 6-dimethylaminopurine strongly inhibited prolactin action of alpha S1-casein gene whereas herbimycin A was only partly inhibitory. An erbstatin analogue, tyrosine phosphate, 1(5 isoquinolylsulphonyl) 2-methylpiperazine and GF 109 203 X did not alter prolactin action. The inhibitors which inhibited prolactin action when cells were directly cultured on collagen I were also those which prevented the induction of alpha S1-casein gene expression when cells were cultured on plastic in the absence of extracellular matrix. The induction of transferrin gene by the extracellular matrix was inhibited slightly by quercetin. Okadaic acid, phenylarsine oxide and sodium pervanadate which inhibit Ser/Thr and Tyr phosphatase inhibitors were unable to mimic prolactin action on alpha S1-casein gene expression. On the contrary, these inhibitors prevented prolactin action. These data suggest that a cascade including protein kinases and phosphatases for Ser/Thr and Tyr phosphate is involved in the transduction of the prolactin message from its receptor to casein genes. The signal delivered to the mammary cells by the extracellular matrix is quite different, possibly involving another cascade of protein kinases.

Animals↗

Matrix metalloproteinase-8 is expressed in human chorion during labor.

OBJECTIVE: The purpose of this study was to investigate the expression of matrix metalloproteinase-8 by human fetal membranes during labor. STUDY DESIGN: Fetal membranes were obtained from women who underwent normal labor or elective cesarean delivery at term. Matrix metalloproteinase-8 levels in fetal membranes were determined by enzyme-linked immunosorbent assay and Western blot; the expression of the matrix metalloproteinase-8 gene was detected by reverse transcription-polymerase chain reaction. Immunohistochemistry and in situ hybridization was performed to localize matrix metalloproteinase-8 protein and messenger RNA in intact membranes. RESULTS: Matrix metalloproteinase-8 protein levels were increased 5-fold in fetal membranes from labor compared with membranes that were obtained from cesarean delivery. Western blots confirmed the presence of matrix metalloproteinase-8 in protein extracts. Reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry demonstrated that matrix metalloproteinase-8 messenger RNA and protein were expressed almost exclusively in the chorion after labor. CONCLUSION: We conclude that matrix metalloproteinase-8 is produced primarily by chorionic cells in human fetal membranes and that the level of matrix metalloproteinase-8 protein and messenger RNA expression in fetal membranes increases during labor.

Blotting, Western↗

Adenoviral-mediated expression and local deposition of recombinant tenascin-C perturbs cell-dependent matrix contraction.

BACKGROUND: To mimic the wound environment, we have developed a three-dimensional (3-D) fibrin-fibronectin (FN) matrix model that is formed in vitro from purified proteins and approximates the provisional matrix. Tenascin-C, a large extracellular matrix (ECM) glycoprotein, is expressed transiently in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. We have constructed a novel recombinant adenovirus vector (Ad-70Ten) to up-regulate local expression and secretion of a recombinant form of tenascin-C. METHODS: Ad-70Ten and a control vector were constructed and used to infect cultured mammalian cells. Post-infection monitoring of expression was accomplished by immunoblot and immunohistochemical techniques. Local protein deposition was examined by immunofluorescence. Cell contractility was assessed by ability of infected cells to contract 3-D fibrin-FN matrices. Some matrices also contained lysophosphatidic acid (LPA), an activator of Rho GTPase. RESULTS: Adenovirus-infected cells demonstrated high recombinant tenascin-C expression and deposited protein at sites of cell-matrix contacts resulting in significantly reduced contractility with 2.5-fold lower contraction of the matrix compared with control cells. Matrix contraction could be restored by treatment with LPA. CONCLUSION: These results show that endogenous expression of tenascin-C down-regulates cell contractility and strongly suggest that it exerts its effects via a Rho GTPase signaling pathway. Taken with previous findings, these results suggest that tenascin-C acts in both a paracrine and autocrine manner via Rho GTPase pathways. This report demonstrates that recombinant adenovirus infection is a feasible method to induce high expression of large matrix proteins in mammalian cells, allowing better approximation of in vivo circumstances for investigations of locally secreted matrix protein. While the current vector has been constructed for research purposes, it also represents a proof in principle that adenoviral vectors encoding large proteins may have potential benefit in clinical applications.

Adenoviridae↗

The cell clone ecology hypothesis and the cell fusion model of cancer progression and metastasis (II): three pathways for spontaneous cell-cell fusion and escape from the intercellular matrix.

The two-stage initiation-progression model of cancer is widely accepted. Initiation appears to result most often from accumulation of damage to the DNA expressed as multiple mutations in the phenotype. Unsymmetrical chromosome segregation during mitosis of normal or mutated cells produces aneuploid cells and also contributes to the evolution of neoplasia. However, it has been pointed out (Parris GE. Med Hypotheses 2005;65:993-4 and 2006;66:76-83) that DNA damage and loss of chromosomes are much more likely to lead the mutant clones of cells to extinction than to successful expansion (e.g., an example of Muller's Ratchet). It was argued that aneuploid neoplasia represent new parasite species that successfully evolve to devour their hosts by incorporating sex-like redistribution of chromosomes through spontaneous or virus-catalyzed cell-cell fusion into their life-cycle. Spontaneous cell-cell fusion is generally blocked by the intercellular matrix to which the cells are bound via surface adhesion molecules (frequently glycoproteins, e.g., CD44). In order for progression of matrix-contained neoplasia toward clinically significant cancer to occur, the parasite cells must escape from the matrix and fuse. Release from the matrix also allows the parasite cells to invade adjacent tissues and metastasize to remote locations. Both invasion and metastasis likely involve fusion of the migrating parasite cells with fusion-prone blast cells. There are at least three pathways through which parasite cells can be liberated from the confining matrix: (i) Their adhesion molecules may be modified (e.g., by hyper-glycosylation) so that they can no longer grip the matrix. (ii) Their adhesion molecules or matrix may be saturated with other ligands (e.g., polyamines). (iii) Their adhesion molecules may be cleaved from the cell surface or the matrix itself may be cleaved (e.g., by MMPs or ADAMs). It is hypothesized that mobilization of parasite cells and cell-cell fusion go hand-in-hand in the progression of neoplasia to clinically significant cancer through invasion and metastasis. The latency between tumor recognition and exposure to mutagens and the increased incidence of cancer with age can probably be related to slow breakdown of the intercellular matrix that provides a barrier to cell-cell fusion.

Animals↗

Destruction of extracellular matrix proteoglycans is pervasive in simian retroviral neuroinfection.

Disruption of the perineuronal matrix has been reported in human immunodeficiency virus (HIV) encephalitis. To better understand the extent of matrix disruption during lentiviral encephalitis, we characterized the extracellular matrix (ECM) damage in brains of 12 macaques infected with simian immunodeficiency virus (SIV). Matrix integrity was assessed by Wisteria floribunda lectin histochemistry. Confocal microscopy was used to quantify matrix loss, macrophage infiltration, and synaptic damage. Disruption of brain ECM was present shortly after retroviral infection, preceding parenchymal macrophage infiltration. In agreement with previous observations, reduced staining of presynaptic and postsynaptic proteins in SIV encephalitis occurred concurrently with matrix abnormalities. Lentiviral infection induced microglial and macrophage expression of two disintegrins and metalloproteinases with thrombospondin motifs (ADAMTS-1 and ADAMTS-4), with high substrate specificity for matrix proteoglycans. Matrix damage is pervasive during SIV neuroinfection, which suggests interventions to conserve brain matrix proteoglycans might avert or delay retroviral-induced neurodegeneration.

ADAM Proteins↗

A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes.

OBJECTIVES: Preterm premature rupture of fetal membranes is responsible for 30% to 40% of preterm deliveries. Fetal membranes are composed primarily of collagen. Matrix metalloproteinases are enzymes capable of degrading extracellular matrix macromolecules, including collagens. Expression of matrix metalloproteinase-9 (gelatinase B, 92 kd) and its tissue inhibitor (tissue inhibitor of metalloproteinase-1) has been localized in amnion and chorion. The objective of this study was to determine whether rupture of fetal membranes and intrauterine infection are associated with changes in the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1. STUDY DESIGN: Two hundred one women in the following categories had amniotic fluid retrieved: (1) preterm labor and intact membranes in the presence (n = 42) or absence (n = 21) of microbial invasion of the amniotic cavity, (2) preterm premature rupture of the membranes with (n = 29) or without (n = 23) microbial invasion of the amniotic cavity, and (3) term gestation with intact membranes (n = 50) or with premature rupture of the membranes (n = 40). Women in groups 1 and 2 were matched for gestational age at amniocentesis. Microbial invasion of the amniotic cavity was defined by a positive amniotic fluid culture for micro-organisms. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 were measured with use of sensitive and specific immunoassays that were validated for amniotic fluid. RESULTS: Spontaneous rupture of membranes at term is associated with a significant increase in the amniotic fluid concentrations of matrix metalloproteinase-9 (premature rupture of membranes, no labor: median 3.9 ng/mL, range 2. 7 to 11.1 ng/mL vs no premature rupture of membranes, no labor: median <0.4 ng/mL, range <0.4 to 22.4 ng/mL; P <.001). Patients with preterm premature rupture of the membranes had higher median matrix metalloproteinase-9 concentrations than those with preterm labor and intact membranes who were delivered at term (7.6 ng/mL, range <0.4 to 230.81 ng/mL vs <0.4 ng/mL, range <0.4 to 1650 ng/mL; P =.06). Women with microbial invasion of the amniotic cavity had higher median matrix metalloproteinase-9 concentrations than did those without microbial invasion regardless of membrane status (preterm labor: 54.5 ng/mL, range <0.4 to 3910 ng/mL vs <0.4 ng/mL, range <0. 4 to 1650 ng/mL; P <.01; preterm premature rupture of membranes: 179. 8 ng/mL, range <0.4 to 611 ng/mL vs 7.6 ng/mL, range <0.4 to 230.81; P <.001). CONCLUSION: Our data support a role for matrix metalloproteinase-9 in the mechanisms responsible for membrane rupture in term and preterm gestations.

Amnion↗

Extracellular matrix proteoglycans and cell-substratum adhesion of human endothelial cells: the effect of methyl beta-D-xylopyranoside.

The influence of methyl beta-D-xylopyranoside on human endothelial cell proteoglycans isolated from the medium and extracellular matrix was investigated. Confluent cultures of human endothelial cells incorporate significant amounts of heparan sulfate (78%), chondroitin sulfate (10%), and dermatan sulfate (12%) into the extracellular matrix. Chondroitin sulfate (35%) and dermatan sulfate (37%) were the major glycosaminoglycans present in the medium. In the presence of methyl beta-D-xylopyranoside, incorporation of labeled proteoglycans into extracellular matrix was diminished by approximately 70%. Heparan sulfate comprised the major proteoglycan present in extracellular matrix (89%) in cells grown in the presence of methyl beta-D-xylopyranoside. In contrast to the incorporation of proteoglycan into extracellular matrix, methyl beta-D-xylopyranoside stimulated the secretion of labeled glycosaminoglycan chains into the medium 2.5-fold. In the presence of methyl beta-D-xylopyranoside, secretion of chondroitin sulfate into the medium was markedly stimulated, with a slight increase in secretion of heparan sulfate. Chondroitin sulfate (62%) and heparan sulfate (34%) were the major labeled glycosaminoglycans present in medium from methyl beta-D-xylopyranoside-treated cultures. The effect of methyl beta-D-xylopyranoside on cell adhesion and detachment was investigated. Cell detachment from extracellular matrix depleted of proteoglycan was significantly faster than detachment from normal matrix. Conversely, human endothelial cells adhered faster to normal matrix than to matrix depleted of proteoglycan.

Cell Adhesion↗

The insulator binding protein CTCF associates with the nuclear matrix.

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.

Amino Acid Sequence↗

Sinusoidal obstruction syndrome (veno-occlusive disease) in the rat is prevented by matrix metalloproteinase inhibition.

BACKGROUND & AIMS: The mechanical origins of the obstruction in sinusoidal obstruction syndrome are initiated by dehiscence of sinusoidal endothelial cells from the space of Disse. The biochemical changes that permit the dehiscence of the sinusoidal endothelial cells were investigated. METHODS: In vitro and in vivo studies examined changes induced by monocrotaline, a pyrrolizidine alkaloid that induces sinusoidal obstruction syndrome in both humans and experimental animals. RESULTS: In the monocrotaline-induced rat model of sinusoidal obstruction syndrome, there was an early increase of matrix metalloproteinase-9 and a later, lower-magnitude increase of matrix metalloproteinase-2 in the liver. In vitro studies of sinusoidal endothelial cells, hepatocytes, stellate cells, and Kupffer cells showed that sinusoidal endothelial cells are the major source of both basal and monocrotaline-induced matrix metalloproteinase-9/matrix metalloproteinase-2 activity. Monocrotaline caused depolymerization of F-actin in sinusoidal endothelial cells, and blocking of F-actin depolymerization prevented the increase in matrix metalloproteinase activity. Administration of matrix metalloproteinase inhibitors prevented the signs and histological changes associated with sinusoidal obstruction syndrome. CONCLUSIONS: Monocrotaline causes depolymerization of F-actin in sinusoidal endothelial cells, which leads to increased expression of metalloproteinase-9 and matrix metalloproteinase-2 by sinusoidal endothelial cells. Inhibition of matrix metalloproteinase-9 and matrix metalloproteinase-2 prevents the development of sinusoidal obstruction syndrome, establishing that matrix metalloproteinase inhibitors may be a therapeutically viable strategy for prevention.

Actins↗

Amniotic fluid matrix metalloproteinase-9 and interleukin-6 in predicting intra-amniotic infection.

OBJECTIVE: To assess the potential role of amniotic fluid (AF) matrix metalloproteinase-9 and interleukin-6 in predicting intra-amniotic infection. METHODS: Eighty-four women with singleton gestations with preterm contraction, preterm labor, preterm premature rupture of membranes, or clinical suspicion of intra-amniotic infection were studied. Amniotic fluid was obtained by transabdominal amniocentesis before starting any treatment. Intra-amniotic infection was defined as the presence of a positive AF culture. Amniotic fluid glucose concentration, leukocytes, matrix metalloproteinase-9, and interleukin-6 were determined. RESULTS: Amniotic fluid matrix metalloproteinase-9 and interleukin-6 levels were significantly higher in women with intra-amniotic infection than in those without. With intra-amniotic infection, levels of matrix metalloproteinase-9 significantly correlated with interleukin-6 (r = 0.813, P <.001). Each of matrix metalloproteinase-9 and interleukin-6 significantly correlated with AF leukocytes and inversely correlated with AF glucose. Using AF cutoff levels of 13.6 ng/mL for matrix metalloproteinase-9 and 11.4 ng/mL for interleukin-6, the sensitivity, specificity, and positive and negative predictive values for diagnosing intra-amniotic infection were 77% versus 73%, 100% versus 79%, 100% versus 61%, and 90% versus 86%, respectively. Combining AF matrix metalloproteinase-9 with interleukin-6 slightly improved the sensitivity and the negative predictive values in diagnosing intra-amniotic infection. CONCLUSIONS: Amniotic fluid matrix metalloproteinase-9 and interleukin-6 are significantly elevated in women with intra-amniotic infection. Amniotic fluid matrix metalloproteinase-9 is an accurate biochemical marker in predicting intra-amniotic infection with better sensitivity, specificity, and positive and negative predictive values than interleukin-6.

Adolescent↗