Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Vitronectin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMed2005

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion.

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.

Cell Movement↗
PubMed2004

Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies.

Cell shapes induced by cell-substratum interactions are linked with proliferation, differentiation or apoptosis of cells. To clarify the relevance of specific surface characteristics, we applied self-assembled monolayers (SAM) of alkyl silanes exhibiting a variety of terminating functional groups. We first characterised the SAMs on glass or silicon wafers by measuring wettability, layer thickness and roughness. Water contact angle data revealed that methyl (CH(3)), bromine (Br), and vinyl (CH=CH(2)) groups lead to hydrophobic surfaces, while amine (NH(2)) and carboxyl (COOH) functions lead to moderately wettable surfaces, and polyethylene glycol (PEG) and hydroxyl (OH) groups created wettable substrata. The surfaces were found to be molecular smooth except for one type of NH(2) surface. The SDS-PAGE analysis of proteins adsorbed from bovine serum to the SAMs showed less protein adsorption to PEG and OH than to CH(3), NH(2) and COOH. Immunoblotting revealed that a key component of adsorbed proteins is vitronectin while fibronectin was not detectable. The interaction of human fibroblasts with CH(3), PEG and OH terminated SAMs was similarly weak while strong attachment, spreading, fibronectin matrix formation and growth were observed on COOH and NH(2). The strong interaction of fibroblasts with the latter SAMs was linked to an enhanced activity of integrins as observed after antibody-tagging of living cells.

Adsorption↗
PubMed2005

The enhancement of recombinant protein production by polymer nanospheres in cell suspension culture.

Recombinant Chinese hamster ovary (rCHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins. Three-dimensional suspension culture is preferred to two-dimensional monolayer culture for the efficient large-scale culture of rCHO cells and subsequent mass production of recombinant proteins. Previously, we have demonstrated that the use of plain polymer nanospheres enhances the growth of anchorage-dependent animal cells (human embryonic kidney 293 cells) in suspension culture in serum-containing medium. Vitronectin and fibronectin were adsorbed onto poly(lactic-co-glycolic acid) (PLGA) nanospheres (696 nm in average diameter) by immersing the nanospheres in fetal bovine serum. In this study, we investigated if the use of vitronectin/fibronectin-adsorbed polymer nanospheres enhances recombinant protein production in rCHO cell suspension culture in serum-free medium. Cell aggregate formation may be critical for the survival and growth of anchorage-dependent animal cells in suspension culture, and cells in single cell suspension may result in cell death. The addition of vitronectin/fibronectin-adsorbed nanospheres to rCHO cell suspension culture promoted the rate and efficiency of cell aggregate formation. The nanospheres enhanced cell growth (2.9 folds on day 10) and, importantly, recombinant antibody production (1.8 folds on day 14), compared to suspension culture without nanospheres. The viability of cells in the aggregates in the nanosphere-added culture was high for the entire culture period of 14 days. Apoptotic activity of cells was much lower in the nanosphere-added culture than in the culture without nanospheres on day 5. The nanosphere suspension culture method developed in this study may be useful for the mass production of recombinant proteins through large-scale suspension culture of anchorage-dependent animal cells.

Adsorption↗
PubMed2005

Conformational analysis of heparin binding peptides.

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339-364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

Amino Acid Sequence↗
PubMed2006

Adhesion of eukaryotic cell lines on the gold surface modified with extracellular matrix proteins monitored by the piezoelectric sensor.

The piezoelectric sensor (quartz crystal microbalance, QCM) was used to monitor cell adhesion in real time. Two cell lines, rat epithelial cells (WB F344) and lung melanoma cells (B16F10) were used. The cells were adhered and grown on the gold surface of the sensor pre-coated with adsorbed layer of extracellular matrix proteins as vitronectin and laminin. The process of cell attachment and spreading on the gold surface was continuously monitored and displayed by changes of the resonant frequency Deltaf and resistance DeltaR values of the piezoelectric resonators. The initial phase of cell attachment and spreading induced a decrease of frequency and increase of resistance relating viscoelastic properties of the cell monolayer on the sensing surface. The steady-state of both shifts was achieved after a few hours. The presence and state of cells on the surface was confirmed by fluorescent microscopy. The obtained results demonstrate that the piezoelectric sensor is suitable for studies of the cell adhesion processes. Thus obtained cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment.

Animals↗
PubMed2005

Expression of urokinase-type plasminogen activator receptor is correlated with metastases of lingual squamous cell carcinoma.

Lingual squamous cell carcinoma is common and the survival rate is relatively low. The invasion of cancer cells from the primary tumour into the surrounding tissue is an early step in the process of metastasis and urokinase-type plasminogen activator receptor (uPAR) is a vital mediator of cellular migration in some carcinomas. By binding urokinase-type plasminogen activator, uPAR localises proteolytic activity to the leading edge of the cells, thereby facilitating cellular migration and penetration through tissue boundaries. uPAR also binds directly to vitronectin and associates with integrins within the plasma membrane, which alters the strength of cellular adhesion. In this study we used reverse transcription polymerase chain reaction, immunocytochemistry, and Western-blot to examine the expression of uPAR mRNA and protein in Ts and Tca 8113 cell lines of lingual squamous cell carcinoma and in normal oral mucosal cells. uPAR mRNA and protein were expressed in Ts cells, but not in Tca 8113 cells or in normal oral epithelial cells. Ts cells have higher metastatic potential than Tca 8113 cells. The results suggest that uPAR has an important role in the aggressiveness of lingual squamous cell carcinoma.

Blotting, Western↗
PubMed2006

A conjugate of camptothecin and a somatostatin analog against prostate cancer cell invasion via a possible signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9.

Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.

Amino Acid Sequence↗
PubMed2006

Identification of novel short chain 4-substituted indoles as potent alphavbeta3 antagonist using structure-based drug design.

The vitronectin receptor alpha(v)beta(3) has been identified as a promising potential target for the treatment of osteoporosis, diabetic retinopathy and cancer. We have recently reported 5-substituted indoles 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3-pyridyl)propionic acid 3 and 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3,4-methylenedioxyphenyl)propionic acid 4, as an original series of potent alpha(v)beta(3) antagonists with subnanomolar activity. Ligand-protein docking analyses have been performed to generate binding models of three different chemical classes of known alpha(v)beta(3) antagonists with alpha(v)beta(3). Results of this docking study suggested that indoles bearing the basic tetrahydronaphthyridine group at position 4 can easily adopt the correct binding conformation and should be as potent as our current 5-substituted indole leads 3 and 4. This hypothesis was nicely demonstrated by the synthesis of a series of 1,4-disubstituted indoles through a tandem of reactions involving: (i) the N-alkylation of indoles 15 and 22 with propargyl esters and cesium fluoride, and (ii) a Heck coupling reaction between 4-bromoindole and 7-vinyl-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid tert-butyl ester 12, or (iii) a reductive amination involving the N-substituted-4-aminoindole 23 and the BOC-protected tetrahydro[1,8]naphthyridine aldehyde 13. Among the compounds assayed, 3-(3-pyridyl)-3-[4-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethyl]indol-1-yl]propionic acid 21 showed the most promising activity on alpha(v)beta(3) (IC(50)=0.5 nM), and was found to have the same potency as our current leads 3 and 4, while maintaining selectivity over alpha(IIb)beta(IIIa). Moreover, based on the reasonable apparent permeability coefficient in an in vitro CACO-2 cell monolayer assay (P(app) apical/basolateral=2.2 x 10(-6)cm/s, P(app) basolateral/apical=2.5 x 10(-6)cm/s), compound 21 is expected to be absorbed through the intestine in human. Thus, 1,4-disubstituted indole 21 represents a new lead for this novel class of conformationally restricted alpha(v)beta(3) antagonists. Additionally, this study validates the pharmacophore model previously postulated and provides an improved basis for further structure-based drug design in the field of alpha(v)beta(3).

Drug Design↗
PubMed2005

GPIIb (CD41) integrin is expressed on mast cells and influences their adhesion properties.

OBJECTIVES: GPIIb integrin expression has been found on platelets and megakaryocytes, and more recently on immature hematopoietic progenitors. We set out to investigate expression of GPIIb in other hematopoietic cell lineages and, having detected it on mast cells, aimed to determine what possible role it might perform. METHODS: We have made use of cultured human and murine bone marrow mast cells (BMMC) in order to characterize the expression of GPIIb. Further, BMMC cultures from wild type and GPIIb deficient (gpIIb-/-) mice were used for comparison of the adhesive properties mediated by this receptor. Finally, peritoneal mast cells were analyzed from both wild type and (gpIIb-/-) mice. RESULTS: We demonstrate expression of GPIIb on cultured BMMC. Using cells derived from mice homozygous for a null allele of gbIIb we show that the absence of GPIIb has no effect on mast cells with respect to a number of measures of cell growth and differentiation. However, loss of GPIIb on BMMC results in an increase in surface expression of aV integrin, the alternative partner of GPIIIa. CONCLUSION: The results in this study demonstrate that GPIIb is expressed in human and murine mast cells. A function for GPIIb on mast cells is suggested by the altered adhesion of gbIIb-/- BMMC to fibronectin- and vitronectin-coated surfaces. Moreover, comparison of mast cells from the peritoneal cavity of wild type and gbIIb-/- mice indicates that GPIIb could influence the in vivo differentiation or homing of tissue mast cells.

Animals↗
PubMed2005

The role of subchondral bone resorption pits in osteoarthritis: MMP production by cells derived from bone marrow.

OBJECTIVE: The vascular invasion of bone marrow tissue into the subchondral plate is often observed in articular cartilage and we named it the subchondral bone absorption pit; however, its implication in the pathogenesis of osteoarthritis (OA) has been poorly understood. The purpose of this study was to evaluate its characteristics and roles in osteoarthritic conditions. METHODS: Articular cartilage specimens from 11 patients with medial type knee OA and 7 non-arthritic cadavers were analyzed with HE staining. OA sections were stained with safranin-O, TRAP (tartrate resistant acid phosphatase) and immunostained with anti-MMP-1, MMP-3, MMP-13, vitronectin receptor (VNR)-alpha chain, vimentin and bone morphogenic protein (BMP) 2/4 antibodies. RESULTS: Subchondral bone resorption pits were classified according to the extent of invasion: pits with bone marrow tissue were located within uncalcified cartilage below the tidemark in grade I and invaded beyond the tidemark in grade II, while no invasion was seen in grade 0. Grade II pits were dominant in OA compared to non-arthritic joints, especially medial condyles. Proteoglycan detected with safranin-O staining was lost around the tip of grade II pits and the density of pits was related to the modified Mankin Score. Cells in pits expressed vimentin, MMP-1, MMP-3 and MMP-13. Some polynuclear cells co-expressed VNR-alpha chain and MMP-13, whereas pits showed reparative features expressing BMP. CONCLUSION: These results suggest that subchondral bone resorption pits contribute to cartilage degradation by expressing matrix metalloproteinases in OA.

Aged↗
PubMed2004

The basement membrane protein laminin-5 acts as a soluble cell motility factor.

One of the basement membrane (BM) proteins, laminin-5 (LN5), is known to support efficient cell adhesion and migration through interaction with integrins on the basal plasma membrane. Here, we show that a soluble form of LN5 induced migration of human epithelial cells and carcinoma cells by interacting with integrins on the apical cell surface. Although both LN5 and laminin-10/11 (LN10/11) promoted cell migration when coated onto a plastic surface as insoluble substrata, only LN5 stimulated cell migration in its soluble form on other substrata such as fibronectin (FN), vitronectin (VN) and collagen. Soluble LN5 interacted with integrins alpha3beta1 and alpha6beta1 on the apical cell surface and stimulated cell migration, while the cell morphology was largely dependent on the underlying substratum. Thus, integrin signals from the apical surface and the basal surface synergistically regulated cytoskeletal organization and cell motility. Soluble and insoluble LN5 induced cell motility by activating signal pathways via protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K) and MAP kinase. The PKC dependency was more prominent for soluble LN5 than insoluble LN5, and was absent in the stimulation by insoluble LN10/11. In vitro scratch assays with keratinocytes, self-produced soluble LN5 bound to the apical cell surface of migrating cells at the scratched edges, suggesting that soluble LN5 may contribute to cell migration in pathological conditions such as wound healing and tumor invasion.

Animals↗
PubMed2006

Carboxypeptidase cathepsin X mediates beta2-integrin-dependent adhesion of differentiated U-937 cells.

Cathepsin X is a lysosomal carboxypeptidase with a potential role in processes of inflammation and immune response. The integrin-binding motifs RGD and ECD, present in the pro- and in mature forms of cathepsin X, respectively, suggest that this enzyme might have a function in cell signaling and adhesion. In this study, we report that cysteine protease inhibitors E-64 and CA-074 and 2F12 monoclonal antibody, all of which inhibit cathepsin X activity, significantly reduced adhesion of differentiated U-937 cells to polystyrene- and fibrinogen-coated surfaces via Mac-1 integrin receptor, whereas their binding to vitronectin, fibronectin or Matrigel was not affected. On the other hand, cathepsin X, added to differentiating U-937 cells, stimulated their adhesion. Using confocal microscopy, we demonstrated that the pro-form of cathepsin X was co-localized with beta(2) and beta(3) integrin subunits and its mature form solely with the beta(2) integrin subunit with the most intense signal in cell-cell junctions in differentiated U-937 cells and in co-cultures with endothelial cells. Our results indicate that active cathepsin X mediates the function of beta(2) integrin receptors during cell adhesion and that it could also be involved in other processes associated with beta(2) integrin receptors such as phagocytosis and T cell activation.

Antibodies, Monoclonal↗
PubMed2006

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells.

The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.

Base Sequence↗
PubMed2005

Targeting ECM-integrin interaction with liposome-encapsulated small interfering RNAs inhibits the growth of human prostate cancer in a bone xenograft imaging model.

The intricate intracellular communication between stromal and epithelial cells, which involves cell-cell-, cell-insoluble extracellular matrix- (ECM), and cell-soluble factor-mediated signaling processes, is an attractive target for therapeutic intervention in hormone-refractory and bone-metastatic prostate cancer. In the present study we demonstrated that androgen-independent PC3 prostate cancer cells adhered to and migrated on vitronectin (VN), a major noncollagenous ECM in mature bone, through the expression of alphav-containing integrin receptors alphavbeta1 and alphavbeta5 on the cell surface, as determined by antibody function blocking assay and flow cytometry analysis. Small interfering RNAs (siRNAs) targeting human integrin alphav markedly reduced their respective mRNA and protein expression in cells, resulting in nearly complete reduction in VN-mediated cancer progression in vitro. In vivo quantitative bioluminescence analysis of human prostate cancer bone xenografts demonstrated for the first time that intratumoral administration of liposome-encapsulated human alphav-siRNAs significantly inhibits the growth of luciferase-tagged PC3 tumors in skeleton, which was associated with decreased integrin alphav expression and increased apoptosis in tumor cells. This integrin-based gene therapy is particularly suitable for the treatment of prostate cancer bone metastasis.

Animals↗
PubMed2005

New multifunctional molecular conjugate vector for targeting, imaging, and therapy of tumors.

We report the in vitro and in vivo characteristics of a new molecular conjugate vector for targeting and imaging of tumors. Its core is a cyclodecapeptide platform named RAFT, onto which two spatially independent functional domains can be covalently and stereospecifically linked: a cell-targeting domain for tumor targeting and a labeling domain able to carry two drugs and/or labeling agents. To prove the interest of this carrier, we used a well-known cRGD cyclopeptide, a ligand for the alphavbeta3 integrin. We demonstrate that this vector presenting four cRGD motifs very efficiently prevents alphavbeta3-mediated cell adhesion to vitronectin. Furthermore, it is actively endocytosed because of the multivalent cRGD presentation, a major advantage for drug delivery. In vivo experiments in nude mice reveal that repeated intratumoral injections of low doses of RAFT(cRGD)4 reduce tumor growth. Furthermore, RAFT(cRGD)4 significantly improves the targeting specificity of subcutaneous tumor masses as well as that of disseminated metastasis after intravenous injection. Thus, RAFT(cRGD)4 is specific, internalized, and perfectly controlled and can carry multiple biological functions on a single, spatially defined backbone, making it a powerful and versatile synthetic vector for drug delivery, molecular imaging, or both.

Animals↗
PubMed2000

IIb's are not IIb's.

Platelet glycoprotein (GP) IIb/IIIa receptor blockade improves clinical outcomes after percutaneous coronary intervention (PCI) and for patients who present with non-ST-segment elevation acute coronary syndromes. Although this class of therapeutic agents has been defined by a common affinity for the platelet GP IIb/IIIa receptor, the 3 currently available agents differ markedly in pharmacodynamic and pharmacokinetic profile as well as receptor affinity. Differential (separate) binding sites on the GP IIb/IIIa receptor explain the observation that abciximab binding to platelets is not influenced by either tirofiban or eptifibatide. Abciximab (ReoPro, chimeric 7E3 Fab) is a low K(d) (high affinity) agent with a very short plasma t(1/2) and a prolonged duration of action at the platelet target receptor. Eptifibatide and tirofiban are high K(d) (low affinity) agents with a relatively long plasma t(1/2) and short duration of action at the platelet target receptor. These pharmacodynamic differences underlie the phenomena of gradual redistribution in abciximab binding and smooth tapering of abciximab antiplatelet effect after discontinuation of therapy. Furthermore, abciximab demonstrates affinity for both the CD11b/18 (alpha(m)beta(2) or MAC 1) and alpha(V)beta(3) (vitronectin) receptors. Although a survival advantage in favor of abciximab has been observed after PCI in both randomized controlled trials and high-volume clinical practice, no survival benefit has been observed to date after eptifibatide or tirofiban therapy for PCI. The mechanism of survival advantage after abciximab therapy has not been defined but may be distinct from the degree of platelet GP IIb/IIIa receptor inhibition during the duration of intravenous treatment. Although this important new "class" of therapeutic agent was simplistically defined by a common affinity for the GP IIb/IIIa receptor, this solitary unifying attribute may not define agent-specific benefit.

Abciximab↗
PubMed1999

Beta3-integrin-mediated focal adhesion complex formation: adult cardiocytes embedded in three-dimensional polymer matrices.

In vivo studies show that beta3-integrin-mediated focal adhesion formation (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskeleton in pressure-overloaded myocardium. To define the mechanism of beta3-integrin-mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta3-integrin-mediated focal adhesion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultured in a 3-dimensional matrix formed focal adhesions consisting of both beta3-integrin and the muscle-specific isoform, beta1-integrin (beta1D). The focal adhesions were associated with focal adhesion kinase on both costameres and intercalated disks. To determine the cause of beta1D-integrin-mediated focal adhesion kinase in this model, time course studies were done. Beta3-integrin-mediated focal adhesion kinase occurred within 30 minutes after embedding cardiocytes and persisted for >24 hours, whereas beta1D-integrin-mediated focal adhesion kinase was present from the outset. Because confocal microscopy showed that laminin was present on the surface of freshly isolated cardiocytes, we hypothesized that this was causative of beta1D-integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then embedded in a 3-dimensional matrix showed complete absence of beta1D-integrin-mediated focal adhesion kinase, but beta3-integrin-mediated focal adhesion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of beta1D-integrin, because acid-washed cardiocytes in the presence of laminin showed beta1D-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta3-integrin-mediated focal adhesion kinase and provide an in vitro model to study beta3-integrin-mediated signaling in response to hemodynamic cardiac loading.

Animals↗
PubMed2001

Coordinated induction of extracellular proteolysis systems during experimental autoimmune encephalomyelitis in mice.

Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) are considered to play an important role in the pathogenesis of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is widely used as an animal model of multiple sclerosis. Whereas several studies have addressed the expression of various MMPs and their inhibitors in the pathogenesis of EAE, the expression of the molecules of the PA system during EAE has not been reported previously. The present study was undertaken to investigate the expression of the molecules of the PA system (tPA, uPA, PAI-1, uPAR, LRP), as well as several members of the MMP family and their inhibitors in the course of actively induced EAE in BALB/c mice. During clinical EAE, the PA system was up-regulated in the central nervous system at several levels. Induction of expression of tPA and PAI-1 transcripts was detected in activated astrocytes in the white matter. Inflammatory cells expressed uPA receptor, uPAR. In situ zymography demonstrated the presence of increased tPA and uPA activities in the areas of the inflammatory damage. Accumulation of fibrin, fibronectin, and vitronectin immunoreactivity was seen in perivascular matrices of symptomatic animals. In addition, transcription of MT1-MMP and metalloelastase (in inflammatory cells), and TIMP-1 (in activated astrocytes) was induced during EAE. Increased gelatinolytic activity was detected at the sites of inflammatory cell accumulation by in situ zymography of fluorescently labeled gelatin; substrate gel zymography identified the up-regulated gelatinolytic activity as gelatinase B. Overall, our study demonstrates concurrent induction of PA and MMP systems during active EAE, supporting further the concept that the neuroinflammatory damage in EAE involves altered balance between multiple extracellular proteases and their inhibitors.

Animals↗