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Biomedical subjects

P A Underwood

Publications and source records attributed to P A Underwood.

At least 19 recordsLinked to original sources
PubMed2001

Specific affinity depletion of cell adhesion molecules and growth factors from serum.

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.

Animals↗
PubMed2000

Low density lipoproteins in human plasma make vascular smooth muscle cells resistant to growth inhibition by heparin.

OBJECTIVE: Vascular smooth muscle cell hyperplasia plays a role in atherosclerosis and restenosis. While heparin has shown promise as an inhibitor of smooth muscle cell proliferation in vitro and in some animal models, it has failed to reduce restenosis in clinical trials. We have previously shown that human smooth muscle cells grown in the presence of human serum are heparin resistant, whereas in the presence of bovine serum they are heparin sensitive. In this report, we demonstrate that the heparin resistance factor is present in human plasma as well as serum, and characterise it further. METHODS: Human vascular smooth muscle cells were cultured as explants from the media of redundant adult internal mammary or umbilical cord arteries. They were tested for sensitivity to heparin at 100 microg/ml in the growth medium, in the presence of foetal bovine serum, human-plasma-derived serum, human whole blood serum, or fractions derived from these. RESULTS: In the presence of foetal bovine serum, heparin inhibited cell proliferation, while human-plasma-derived or whole blood sera conveyed heparin resistance. This activity was contained within the fraction of plasma/serum which bound to heparin Sepharose, and the sub-fraction which was retained by a membrane filter of molecular weight cut off of 100000. All the heparin resistance in this latter fraction was supplied by lipoproteins. LDL prepared directly from human plasma conveyed similar heparin resistance to the lipoproteins from the above sub-fraction. CONCLUSION: LDL in human plasma/serum conveys resistance to the anti-proliferative effects of heparin upon vascular smooth muscle cells. This activity may interfere with potential therapeutic effects of heparin as an anti-restenosis agent.

Adult↗
PubMed2000

The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture: the effect of beclomethasone.

Airway remodeling is a key feature of persistent asthma. Part of the remodeling process involves the laying down of extracellular matrix (ECM) proteins within the airways. In this study we compared the production of ECM proteins by human airway smooth-muscle (ASM) cells in culture after exposure to 10% serum from an asthmatic individual or 10% serum from a nonasthmatic individual with or without beclomethasone (0.01 to 100 nM). Enzyme-linked immunosorbent assays were done with antibodies to human fibronectin; perlecan; elastin; the laminin beta(1), gamma(1), beta(2), alpha(1) chains; thrombospondin; chondroitin sulfate; collagen types I, III, IV, and V; versican; and decorin. Serum from the asthmatic individual, when compared with that from the nonasthmatic individual, caused a significant increase in the production of fibronectin, perlecan, laminin gamma(1), and chondroitin sulfate. Beclomethasone caused a significant reduction in the number of cells exposed to serum from either the asthmatic or nonasthmatic individual, but did not reverse the increase in ECM protein induced by the former. These results suggest an interaction between the ASM and the allergic process that may alter components of the airway wall in asthma, and that corticosteroids may not prevent the fibrosis induced by resident cells within the airways.

Adult↗
PubMed1999

Expression of human perlecan domain I as a recombinant heparan sulfate proteoglycan with 20-kDa glycosaminoglycan chains.

Recombinant forms of human perlecan domain I were secreted as proteoglycans by stably transfected human 293 cells. A recombinant domain I-only proteoglycan spanned the 95- to 265-kDa region in SDS-PAGE and appeared to be 160 kDa in denaturing gel filtration. Its glycosaminoglycan (GAG) content was approximately 67% heparan sulfate, and its average GAG chain size of 20 kDa suggested that the true molecular mass of the proteoglycan was 90 kDa. Domain I with enhanced green fluorescent protein fused to its C-terminus had an apparent molecular mass of 210-220 kDa and contained approximately 100% heparan sulfate. Its average GAG chain size (also 20 kDa) suggested a true molecular mass of 117 kDa for this proteoglycan. Its sulfate content of 53-77 mol SO2-4 per mole of protein indicated the presence of one sulfate group per 4-7 GAG sugar residues.

Amino Acid Sequence↗
PubMed1999

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.

Animals↗
PubMed1999

Human endothelial cell attachment to and growth on polypyrrole-heparin is vitronectin dependent.

Composite materials comprised of the electrically conducting polymer, polypyrrole, with a variety of biologically active molecules, e.g. proteins or polysaccharides, are emerging as a novel class of "smart" biomaterials. In the present work we have studied the utility of a heparin-polypyrrole composite as a substrate for human umbilical vein endothelial cell (HUVEC) growth. We found that the polymer composites were well suited to support cell attachment and growth; displaying low surface hydrophobicity (water contact angle of approximately 20 degrees ) and roughness, Rq of approximately 10-12 nm. Doubling times for HUVEC on heparin-polypyrrole were greater than observed for gelatin-coated tissue culture polystyrene (44 and 36 h, respectively), however, the cells did proliferate to cover the polymer in an even monolayer. The initial mechanism of attachment and subsequent proliferation of HUVEC on heparin-polypyrrole was critically dependent on the presence of the serum adhesion glycoprotein vitronectin. Polymers that were composed of polypyrrole and sodium nitrate were more hydrophobic than heparin-polypyrrole and they did not support HUVEC growth. Given the relative ease with which these polymer composites can be electrochemically synthesized, the diverse range of cellular "signal agents", e.g. growth factors, that can be incorporated within them, and the high degree of control that can be achieved in the release-surface exposure of these agents, we suggest that polypyrrole composites could serve a useful role as "smart" biomaterials in the near future.

Journal Article↗
PubMed1998

Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen.

Endothelial cells recovering from damage due to disease or surgical procedures come into close contact with extracellular matrix (ECM) secreted by intimal vascular smooth muscle cells (VSMCs). We have investigated these relationships using human umbilical artery endothelial cells (HUAECs) and human mammary artery VSMC in vitro. HUAEC adhesion and proliferation were significantly lower on ECM secreted by VSMC compared with HUAEC ECM or surface-coated fibronectin. Characterisation of the ECM of both cell types with monoclonal antibodies showed that the ECM secreted by VSMC contained significantly more elastin, chondroitin sulphate and collagen types I, III and V than that from HUAECs. HUAECs adhered poorly to collagen type V coated on plastic and not at all to elastin. When these proteins were co-coated with fibronectin, elastin did not inhibit migration or proliferation compared to the response on fibronectin but collagen type V significantly inhibited both. Treatment of VSMC ECM with enzymes which selectively depleted the matrix of collagen types I, III and IV, or chondroitin sulphate, had no effect on HUAEC responses to the ECM, suggesting that these molecules did not contribute to the inhibition of HUAECs. Treatment of VSMC ECM with a mixture of collagenases, selectively depleted the matrix of collagen type V, as well as types I, III and IV. Such depleted ECMs supported increased proliferation of HUAECs compared to buffer controls. Overall these results suggest that collagen V secreted into the ECM of VSMC may inhibit the recovery of adjacent endothelium.

Cell Adhesion↗
PubMed1998

Albumin-binding surfaces: synthesis and characterization.

The nature of the proteinaceous film deposited on a biomaterial surface following implantation is a key determinant of the subsequent biological response. To achieve selectivity in the formation of this film, monoclonal antibodies have been coupled to a range of solid substrates using avidin-biotin technology. Antibody clones varied in their antigen-binding activity following insertion of biotin groups into lysine residues. Biotinylated antibodies coupled to solid substrates via an immobilized avidin bridge retained their biological activity. During immobilization of avidin a significant proportion of the protein molecules were passively adsorbed rather than covalently attached to the surface. This loosely bound material could be removed by stringent elution procedures which resulted in a surface density of 5.4 pmol avidin cm(-2). Although these conditions would be harsh enough to denature monoclonal antibodies, they did not destroy the biotin-binding activity of the residual surface-coupled avidin, enabling the subsequent immobilization of biotinylated antibodies. The two-step immobilization technique allowed the use of gentle protein modification procedures, reduced the risk of surface-induced denaturation and removed loosely bound material from the surface. The versatility of the technique encourages its application to a wide range of immobilization systems where retention of biological activity is a key requirement.

Adsorption↗
PubMed1997

The effect of human endothelial cell-derived proteoglycans on human smooth muscle cell growth.

Extracellular proteoglycans (PGs) purified from cultured human arterial endothelial cells were tested for their effects on the proliferation of human vascular smooth muscle cells (VSMC). Fractions containing perlecan, the basement membrane heparan sulphate (HS) PG, the large chondrotin sulphate (CS) proteoglycan from connective tissue and other immunoreactive CS did not inhibit the proliferation of human VSMC. Native endothelial extracellular matrix, which was shown to contain the same PGs, demonstrated a pronounced stimulatory effect on the proliferation of human VSMCs. This stimulatory effect was not removed by pre-incubation of the matrix with 1 M NaCl, heparin, platelet extract or plasmin. These experiments demonstrate that PGs produced by human arterial endothelial cells do not inhibit the proliferation of VSMC. These data do not support the hypothesis that human endothelial cells, in vivo, control the activation or proliferation of VSMCs directly by the secretion of a non-proliferative molecule. Instead they support the hypothesis that the endothelial cells counteract intimal hyperplasia of VSMC indirectly by providing a barrier from activating factors in the plasma.

Arteries↗
PubMed1997

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide.

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 microM inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited approximately 80% by 5 microM LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited approximately 70% by 0.3 microM and 5 microM LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality.

Animals↗
PubMed1997

Mechanism of initial attachment of corneal epithelial cells to polymeric surfaces.

The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: 'tissue culture' polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)-80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85-95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture neophytes can employe endogenous mechanisms for the initial attachment to TCPS, whereas cells established in culture are dependent on exogenous vitronectin in order to attach.

Animals↗
PubMed1996

Identification of monoclonal antibodies that recognize different disulfide bonded forms of thrombospondin 1.

Thrombospondin 1 (TSP1) is a multidomain glycoprotein from platelets and cells which functions in cell-cell and cell-matrix interactions. The structure of TSP1 is regulated by sulfhydryl-disulfide interchange in the carboxy-terminal Ca2(+)-binding loops and globular domain which markedly influence its interaction with cell surface integrins and its inhibition of neutrophil enzymes. We have identified murine monoclonal antibodies that recognized different disulfide-bonded forms of TSP1, made by preparing TSP1 in buffers containing either 0.1 mM or 2 mM Ca2+. Antibody HB8432 recognizes TSP1 prepared in buffers containing either 0.1 or 2 mM Ca2+, while antibodies D4.6 and A65M recognized only TSP1 prepared in buffers containing 0.1 mM Ca2+. The antibodies recognize these different TSP1 preparations either adsorbed to plastic or extracellular matrix. Immunohistochemistry of human rheumatoid synovial tissue using HB8432 resulted in staining of numerous blood vessel walls and matrix cells, while D4.6 and A65M stained a subset of the HB8432 positive blood vessels and only occasionally stained matrix cells. These results suggested that different disulfide-bonded forms of TSP1 were being expressed in different areas of inflamed tissue.

Adsorption↗
PubMed1996

The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases.

Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the approximately 450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of approximately 300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.

Cells, Cultured↗
PubMed1996

Comparison of the heparanase enzymes from mouse melanoma cells, mouse macrophages, and human platelets.

The intracellular heparanases from mouse macrophage and melanoma cells are very similar in terms of their size (60-80 kDa), pI (5.3-4.1), pH optimum (< or = 5.5), and interactions with heparin. These proteins are therefore likely to be identical, suggesting that tumour and blood cells utilise the same heparanase enzyme. The human platelet enzyme is similar to the mouse enzymes in terms of pH optimum (< or = 5.5) and pI value (5.3-4.8), but appears to be smaller in size (40-60 kDa). It also seems to differ from the mouse enzymes in aspects of its surface charge, and in its interactions with heparin. There was no indication that proteolysis was of significance for the enzymes, nor that they contained any sialic acid residues.

Animals↗
PubMed1996

Measurement of cell adhesion and migration using phosphor-screen autoradiography.

This study demonstrates the use of phosphor-screen autoradiography as a means of measuring cell adhesion and cell expansion on polymer surfaces. The method has particular merit in cases where a specific substrate may be opaque or biochemically incompatible with colorimetric assay methodologies. With the phosphor-screen autoradiography method, there was a linear relationship between cell number and quantitated radioactivity. The technique has also been validated by comparison with a colorimetric assay of adhesion conducted for attachment to conventional culture substrata. The data supported the view that the use of phosphor-screen autoradiography was a valid method for detecting cell attachment, and it gave equivalent results to the colorimetric assay. Furthermore, a comparison between phosphor-screen autoradiography and a dye-staining method showed that this technique can be used as a means of quantifying cellular expansion over surfaces.

Autoradiography↗
PubMed1995

Evidence for the location of a binding sequence for the alpha 2 beta 1 integrin of endothelial cells, in the beta 1 subunit of laminin.

To date no specific location on laminin 1 for the binding of alpha 2 beta 1 integrin has been described, although recent evidence supports a location in the E1XNd fragment of the cross region. We have identified a peptide sequence from this region, in the beta 1 chain of laminin 1, YGYYGDALR, which inhibits the adhesion of endothelial cells to laminin 1 and type-IV collagen. A structurally related sequence from the CNBr-cleaved fragment CB3 of the alpha 1 chain of collagen type IV, FYFDLR, inhibits endothelial cell adhesion to both collagen types I and IV and laminin 1. The CB3 fragment containing the FYFDLR sequence has been shown to contain binding sites for both alpha 1 beta 1 and alpha 2 beta 1 integrins. Present experiments with anti-integrin antibodies indicate that the alpha 2 beta 1 integrin on endothelial cells can account for all the cell binding to collagen types I and IV, and that this integrin makes a major contribution towards the adhesion of these cells to laminin 1. We therefore propose that the peptide FYFDLR participates in alpha 2 beta 1 binding to collagen type IV and that the putatively structurally similar peptide, YGYYGDALR, participates in alpha 2 beta 1 binding to laminin 1. This is the first account of structurally related peptide sequences from laminin 1 and type-IV collagen which show reciprocal inhibition of cell adhesion to either ligand and which might form part of a common integrin-binding site, as well as the first suggestion of a precise location contributing to the alpha 2 beta 1 integrin binding site on laminin 1.

Amino Acid Sequence↗
PubMed1995

Effect of extracellular matrix proteins on vascular smooth muscle cell phenotype.

The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (Vvmyo) in their cytoplasm. Laminin was best at maintaining SMC with a high Vvmyo (Vvmyo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest Vvmyo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high Vvmyo phenotype.

Actin Cytoskeleton↗