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Biomedical subjects

L Terracio

Publications and source records attributed to L Terracio.

At least 55 records · Page 3Linked to original sources
PubMed1990

Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF.

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.

Actins↗
PubMed1989

Identification of integrin-like matrix receptors with affinity for interstitial collagens.

Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors.

Amino Acid Sequence↗
PubMed1989

Expression of collagen adhesion proteins and their association with the cytoskeleton in cardiac myocytes.

Previous investigations have shown that specific cell surface glycoproteins on rat hepatocytes (COLL-CAM) are involved in the recognition of interstitial collagens (Rubin et al., Exp. Cell Res., 164:127-138, 1986). Western blot analysis with anti-COLL-CAM antibodies revealed the presence of a variable but restricted number (two) of glycoproteins in detergent-extracted membranes from rat hearts at various developmental stages. Using antibodies against these collagen adhesion proteins, we show an expression of the antigens during different developmental stages of the rat heart and during cardiac hypertrophy. This expression is described morphologically by immunohistochemical staining of cell surfaces of freshly isolated myocytes from neonates, normal adults, and hypertrophied adult hearts. Antibodies made against COLL-CAM were localized on the cell surface of cardiac myocytes and antibodies against talin and vinculin co-localized in a similar position on the inside of the cell. Antibody staining appears to be increased at times when collagen synthesis is high (neonate and cardiac hypertrophy) and low when collagen synthesis is low, as in the normal adult. These results indicate that collagen adhesion proteins may play an important role in linking the extracellular matrix to the cytoskeleton in the heart.

Animals↗
PubMed1989

Platelet-derived growth factor receptors in the kidney--upregulated expression in inflammation.

Major features of a long-standing inflammation in the kidney are vascular proliferation, glomerulosclerosis, interstitial fibrosis and tubular atrophy, leading to a gradual deterioration of the renal function. In this study we have investigated the expression of B-type receptors for platelet-derived growth factor (PDGF) in frozen sections from normal and inflamed kidneys. Immunohistochemical techniques, employing two monoclonal antibodies specific for PDGF B-type receptors, were used. The specimens investigated were 15 kidneys removed by transplantectomy because of chronic rejection, 20 cases of glomerulonephritis with crescent formation, mesangial proliferation or non-proliferative glomerulonephritis, and six normal kidneys. In parallel we characterized cellular infiltrates and class II transplantation antigen expression in the inflamed kidneys. An enhanced PDGF receptor expression was found on intimal cells and on smooth muscle cells of the proliferating vessels, on glomerular cells in glomeruli with mesangial proliferation, and on fibroblast-like cells in the proximity of clusters of infiltrating macrophages and T-lymphocytes of the interstitial tissue. Induction of PDGF receptor expression may render cells responsive to stimulation by PDGF, released from PDGF-producing cells, such as activated macrophages and from platelets. Our data suggest that PDGF is involved in the proliferation of mesenchymal cells that is seen in rejected kidney transplants and glomerulonephritis.

Antibodies, Monoclonal↗
PubMed1989

Effects of extracellular matrix on cytoskeletal and myofibrillar organization in vitro.

The distribution and three-dimensional relationship of myofibrillar and cytoskeletal components during myofibrillogenesis were examined in preparations of neonatal rat cardiac myocytes processed in parallel for scanning electron microscopy (SEM), intermediate voltage transmission electron microscopy (IVEM) and immunofluorescence (IF). Of the various methods used for processing, optimal results were achieved by pre-extraction with Triton X-100 in an actin-stabilizing buffer. This procedure effectively removed the surface membrane, as viewed by SEM images, while preserving myofibrillar and cytoskeletal structure, as evidenced by IF for actin, alpha-actinin and vinculin. Cytoskeletons in SEM images consisted of a cortex of anastomosing filaments through which ran parallel filament bundles oriented in the long axis of the cell and attached along their length to the substrate by numerous fine filaments. In IVEM images, myofibrils were laterally connected at the level of the Z bands. Myocytes grown on different extracellular matrices showed different patterns and distributions of both striated myofibrils and focal adhesions, as determined by IF for alpha-actinin and vinculin, respectively. Cells on collagen I and III contained striated myofibrils which extended to the cell perimeters where focal adhesions were predominately located. Cells on laminin and fibronectin matrices exhibited myofibrils and focal adhesions more centrally located. In addition, cells on laminin contained circumferential arcs of filaments near the cell periphery.

Animals↗
PubMed1988

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor.

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.

Animals↗
PubMed1988

Induction of B-type receptors for platelet-derived growth factor in vascular inflammation: possible implications for development of vascular proliferative lesions.

Expression of B-type receptors for platelet-derived growth factor (PDGF) in frozen sections of blood vessels from tissues affected by abnormal vascular cell proliferation was investigated by immunohistochemical techniques and compared with expression of these receptors in blood vessels of normal tissues. Receptors were not expressed, or expressed at low levels, in vessels of normal tissues. In contrast, a pronounced expression of PDGF B-type receptors was seen on vascular smooth muscle cells in atherosclerotic plaques, rejected kidneys, and chronic synovitis. These observations suggest induction of PDGF B-type receptors on vascular smooth muscle cells in inflamed tissues, which would render such cells responsive to growth stimulation by PDGF released from captured platelets, or produced locally (eg, by inflammatory cells or smooth muscle cells). Autocrine or paracrine stimulation of cell growth caused by the effect of PDGF on cells with induced receptors may be important in the formation of the proliferative lesions found in atherosclerosis and in certain forms of chronic inflammation.

Antibodies, Monoclonal↗
PubMed1988

In vitro studies on adult cardiac myocytes: attachment and biosynthesis of collagen type IV and laminin.

The interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P-CIV) or to collagen type I. However, when laminin was used to coat P-CIV, attachment was enhanced. Affinity-purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface-bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14-day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment.

Adult↗
PubMed1988

Oncogenicity of rat prostate cells transformed in vitro with cadmium chloride.

Three rat ventral prostate (RVP) cell lines transformed after in vitro treatment with cadmium chloride (CdCl2) and one control untreated cell line were tested for tumorigenicity in newborn rats. All three cadmium-transformed RVP cell lines induced tumors at the site of inoculation in 95-100% of animals. The fibroblastoid RVP56Cd cell line induced sarcomas, whereas the epithelial cell lines RVP47-3G and RVP47-3F produced highly differentiated squamous cell carcinomas. About 20% of animals injected with RVP47-3G developed lung and splenic metastases. The tumors could be further passaged into young rats. The sarcomas had a hyperdiploid modal chromosome number similar to that of the RVP56Cd cell line. Carcinomas induced by the RVP47-3G cell line had a large proportion of stromal metaphases. The modal chromosome number of these carcinomas was in the hypertriploid-hypotetraploid range, similar to that of the parental cell line. These results demonstrate that treatment of RVP cells with CdCl2 in vitro results in neoplastic transformation. Since both fibroblastoid and epithelial prostate cells have undergone transformation, it seems possible that cadmium acted as a carcinogen without cell specificity. The susceptibility of these cells to the carcinogenic effect may be related to their resistance to cadmium. In the process of neoplastic transformation induced by CdCl2 in RVP epithelial cells changes of squamous metaplasia occur, and probably precede acquisition of tumorigenicity.

Animals↗
PubMed1988

Modulation of beta-receptors as adult and neonatal cardiac myocytes progress into culture.

Modulation of beta-adrenergic receptors and their ability to respond to beta-receptor stimulation was studied in cultures of adult and neonatal rat cardiac myocytes. The radioligand iodocyanopindolol (125I-CYP) was used to identify beta-adrenoceptors on the intact cells. 125I-CYP was found to bind to the receptors in a stereospecific and saturable manner. Freshly isolated neonatal and adult myocytes both had a receptor density of approximately 50 fmol/mg protein. The number of beta-receptors per milligram protein was similar during a 10-d culture period for adult myocytes but increased after a 5-d culture period for neonatal myocytes. Both cell types responded to beta-receptor stimulation with isoproterenol by a twofold increase in the concentration of cAMP and this response increased with time in culture. The number of receptors as well as the response to isoproterenol was similar for neonatal myocytes cultured on laminin, collagen type I, or on uncoated culture dishes. From these data we conclude that cultured cardiac myocytes maintain functional beta-receptors as they progress into culture, and the expression of beta-receptors is not influenced by culture substrates.

Animals↗
PubMed1988

Effects of cyclic mechanical stimulation of the cellular components of the heart: in vitro.

The response of the cellular components of the heart to cyclic mechanical stimulation is of particular importance because these cells are continually subjected to mechanical forces as a result of changes in blood volume and pressure. To directly investigate how mechanical tension affects these cellular components of the heart, an in vitro system that exposes the particular cell type (cardiac myocytes, endothelial cells, or fibroblasts) to a calibrated increase in cyclical linear stretch was developed. Cells were grown on silastic membranes coated with laminin and subjected to a 10% cyclical distention 10 times a minute for 72 h. Within 24 h of being exposed to the mechanical stretch, the cells became elongated and oriented perpendicular to the direction of the stretch. These results indicate that cyclical mechanical stimulation directly influences the cellular organization of the heart cells in vitro.

Animals↗
PubMed1988

Membrane glycoproteins involved in hepatocyte adhesion to collagen type I.

Liver membrane glycoproteins with affinity for immobilized collagen type I were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution of the separated proteins. Electroeluted glycoproteins with ability to neutralize the inhibitory effect of anti-CollCAM antibodies on hepatocyte adhesion to collagen were collected from several consecutive runs and used to raise a high titer antiserum, denoted anti-CollCAM II. IgG from this antiserum inhibited the attachment of hepatocytes to dishes coated with collagen type I, but not to fibronectin- or collagen type IV-coated dishes. When the antibodies were immobilized to Sepharose CL-4B they bound three sets of glycoproteins with apparent Mr's of 105,000, 115,000, and 130,000 as analyzed by SDS-PAGE under nonreducing (NR) conditions. Upon reduction (R) the glycoproteins migrated with apparent Mr's of 115,000, 130,000, and 160,000, respectively. The Mr 105,000-115,000 (NR) glycoproteins effectively neutralized the inhibitory effect exerted by both anti-CollCAM and anti-CollCAM II antibodies, on hepatocyte spreading and attachment to collagen type I substrates. Peptide mapping suggested the Mr 160,000 (R) species to be different from the Mr 115,000 (R).

Animals↗
PubMed1988

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing.

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.

Animals↗
PubMed1988

Expression of platelet-derived growth factor receptors is induced on connective tissue cells during chronic synovial inflammation.

The tissue distribution of the receptor for platelet-derived growth factor (PDGF) was investigated by immunohistochemistry on frozen sections from normal and inflamed synovial tissue using monoclonal antibodies to the receptor. Non-inflamed synovial tissue showed no staining, indicating that PDGF receptor expression is low or absent in normal tissue. In contrast, tissue from synovitis with prominent neovascularization showed a strong staining in the tunica media of the proliferating blood vessels as well as on connective tissue cells in the stroma. Tissue from synovitis with prominent proliferation of synovial lining showed intense staining for PDGF receptors in fibroblast-like cells of the lining and a less intense staining on vascular and connective tissue cells deeper in the stroma. Staining for PDGF receptors was also intense in the pannus tissue close to infiltrated bone and cartilage. In all these forms of synovitis, PDGF receptor staining was associated with increased HLA-DR staining and infiltration of macrophages and T lymphocytes. The finding that PDGF receptor expression is induced in conjunction with the chronic synovial inflammation associated with rheumatoid arthritis and some other forms of arthritides suggests that PDGF may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory disease.

Chronic Disease↗
PubMed1987

Transformation of rabbit arterial smooth muscle cells with simian virus 40.

In vitro studies were carried out to induce viral transformation of vascular smooth muscle cells. Cultured rabbit arterial smooth muscle cells were infected with simian virus 40 (SV 40), and transformed cultures were produced that exhibit altered morphology, increased growth rate and plating efficiency, growth on semi-solid substrate, and chromosomal abnormalities. Nuclear SV 40 T-antigen was detected in all cells of these cultures. Muscle-specific actin was identified by a specific monoclonal antibody suggesting retention of smooth muscle cell characteristics by the transformed cells. Significant cytoplasmic lipid accumulation occurred in transformed cells incubated with beta-very low density lipoprotein, as revealed both by chemical analyses and Nile Red lipid staining of the culture. The transformed smooth muscle cells grow permanently in cell culture. Our investigations show that arterial smooth muscle cells transformed with SV 40 virus exhibit altered phenotypic properties distinct from that of normal arterial smooth muscle cells.

Animals↗