Faculty Bibliography

Residualizing labels are tracers which remain in lysosomes after uptake and catabolism of the carrier protein and have been especially useful for studies on the sites of plasma protein degradation. Thus far these labels have contained radioactive reporters such as 3H or 125I. In the present paper we describe a fluorescent residualizing label, NN-dilactitol-N'-fluoresceinylethylenediamine (DLF). Modification of asialofetuin (ASF) or rat serum albumin (RSA) with DLF affected neither their normal kinetics of clearance from the rat circulation nor their normal tissue sites of uptake and degradation. After injection of DLF-ASF, fluorescent degradation products were recovered nearly quantitatively in liver and retained with a half-life of about 2 days. Fluorescent degradation products from DLF-RSA were recovered in skin and muscle, and were localized in fibroblasts by fluorescence microscopy. These results confirm previous studies with radioactive residualizing labels in which fibroblasts in peripheral tissues were identified as primary sites of albumin degradation. Fluorescent catabolites also accumulated in fibroblasts incubated with DLF-RSA in vitro, and residualized with a half-life of about 2 days. Overall, the data establish that DLF functions efficiently as a fluorescent residualizing label both in vivo and in vitro. The advantages of fluorescent, compared with radioactive, residualizing labels should make them valuable tools for studies on protein uptake and catabolism in biological systems.

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.

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 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.

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.

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.

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.

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.