Faculty Bibliography

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.

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.

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).

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.

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.

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.

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.

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.

Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were evaluated by using purified adult Leydig cells from Sprague-Dawley rats. Purified Leydig cells were found to contain both high and low affinity binding sites for insulin, with Ka values of 1.08 X 10(9) and 1.1 X 10(7) M-1, respectively. Using affinity cross-linking of [125I]iodoinsulin to plasma membrane insulin receptor, several bands were identified by autoradiography under nonreduced conditions with mol wt of 230,000, 280,000, and 300,000. After reduction with 50 mM dithiothreitol, only one band was identified with a mol wt of 130,000, consistent with the alpha-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 X 10(9) M-1. Multiple high mol wt bands (greater than 250,000) were identified under nonreduced conditions by affinity cross-linking. Under reduced conditions, one band with an approximate mol wt of 135,000 was identified. Purified Leydig cells (10(5) cells/ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 Nutrient Mixture (1:1) containing 0.1% fetal calf serum at 37 C in a humidified atmosphere of 5% CO2-95% air. Insulin and IGF-I stimulated testosterone formation as early as 3 h after administration, and their effects were completely blocked by the addition of a protein synthesis inhibitor, cycloheximide (1 microgram/ml). Insulin and IGF-I also significantly potentiated hCG-and 8-bromo-cAMP-induced testosterone formation. Furthermore, insulin and IGF-I potentiated hCG-stimulated cAMP formation. This suggests that insulin and IGF-I have effects at both the LH receptor sites and the steps beyond adenylate cyclase. The ED50 values of insulin and IGF-I-stimulated testosterone formation were comparable (25 ng/ml). In conclusion, we found that Leydig cells contain specific insulin and type I IGF receptors, and both insulin and IGF-I are capable of modulating Leydig cell steroidogenesis.