Faculty Bibliography

The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I+III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN greater than LN greater than C I+III greater than plastic greater than BSA. Addition of anti-beta 1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration. Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of alpha-actinin aggregates into Z bands. At later times, vinculin at 8 hours and beta 1 integrin at 22 hours became co-localized with alpha-actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM-integrin-cytoskeleton associations.

The study of the deep pineal gland of the Mongolian gerbil and other neuronal tissue from the rat by means of confocal laser scanning microscopy (CLSM) is described. Opical serial sectioning was performed on thick (100-200 microns) sections of the deep pineal gland of the Mongolian gerbil stained immunohistochemically using antisera to S-antigen and tyrosine hydroxylase (TH). Both dual-stained and single-stained material was examined using the fluorochromes fluorescein isothiocyanate (FITC) and Texas Red. High resolution images were obtained showing that pinealocytes have 1-3 processes that extend primarily to other pinealocytes or presumptive pinealocytes. Pinealocytes are located within the deep pineal gland as well as adjacent to the posterior aspect of the medial habenular nuclei. Pinealocyte processes were not seen extending into the habenular nuclei, but rather ended within the deep pineal gland a significant distance from their perikarya. The TH-immunopositive fibers were distributed throughout the deep pineal gland, often forming "baskets" of fibers around pinealocytes rather than being associated primarily with blood vessels. Other uses of the confocal microscope are demonstrated on rat neural tissue reacted with peroxidase/diaminobenzidine (DAB) immunohistochemistry and FITC fluorescence immunohistochemistry (paraventricular nucleus) as well as Golgi-stained neuronal tissue (cerebral cortex). The HRP/DAB and Golgi-stained images were visualized using the reflected image mode of the confocal system.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to determine the feasibility of using a positive reinforcement protocol to motivate weight-lifting exercise in rats. Intracranial self-stimulation was used to induce weight-lifting exercise. Bipolar electrodes were implanted in the ventral tegmental area of rats, and the animals were trained to bar press on a continuous reinforcement schedule for electrical brain stimulation. Animals with response rates of 1,200-1,500 presses/h were then trained with a discriminative light stimulus to alternate between a normally positioned bar and an elevated bar that could be reached only by standing on the hindlimbs. The animals were fitted with a weighted jacket at a starting resistance of 5-10% of their body weight. Weight-training sessions were conducted 5 days/wk for 10 wk. Training consisted of 600 presses/session, alternating every 15 presses between the low and high bars. At the beginning of each subsequent week, the resistance was progressively increased, with some animals eventually training at resistances greater than 50% of their body weight. At the end of the training period, the rats were lifting over 550% of the starting weight. Gastrocnemius size and mean fiber diameter were increased in the weight-lifting animals. This model combines exercise with positive incentive and has the advantages of being relatively easy to implement and not producing any apparent physical or mental trauma in the animal.

The cardiac extracellular matrix, composed predominantly of collagenous fibers, forms a stress-tolerant network that facilitates the distribution of forces generated in the heart and provides for proper alignment of cardiac myocytes. Although considerable information exists regarding the morphological organization of the heart extracellular matrix, little is known about the regulation of the synthesis and accumulation of extracellular matrix components. A potentially significant factor in the cardiovascular system is mechanical stimulation including changes in physical tension and pressure. We recently have developed an in vitro model system to elucidate the effects of mechanical stretch on isolated populations of heart cells. In the present study, we have used biochemical and molecular biological techniques to analyze changes in collagen synthesis by cardiac fibroblasts in response to mechanical stretch. These studies show that the ratio of collagen type III to collagen type I increases in mechanically stretched cells. They also show that type III collagen mRNA levels are increased in response to cyclic mechanical stretch for durations as short as 12 hours. Type I collagen mRNA levels were not found to change under the stretch conditions used in this study. Our results emphasize the potential regulatory role of mechanical stimulation in the expression of specific genes in the heart and support previous studies indicating this to be an intriguing in vitro model of cardiac hypertrophy.

Neonatal rat cardiomyocytes were cultured on extracellular matrix components laminin and collagens I + III to examine effects of extracellular matrix on the assembly of cytoskeletal proteins during myofibrillogenesis. Myofibril assembly was visualized by immunofluorescence of marker proteins for myofibrils (f-actin for I bands and alpha-actinin for Z bands), focal adhesions (vinculin), and transmembrane extracellular matrix receptors (beta 1 integrin) as cells spread for various times in culture. By 4 h in culture, f-actin appeared organized into nonstriated stress-fiber-like structures while alpha-actinin, vinculin and beta 1 integrin were localized in small streaks and beads. Subsequently, striated patterns were observed sequentially in the intracellular cytoskeletal components alpha-actinin, vinculin, f-actin, and then in the transmembrane beta 1 integrin receptor. These data support an earlier model for sarcomerogenesis in which stress-fiber-like structures serve as initial scaffolds upon which alpha-actinin and then vinculin-containing costameres are assembled. This sequential and temporal assembly was the same on both laminin and collagens I + III. A quantitative difference, however, was apparent on the 2 matrices. There was an increased appearance on collagens I + III of rosettes (also called podosomes or cortical actin-containing bodies in other cells) which consisted of an f-actin core surrounded by alpha-actinin, vinculin and beta 1 integrin rims. The increased incidence of rosettes in neonatal myocytes on collagens I + III suggests that these cytoskeletal complexes are involved in recognition and interaction with extracellular matrix components.

The interaction between components of the extracellular matrix and the cell surface of cardiac myocytes appears to be regulated in part by receptors belonging to the integrin superfamily. The expression of the integrins was investigated at different stages of development of the heart as well as during cardiac hypertrophy. The characterization of the membrane proteins showed that a beta 1-integrin and associated alpha-chains were responsible for the interaction with collagen, laminin, and fibronectin. Immunoprecipitation data indicated that the presence of specific alpha-chains varied with development. These data were correlated with the ability of the isolated myocytes to attach to specific components of the extracellular matrix. The expression of the alpha 1-chain was prominently associated with the recognition of interstitial collagens. The presence of the alpha 1-chain was also associated with stages when collagen synthesis was increased, especially during fetal and neonatal growth and cardiac hypertrophy. Immunohistochemical localization with the antiserum against beta 1-integrin demonstrated its specific localization near the Z lines of cardiac myocytes. The localization both in vitro and in vivo indicated that the beta 1-integrin may play a role in myofibrillogenesis during development. The present immunohistochemical, cell adhesion, and biochemical data clearly indicate that integrins play a major role in the regulation of the interaction between cardiac myocytes and the extracellular matrix during development and disease.

Platelet-derived growth factor (PDGF) beta-receptor expression in normal and rheumatoid synovia was investigated by double immunofluorescence staining of frozen sections and by in situ hybridization. In the inflamed synovia, PDGF beta-receptor mRNA was present in vascular cells, as well as in discrete stromal cells. PDGF beta-receptor expressing cells in rheumatoid synovia were characterized by double immunofluorescence staining using the PDGFR-B2 monoclonal antibody at a concentration at which this antibody merely stained granular accumulations of PDGF beta-receptors. Granular accumulations of PDGF beta-receptors were articulate in blood vessel cells, but also appeared in discrete stromal cells. Thus, the overall distribution of cells having granular accumulations of PDGF beta-receptors was similar to the distribution of cells expressing PDGF beta-receptor mRNA. Double immunofluorescence stainings showed that: (a) a majority (greater than 90%) of resident macrophages did not express granular PDGF beta-receptor staining, but macrophages were often juxtaposed to PDGF beta-receptor-positive cells; (b) T lymphocytes did not express PDGF beta-receptors, but these cells were frequently found in the proximity of cells stained by PDGFR-B2; (c) in some blood vessels both HLA-DR expressing cells and PDGF beta-receptor expressing cells could be visualized, whereas in other blood vessels, cells expressing only one of these activation markers could be detected; (d) smooth muscle cells in blood vessels contained PDGF beta-receptors; and (e) capillary endothelial cells in the inflamed synovia recurrently displayed granular PDGF beta-receptor staining. The granular accumulations of PDGF beta-receptors may reflect internalization of the receptor as a result of paracrine or autocrine ligand stimulation. In support of such a possibility are the findings that elevated levels of PDGF B chain mRNA were detected by in situ hybridization in the inflamed synovia, and that cells expressing PDGF B chain mRNA were distributed similarly to cells expressing PDGF beta-receptor mRNA. Taken together, the results indicate that PDGF has a role in the inflammatory process in rheumatoid synovitis, most likely by stimulating proliferative events in the vasculature.

Mouse glucocorticoid receptors (GR) that are over-expressed in Chinese hamster ovary (CHO) cells behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine the localization of the overexpressed GR within the CHO cell nucleus by confocal microscopy. In hormone-free cells the receptor distributes in a mottled pattern throughout all planes of the nucleus. The receptor is not present in nucleoli and shows no preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each plane of the nucleus demonstrates clearly that there are regions that do not contain receptor; thus, the distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is no detectable change in receptor distribution. Overexpressed receptors that have either no hormone-binding activity or no DNA-binding activity because of point mutations localize in the same mottled pattern as the wild-type receptor. These observations are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded state and proceed to loci distributed throughout the nucleus, where it is retained in an inactive docking complex until the binding of hormone triggers its progression to high affinity sites where the primary events in transcriptional activation occur. As there is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site where the primary events in transcriptional activation occur.

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis.

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.