Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

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.

Study of the surface secretion of the bronchiole using radioautography.

The purpose of the present study was to obtain further information regarding the chemical nature and cell of origin of the surface secretion of the bronchiole using radioautography after the injection of labeled leucine, glucosamine, galactose. Rat lungs were perfused with mammalian Ringer's solution with added albumin. A 10-min pulse of L-leucine labeled with hydrogen-3 was administered, followed by perfusion with nonradioactive solution for 15 min to 4 hours. Similar studies were performed with [3H] glucosamine and [3H] galactose. Heavy labeling of the Clara cells of bronchioles was obtained after injection of [3H] L-leucine. Labeling of alveolar wall cells was also obtained. Labeling of the surface of the bronchiole was much heavier than the labeling of the surface of the alveoli. No specific labeling of bronchiolar cells was obtained with [3H] glucosamine and [3H] galactose. The results provide added evidence for the protein nature of the surface layer of the bronchiole and for the Clara cell as its cell of origin.

Observation of the secretion on the surface of the bronchioles with the scanning electron microscope.

The secretion covering the surface of the bronchioles can be demonstrated with the scanning electron microscope if fixative is introduced into the pulmonary artery or directly into the lung rather than through the trachea. Buffered glutaraldehyde, ethanol, or an ethanol-ether mixture can be used as a fixative. With this method of preparation the surface secretion appears as a smooth coating covering the cilia and cells. The secretion is insoluble in ethanol ether or chloroform methanol, suggesting that there is little lipid contained in it. Reasons are cited for believing that the major component of the secretion is a protein that arises from the Clara cell.

The bronchiolar epithelium in cigarette smokers. Observations with the scanning electron microscope.

Observations with the scanning electron microscope were made on the bronchiolar epithelium of 25 lungs removed at surgery. In all but 3 cases, the lungs were removed for a malignant tumor. Abundant ciliated cells were present in all lung specimens. In lungs obtained from nonsmokers, numerous Clara cells were present in the small bronchioles, whereas goblet cells were seen, and in most cases, the Clara cells were infrequent or absent. The evidence for the secretory nature of the Clara cells is discussed as is the possible effect on lung function of alteration in the type of secretory cell in the bronchioles.