This report provides evidence from a number of different approaches (i.e., comparison of cell shape in 1-microm sections of photodamaged versus healthy skin at the light microscopic level; comparison of cell shape and apposition to collagen fibrils in ultrathin sections of the same tissues examined by transmission electron microscopy, and fluorescence staining for adhesion site protein expression and actin filament architecture in frozen tissue sections) that dermal cells in healthy skin are attached to collagen fibrils over a large part of the cell border, have a flattened/spread (two-dimensional) appearance and have abundant actin in their cytoplasm. In contrast, cells in photodamaged skin are often in contact with fragmented collagen or amorphous debris rather than intact collagen, have a collapsed/elongated shape, and have a lower amount of actin. Collagen synthesis is reduced in severely photodamaged skin relative to collagen synthesis in corresponding sun-protected skin (N Engl J Med 329:530, 1993). We hypothesize that fibroblasts in severely damaged skin have less interaction with intact collagen and as a result experience a reduction in mechanical tension. Decreased collagen synthesis is (presumed to be) the result.

ABSTRACT

IntroductionVisceral smooth muscle (SM) originates from local mesenchymal cells that in early-midgestation begin to synthesize SM proteins, including SM α-actin, desmin, SM myosin, SM22, and calponin in a specific periairway distribution (1-5). In the mouse developing respiratory system, cells expressing SM proteins are first detected in the trachea on day 11 of gestation (3, 4), and then SM differentiation proceeds in a cranial-tocaudal fashion to form the bronchial musculature (1-5). The other type of visceral SM cells found in the lung are interstitial SM cells, also known as interstitial contractile cells, or myofibroblasts. Interstitial SM cells are originally located at the sites of future alveolar septae, and, in the mature organ, they form part of the septae tips (6). Except for the aorta, the development of the vascular musculature lags behind that of visceral SM by several days (4, 7-9).Unlike striated muscle differentiation, on which considerable information was gathered over the years, the mechanisms and genetic program that control SM myogenesis remain, for the most part, unknown. We and others have observed that lung mesenchymal cell precursors change their shape from round to elongated before undergoing bronchial SM differentiation (ref.3; Y. Yang and L. Schuger, unpublished observations). Based on this observation we recently examined whether changes in cell shape might play a role in airway myogenesis. Unexpectedly, our studies demonstrated that essentially all undifferentiated embryonic mesenchymal cells are potential SM precursors (10-12). These studies also confirmed the critical role of cell shape in myogenesis. Specifically, we found that cell rounding prezvents myogenesis, regardless of the normal fate of the cell in vivo, whereas cell spreading/elongation induces SM differentiation, even in mesenchymal cells from nonmuscular organs (10-12).Developing tubular tissues, such as those of the respiratory, gastrointestinal, and urinary systems, are filled with liquid. As a consequence, the periluminal mesenchymal cells are subjected to mechanical tension/stretch exerted by the liquid's hydrostatic pressure (13). These forces likely represent a significant factor in determining the periluminal mesenchymal cell shape. In the developing lung, cells are additionally subjected to repeated stretch caused by intrauterine breathing (13). The fact that mechanical stretch causes cell elongation and that cell elongation is likely to be sensed by the cell as a mechanical stimulus suggested to us that cell tension/stretch may play an important role in the process of visceral myogenesis.Here we used a combination of lung cell and organ cultures from fetal mouse and human origin to determine the effect of mechanical stretch upon SM myogenesis. Smooth muscle (SM) develops only in organs and sites that sustain mechanical tensions. Therefore, we determined the role of stretch in mouse and human bronchial myogenesis. Sustained stretch induced expression of SM proteins in undifferentiated mesenchymal cells and acc...

. Mesenchymal-epithelial interactions in lung development and repair: are modeling and remodeling the same process? Am J Physiol Lung Cell Mol Physiol 283: L510-L517, 2002; 10.1152/ajplung.00144. 2002We propose that lung morphogenesis and repair are characterized by complex cell-cell interactions of endodermal and mesodermal origin, leading to (or returning back to) an alveolar structure that can effectively exchange gases between the circulation and the alveolar space. We provide the developmental basis for cell/molecular control of lung development and disease, what is known about growth and transcription factors in normal and abnormal lung development, and how endodermal and mesodermal cell origins interact during lung development and disease. The global mechanisms that mediate mesenchymalepithelial interactions and the plasticity of mesenchymal cells in normal lung development and remodeling provide a functional genomic model that may bring these concepts closer together. We present a synopsis followed by a vertical integration of the developmental and injury/repair mechanisms. bronchopulmonary dyplasia; smooth muscle; terminal sac THE PREMISE OF THIS SYMPOSIUM was that lung morphogenesis and repair are characterized by complex cellcell interactions of endodermal and mesodermal origin, leading to (or returning back to) an alveolar structure that can effectively exchange gases between the circulation and the alveolar space. The presenters provided the developmental basis for cellular/molecular control of lung development and disease, what is known about growth and transcription factors in normal and abnormal lung development, and how endodermal and mesodermal cell origin interacts during lung development and disease. The global mechanisms that mediate mesenchymal-epithelial interactions and the plasticity of mesenchymal cells in normal lung development and remodeling provide a functional genomic model that may bring these concepts closer together. The following is a synopsis of each presentation, followed by an integration of the developmental and injury/repair mechanisms.

Laminins, the main components of basement membranes, are heterotrimers consisting of α, β, and γ polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of β1 and γ1 chains and differ from each other on their α chain, which is α1 and α2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial–mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin α1 chain. Synthesis of laminin α1 chain however returns upon re-establishment of epithelial–mesenchymal contact. Cell–cell contact is critical, since laminin α1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial–mesenchymal cocultures in which heterotypic cell–cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin α1 chain upon heterotypic cell– cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin α1, α2, and β/γ chains. Lung explants exposed to monoclonal antibodies to laminin α1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle α actin and desmin. Taken together, our studies suggest that laminin α1 chain synthesis is regulated by epithelial–mesenchymal interaction and may play a role in airway smooth muscle development.

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.

The pattern of deposition and the role of laminin, a major glycoprotein constituent of basement membranes, were investigated during lung morphogenesis in the fetal mouse. Lung primordia were removed from Day 13 embryos, right lower lobes were further dissected and placed in filter membrane assemblies. Explants were then cultured at the liquid-air interface for 3 days in the presence of anti-laminin, anti-thrombospondin (another extracellular matrix constituent), preimmune serum, laminin-neutralized anti-laminin, or medium alone. Cultures were monitored by (direct) phase-contrast microscopy, light microscopy, and immunofluorescence. We found that anti-laminin antibodies altered normal lung morphogenesis in a dose-dependent manner. The anti-laminin-treated explants presented a marked inhibition of branching morphogenesis and a distortion of the bronchial tree. A lower rate of growth was also observed in the explants exposed to this antibody. High concentrations of anti-thrombospondin antibodies, normal rabbit serum, or laminin-neutralized anti-laminin antibodies had no effect on lung morphogenesis. These results were not modified by culturing the explants in submersion culture or on Vitrogen 100-coated surfaces.

Stretch induces lung embryonic mesenchymal cells to follow a myogenic pathway. Using this system we identified a set of stretch-responsive factors, which we referred to as TIPs (tension-induced/inhibited proteins). TIPs displayed signature motifs characteristic of nuclear receptor coregulators and chromatin remodeling enzymes. A genomic BLAST search suggested that the three TIPs identified were isoforms originated by alternative splicing from a single gene. Functional studies revealed that TIP-1 and TIP-3 were involved in the cell's selection of the myogenic or the adipogenic pathway. TIP-1, induced by stretch, promoted myogenesis, while TIP-3, inhibited by stretch, stimulated adipogenesis. The selection involved TIP-mediated chromatin remodeling via a histone acetylation process and depended on TIP-1 and TIP-3 nuclear receptor binding boxes (NRBs). This study, therefore, suggests a new developmental mechanism linking the presence or absence of tension with divergent differentiation pathways.