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Scott Boitano

Publications and source records attributed to Scott Boitano.

8 recordsLinked to original sources
PubMed2006

Laminin-332 alters connexin profile, dye coupling and intercellular Ca2+ waves in ciliated tracheal epithelial cells.

BACKGROUND: Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin alpha3beta3gamma2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC). METHODS: Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca2+ waves. Expression of connexin (Cx) mRNA and proteins were assayed by reverse transcriptase - polymerase chain reaction and immunocytochemistry, respectively. RESULTS: When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular Ca2+ waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca2+waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins. CONCLUSION: Alterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function.

Animals↗
PubMed2005

Extracellular matrix-driven alveolar epithelial cell differentiation in vitro.

During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and re-establish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell-specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell-specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5. Over days 5 to 7 in culture, cells took on an AT1-like phenotype (on collagen/fibronectin alone; collagen alone; or Ln5 alone), an AT2-like phenotype (on collagen/fibronectin/Ln5; or collagen/Ln5), or both AT1-like and AT2-like phenotypes (on collagen/fibronectin matrix with a subsaturating amount of Ln5). Cells transferred between matrices at day 4 of culture retained the ability to alter day 7 phenotype. We conclude that in vitro, (1) AT2 cells exhibited phenotype plasticity that included an intermediate cell type with both AT1 and AT2 cell characteristics independent of day 7 phenotype; (2) both collagen and Ln5 were needed to promote the development of an AT2-like phenotype at day 7; and (3) components of the extracellular matrix (ECM) contribute to phenotypic switching of alveolar cells in culture. The described tissue culture models provide accessible models for studying changes in alveolar epithelial cell physiology from AT2 cell progenitors to the establishment of alveolar epithelial monolayers that represent AT1-like, AT2-like, or a mix of AT1- and AT2-like cells.

Animals↗
PubMed2005

Bordetella bronchiseptica adherence to cilia is mediated by multiple adhesin factors and blocked by surfactant protein A.

In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg- phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg- strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.

Adhesins, Bacterial↗
PubMed2004

Cell-cell interactions in regulating lung function.

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.

Animals↗
PubMed2003

Cell-cell communication in heterocellular cultures of alveolar epithelial cells.

The mammalian alveolar epithelium is composed of alveolar type I (AT1) and alveolar type II (AT2) cells that together coordinate tissue function. We used a heterocellular culture model of AT1 and AT2 cells to determine pathways for intercellular signaling between these two phenotypes. Gap junction protein (connexin) profiles of AT1 and AT2 cells in heterocellular cultures were similar to those seen in rat lung alveolar sections. Dye coupling studies revealed functional gap junctions between and among each cell phenotype. Localized mechanical stimulation resulted in propagated changes of intracellular Ca2+ to AT1 or AT2 cells independent of the stimulated cell phenotype. Ca2+ communication that originated after AT1 cell stimulation was inhibited by gap junction blockers, but not by an inhibitor of extracellular nucleotide signaling (apyrase). Conversely, Ca2+ communication after stimulation of AT2 cells was not significantly reduced by gap junction inhibitors. However, apyrase significantly reduced Ca2+ communication from AT2 to AT1 cells, but not from AT2 to AT2 cells. In conclusion, AT1 and AT2 cells have unique connexin profiles that allow for functional coupling and distinct intercellular pathways for coordination of Ca2+ signaling.

Animals↗
PubMed2003

Functional BvgAS virulence control system in Bordetella bronchiseptica is necessary for induction of Ca2+ transients in ciliated tracheal epithelial cells.

To study initial Bordetella bronchiseptica-tracheal epithelial cell interactions, we coincubated B. bronchiseptica with rabbit tracheal explant cultures and assayed bacterial adherence and host cell Ca(2+) signaling. Wild-type B. bronchiseptica (RB50) preferentially adhered to cilia and induced ciliated host cell Ca(2+) transients within 2 min of coincubation, whereas coincubation with an avirulent strain (RB57) resulted in limited binding and Ca(2+) signaling. The described cell system allows for assessment of initial B. bronchiseptica-host cell interactions that can contribute to pathogenicity or to host cell defense.

Animals↗
PubMed2002

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix.

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.

Animals↗