Our work has focused on t he mechanisms that influence the activities of a key cell in the pulmonary alveolus - the type II (AT2) epithelial cell – that plays a crucial role in repair processes following injury. In addition to producing surfactant, it serves as the precursor for the AT1 cell, which covers over 90% of the alveolar surface area. We suggested that it is the interaction of alveolar epithelial cells with specific components of the extracellular matrix (ECM) that directly affects their ability to proliferate and to synthesize additional effector molecules vital to the repair process, such as cytokines and additional ECM components. Specifically, we proposed that the keys to repair following injury in the alveolar region are sulfated molecules within the alveolar basement membrane (ABM). Specifically, regions of low sulfate in ABM's promote biologic responsiveness to critical growth factor signals (such as fibroblast growth factors -1 and -2) and normal proliferation - a major component of the repair process. Regions of high sulfate in ABM's, however, retard biologic responsiveness by slowing/impeding proliferation leading either to normal, or in some cases to aberrant repair. In previous and preliminary studies we showed that: 1) the ABM associated with AT2 cells in situ is less sulfated than that of ATI cells; 2) desulfated ECMs permit, and sulfated ECMs inhibit, growth factor stimulated-AT2 DNA synthesis; 3) sulfated ECMs down-regulate gene expression of key modifiers of AT2 cell proliferation and differentiation: fibroblast growth factors-1, -2, and -7, and fibroblast growth factor receptors -1 and –2; and 4) sulfated ECMs down-regulate expression of sulfotransferases by isolated AT2 cells - a mechanism for modifying the sulfation of their environment. We concluded from these collective observations that a dynamic relationship between ECMs, growth factors, and cell surfaces defines the AT2 cell's capacity to respond to as well as actively modify its environment in normal and pathologic processes. We proposed that a series of interrelated mechanisms control an orderly sequence of events following injury that insure resolution and which are modulated by sulfated ECMs. These events include growth factor-receptor binding, receptor-related signal transductive events at/near the cell surface, gene expression, expression of sulfotransferases that modify the cell's surface environment, and concludes with restablilization of cell populations and appropriate synthesis of ECM components.
The research highlights of the last five year period included proof that: 1) decreased levels of sulfate in ECM substrata act to up-regulate responses to heparin-binding growth factors by alveolar epithelial cells cultured on laminin substrata; 2) TGFβ-1 and FGF-1 regulate gene and protein expression of FGF-2 in AT2 cells; 3) heparin-binding epidermal growth factor is fibroblast mitogen produced by airway cells that induces an FGF-2 autocrine loop; 4) chondroitin sulfate proteoglycans are widely distributed in rapidly growing lung tissue during development, but are dramatically reduced and more focal at later time points coinciding with known patterns of fibrillar elements of ECM and more differentiated states; 5) FGF-1 synergizes with fibronectin to promote adhesion, cytoskeletal organization, and increased proliferation, and thus positively influences cell-substrata interactions and signaling during alveolar repair; 6) sulfated molecules in the ABM play important regulatory roles in selected AT2 cell activities during normal homeostasis, turnover and repair after lung injury; 7) the development of a basement membrane zone in airways is a postnatal event and involves FGF-2; 8) remodeling of the basement membrane zone following allergen exposure in developing monkeys involves basal cells of the airways and FGF-2; 9) heparin’s ability to alter DNA synthesis and gene expression in AT2 cells involves alteration of multiple signaling pathways; 10) heparin differentially regulates the gene expression of selected surfactant proteins by type II cells in vitro; 11) heparin modifies proteome profiles of lung in vivo.
Equipment
and Techniques:
Molecular biology: RNA preparation and analysis,
Northern blotting, RT-PCR, cDNA cloning and characterization
Protein analysis: Western blotting, gel electrophoresis,
biosynthetic labeling and analysis, radioactive and non-radioactive
probes
Microscopy: light and electron microscopy, immunohistochemistry,
fluorescent probes, cytochemistry
Representative
Publications:
Newman, D.R., Walsh, E., Apparao, K.B.C. and Sannes, P.L. 2007. Fibroblast growth factor-binding protein and
N-deacetylase/N-sulfotransferase-1 expression in type II cells is modulated by heparin and extracellular matrix.
Am.J. Physiol. Lung cell Mol. Physiol. 293:L1314-1320.
Amir A. Gabr, Mathew Reed, Donna R. Newman, Jan Pohl, Jody Khosla and Philip L. Sannes
Respiratory Research 2007, 8:36 (08 May 2007)
http://respiratory-research.com/content/8/1/36
Leiner, K.A., D. Newman, C-M. Li, E. Walsh, J. Khosla, and P.L. Sannes. 2006. Heparin and Fibroblast Growth Factors Affect Surfactant Protein Gene Expression in Type II Cells. Am. J. Respir. Cell Mol. Biol. 35: 611-618.
Wang, J., K. Sakamoto, J. Khosla and P.L. Sannes. 2002.
Detection of Chondroitin Sulfates and Decorin in Developing
Fetal and Neonatal Rat Lung. Am. J. Physiol. Lung Cell. Mol.
Physiol. 282:L484-L490.
Pagan, I., J. Khosla, C-M. Li, and P.L. Sannes. 2002.
Effect of Growth Factor-Fibronectin Matrix Interactions in
Rat Type II Cell Adhesion and DNA Synthesis. Exp. Lung Res.
28:69-84.
Li, C-M., D. Newman, J.Khosla, and P.L. Sannes. 2002.
Heparin Inhibits DNA Synthesis and Selected Gene _Expression
in Rat Alveolar Type II Cells . Am. J. Resp. Cell Mol. Biol.
27:345-352.
Evans, M.J., M.V. Fanucchi, L.S. Van Winkle, G.L. Baker, A.E.
Murphy, S. Nishio, P.L. Sannes, and C.G. Plopper.,
2002. Fibroblast growth factor-2 during postnatal development
of the tracheal basement membrane zone. Am. J. Physiol. Lung
Cell. Mol. Physiol. 283: L1263-L1270.
M.J. Evans, L.S. Van Winkle, M.V. Fanucchi, G.L. Baker, A.E.
Murphy, S. Nishio, E.S. Schelelgle, L.J. Gershwin, P.L.
Sannes, and C.G. Plopper. 2002. Remodeling of the Tracheal
Basement Membrane Zone of Infant Rhesus Monkeys Sensitized
with House Dust Mite Allergen Lab. Invest. 82(12):1747-54.
Evans, M.J., and P.L. Sannes. 2003. Development of the Extracellular Matrix. The Basement Membrane Zone. In THE LUNG: DEVELOPMENT, AGING AND THE ENVIRONMENT. R. Harding, K. Pinkerton, and C. Plopper, eds. Academic Press.
M.J. Evans, M.V. Fanucchi, G.L. Baker, L.S. Van Winkle, L.M. Pantle, S.J. Nishio, E.S. Schelegle, L.J. Gerswhin, L.A. Miller, D.H. Hyde, P.L. Sannes, and C.G. Plopper. 2003. Atypical Development of the Tracheal Basement Membrane Zone of Infant Rhesus Monkeys Exposed to Ozone and Allergen. Am J Physiol Lung Cell Mol Physiol 285: L931-939.
Newman, D., C-M. Li, R. Simmons, J.Khosla, and P.L. Sannes. 2004. Heparin Affects Signaling Pathways Stimulated by FGF-1 and FGF-2 in Type II Cells. Am J Physiol Lung Cell Mol Physiol 287: L191-L200.
Frevert C.W., and P. L. Sannes. 2005. Matrix proteoglycans as effector molecules for epithelial cell function. Eur. Respir. Rev. 14(97):137-144.
Ornitz, D., and P. Sannes. 2006. Fibroblast Growth Factors In Encyclopedia of Respiratory Medicine: Volume II, G. J. Laurent and S. D. Shapiro, editors. Elsevier, London. pp. 210-213.
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