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Sannes, Philip L., Ph.D.

Dr. Philip Sannes

 

 

 

 

 

 

 



Professor of Cell Biology
Ph.D.: The Ohio State University
Post-doctoral Training: Medical University of South Carolina, Division of Pathobiology
Phone: (919) 515-7656
Fax: (919) 515-4237
E-Mail: philip_sannes@ncsu.edu

Research Area:

Epithelial responses to injury and mechanisms of repair in the mammalian lung; the relationships between components of extracellular matrices and soluble growth factors, transcription factors, and signaling molecules which modulate gene and protein expression; factors that influence proliferation and differentiation of adult lung epithelium.

Current Research:

Our work has focused on the mechanisms that influence the activities of a key cell in the adult pulmonary alveolus - the type II (AT2) epithelial cell – that plays a crucial role in repair processes following injury. In addition to producing surfactant, it acts as a facultative stem cell with the capacity to renew itself as well as serve as the precursor for AT1 cells, which cover over 90% of the alveolar surface area.  We’ve developed data suggesting that the interaction of alveolar epithelial cells with specific components of the extracellular matrix (ECM) directly affects their ability to proliferate, to synthesize effector molecules vital to the repair process (such as cytokines and additional ECM components), and to effectively differentiate into type I (AT1) cells. We believe that one of the keys to repair following injury in the alveolar region is the distribution and nature of sulfated molecules that are either shed from cell surfaces in a soluble form or bound to fixed fibrillar components of ECM in insoluble forms, as found within the alveolar basement membrane (ABM). Specifically, the ABM microdomains associated with the AT2 cells are low in sulfate content, which promotes the biologic responsiveness of AT2 cells to critical growth factor signals (such as fibroblast growth factors -1 and -2) and normal proliferation - a major component of the repair process. The adjacent ABM microdomains of AT1 cells are high in sulfate content, which reduces the cell’s biologic responsiveness by slowing or impeding proliferation leading either to normal or, in some cases, aberrant repair.

More recent studies from our lab have confirmed that normal, constitutive maintenance of AT2 cell function is controlled by a balance of 1) gene expression modulated by low level Wnt signaling; and 2) an under-sulfated ABM.  AT1 cell turnover or injury/death leads to liberation of ECM-bound growth factors (e.g. fibroblast growth factors) which initiate DNA synthesis. Following cytokinesis, the differences in sulfation of the ABM microdomains beneath AT2 versus AT1 cells promote the asymmetric differentiation that follows.  The cell remaining in contact with the under-sulfated ABM and retains its AT2 phenotype, while the other moves onto the heavily sulfated ABM (left vacated by the dead AT1 cell) and is exposed to an environment rich in fixed and shed sulfated ECMs.  This triggers rapid, increased levels of forkhead transcription factors (Fox) and Wnt signaling molecules in parallel with increased TGFβ expression and signaling which together drive differentiation of AT2 cells into AT1 cells and reduce proliferation. This process is brief in vivo (~48 hours) but protracted in vitro (4-12 days).

These transient, interconnected events are linear and ordered to constitute essential autocrine and paracrine mechanisms for promoting proliferation, driving differentiation, and assuring stable cell numbers - vital cellular events in normal homeostasis and resolution of injury/disease states in the adult alveolus.

   

Figs. 1 and 2  Foxa1 expression may be an important factor in promoting the differentiation of AT2 cells into AT1 cells – a critical event in lung homeostasis. Figs. 1 and 2  Foxa1 expression may be an important factor in promoting the differentiation of AT2 cells into AT1 cells – a critical event in lung homeostasis. Following chemical injury to the alveolar epithelium, Foxa1 is seen in situ in rat alveolar epithelial cells during the differentiation of AT2 cells to AT1 cells. Immunofluorescence of the AT2 cell marker, surfactant protein-C (SP-C, in orange) in an AT2 cell not actively differentiating is seen next to an AT1 cell with Foxa1 (green) without SP-C (Fig. 1) while differentiating AT2 cells stain with both markers (Fig. 2).

Ongoing studies are expected to support the fundamental hypothesis that fixed (whole) and shed (fragments) of pericellular sulfated components of extracellular matrices alter the microenvironment of alveolar epithelial cells. They constitute critical determinants of cellular interactions, their proliferative potential, and their differentiated fate by influencing defined responses to Fox transcription factors, TGFβ and WNT signaling, and specific gene regulation. They will provide essential information needed to better understand basic cell-cell and cell-ECM relationships in adult lung homeostasis as well as the mechanisms that steer the pathogenesis of fibrosis in the lung as a consequence of injury and/or disease. 

Equipment and Techniques:

Molecular biology: RNA preparation and analysis, Northern blotting, qRT-PCR, cDNA cloning and characterization, inhibition and over-expression of selected gene targets by adenovirus and lentivirus gene transfer, gene arrays

Protein analysis: Western blotting, SDS-PAGE and native gel electrophoresis, biosynthetic labeling and analysis, radioactive and non-radioactive probes, CHiP assay.

Cell Biology and Microscopy: light and electron microscopy, immunohistochemistry, fluorescent probes, cytochemistry, isolation of primary lung cells and culture of cell lines.

Representative Publications:

Frevert C.W., and P. L. Sannes. 2005. Matrix proteoglycans as effector molecules for epithelial cell function. Eur. Respir. Rev. 14(97):137-144.

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.

Gabr, A. A., M. Reed, D. Newman, J. Pohl, J. Khosla, and P.L. Sannes. 2007. Alterations in Cytoskeletal and Immune Function-related Proteome Profiles in Whole Rat Lung Following Intratracheal Instillation of Heparin. Respir Res. 8(1):36

Newman, D., E. Walsh, K.B. C. Apparao, and P.L. Sannes. 2007. Heparin alters DNA Synthesis and 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. 293:1314-1320.

Apparao, K.B.C., D.R. Newman, H. Zhang, J. Khosla, and P.L. Sannes. 2008. Sulfated Extracellular Matrix Modulates Differentiation of Human Alveolar Type II Cells In Vitro. Proc. Amer. Thor. Soc. 5:372.

Newman, D.R., H. Zhang, K. Bortoff, J. Bonner, and P.L. Sannes. 2010. Alveolar epithelial differentiation during repair involves FoxA1, Wnt7A, and TGF-b. Proc. Amer. Thor. Soc. 7:155-156.

Apparao, K.B.C., D. Newman, H. Zhang, J. Khosla, S.H. Randell and P.L. Sannes. 2010. Temporal changes in expression of FoxA1 and Wnt7A in isolated adult human alveolar epithelial cells enhanced by heparin.  Anat. Rec. 293:938–946.

Meuten, T, A. Hickey, K. Franklin, B. Grossi, J. Tobias, H. Zhang, D.R. Newman, S. Jennings, M. Correa, P.L. Sannes. 2012. Wnt7B in fibroblastic foci of idiopathic pulmonary fibrosis. Resp. Res. Jul 28;13(1):62. PMID:22838404; PMCID:3479038

Zhang, H, D. R. Newman, and P. L. Sannes. 2012. HSulf-1 Inhibits ERK and AKT Signaling and Decreases Cell Viability in vitro in Human Lung Epithelial Cells. Respir Res. Aug 8;13(1):69. PMID:22873647

Zhang, H, D. R. Newman, J. C. Bonner and P. L. Sannes. 2012. Over-expression of Human Endosulfatase-1 Exacerbates Cadmium-induced Injury to Transformed Human Lung Cells In Vitro. Tox.& Appl.Pharm.doi.org/10.1016/j.taap.2012.09.008 PMID:23000194

Sannes Lab Personnel:


Huiying Zhang, MS, Graduate Student
zhyfly@hotmail.com

Donna Newman, PhD, Research Associate
donna_newman@ncsu.edu

Jody Khosla, Instructor & Teaching Porfessor
jody_khosla@ncsu.edu

Helena Morales Johansson, PhD, Research Assistant
hmorale@ncsu.edu

Na Young Yi, DVM, PhD, Post Doc Research Associate
nyyi@ncsu.edu


Pictured below: Na Young Yi, Jody Khosla, Huiying Zhang, Helena Morales Johansson, Donna Newman, Dr. Phil Sannes

Sannes Lab personnel