Defense Date

2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physiology and Biophysics

First Advisor

Bruce K. Rubin, M.Engr., M.D., M.B.A., FRCPC

Second Advisor

John F. Hunt, M.D.

Third Advisor

Alison J. Montpetit, R.N., Ph.D.

Fourth Advisor

Judith A. Voynow, M.D.

Fifth Advisor

Roland N. Pittman, M.D.

Sixth Advisor

Vijay Lyall, Ph.D.

Abstract

Rationale: Tissue Factor (TF) is a transmembrane glycoprotein that canonically functions as the initiator of the coagulation cascade. Increased levels of TF have been associated with inflammatory airway diseases. Since lipopolysaccharide (LPS) is known to elicit and inflammatory response in airway epithelium, we hypothesized that airway epithelial cells release TF when exposed to LPS. Since TF aids in local wound healing, we also hypothesized that inhibition of TF would decrease NHBE growth. The specific aim of this work was to evaluate the effects of LPS exposure on TF production and release from airway epithelia and determine the signaling pathways involved. A secondary aim was to evaluate the effects of TF inhibition on NHBE growth.

Methods: Normal human bronchial epithelial cells were grown in submerged cell culture and exposed to LPS as well as several intracellular signaling pathway agonist and inhibitors.

Measurements: Tissue Factor mRNA and protein were measured in culture media and cell lysate by reverse-transcriptase polymerize chain reaction and enzyme-linked immunosorbent assay, respectively. Signaling pathways were evaluated using selective agonists and inhibitors.

Main results: TF protein levels increased nearly two-fold in cell media after exposure to LPS (p < 0.01). This did not occur in the presence of an MEK/ERK inhibitor (PD98059) or a SMAD inhibitor (SB431542). TF protein levels also increased nearly ten-fold in the presence of TGF-beta (p < 0.05). mRNA of TF and TGF-beta was not altered by LPS or TGF-beta exposure. NHBE grown in the presence of Tissue Factor Pathway Inhibitor grew significantly slower than those grown in standard media (P < 0.05).

Conclusions: NHBE release TF when exposed to LPS. This phenomenon is post-translational and may be mediated by an autocrine mechanism involving MEK/ERK signaling that increases TGF-beta which then leads to the release of TF. Our data suggest that this airway epithelium release of TF serves as a local repair function.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-7-2017