UVM Theses and Dissertations
Format:
Print
Author:
Bove, Peter F.
Dept./Program:
Cell and Molecular Biology Program
Year:
2007
Degree:
Ph. D.
Abstract:
The respiratory epithelium is frequently damaged after exposure to infectious or noninfectious environmental agents. Regardless of the source of injury, lesions can lead to the loss and alterations of the surface epithelium, resulting in the shedding of epithelial cells. After injury, the respiratory epithelium initiates a wound healing process to restore its barrier integrity. Nitric oxide (NO) is produced by many diverse cell types, including the airway epithelium, as a cellular or intracellular signaling molecule, by the activation of nitric oxide synthases (NOSs). All three known NOS isoforms are expressed within the respiratory tract and mediate various airway functional properties such as host defense and wound repair. Increased airway NO production in inflammatory respiratory tract diseases, including asthma, may activate additional signaling mechanisms to regulate inflammatory-immune pathways, and epithelial barrier repair.
Development, tissue remodeling, cell migration, inflammation, angiogenesis, and wound healing are just some of the physiological processes in which the matrix metalloproteinases (MMPs) play a crucial role. MMPs are a family of proteolytic enzymes that collectively have the capacity to degrade virtually every component of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9, 92-kD gelatinase B) is induced in conditions of airway inflammation, and participates inepithelial repair following injury. The inflammatory mediator, NO, is known to control the expression and activation of MMP-9 within the epithelium, however, the exact nature of NO-mediated MMP-9 regulation and its significance for airway epithelial repair are unclear.
Using primary or immortalized bronchial epithelial cells, we demonstrate that low, physiological concentrations of NO promote epithelial cell migration and wound repair in an in vitro wound assay, which is associated with increased activation of MMP-9. Endogenous NO from NOS2 stably expressing HBEI cells, or exogenously-generated NO from the NOdonor, diethylenetriamine NONOate (DETA NONOate) (10 [mu]M), demonstrate stimulatory effects on epithelial wound repair, and MMP-9 expression was found to be dependent on a cGMP-mediated pathway. Moreover, pharmacological MMP inhibition and siRNA knockdown of MMP-9 expression demonstrate the significant contribution of MMP-9 to NOmediated wound repair.
In addition to NO promoting epithelial repair, we also reveal a novel participation of the NADPH oxidase homolog, Dual Oxidase (Duox)-I, which may be involved in cellular redox signaling. We demonstrate that ATP-mediated activation of Duox-1 promotes epithelial cell migration and wound repair also through regulation of MMP-9. Collectively, we reveal a strong correlation for NO-mediated epithelial cell migration and wound repair depending on the local environment within the airway, through its regulation of MMP-9 activation. This data suggests a potential important role for NO-mediated epithelial remodeling and inflammatory processes in vivo.
Development, tissue remodeling, cell migration, inflammation, angiogenesis, and wound healing are just some of the physiological processes in which the matrix metalloproteinases (MMPs) play a crucial role. MMPs are a family of proteolytic enzymes that collectively have the capacity to degrade virtually every component of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9, 92-kD gelatinase B) is induced in conditions of airway inflammation, and participates inepithelial repair following injury. The inflammatory mediator, NO, is known to control the expression and activation of MMP-9 within the epithelium, however, the exact nature of NO-mediated MMP-9 regulation and its significance for airway epithelial repair are unclear.
Using primary or immortalized bronchial epithelial cells, we demonstrate that low, physiological concentrations of NO promote epithelial cell migration and wound repair in an in vitro wound assay, which is associated with increased activation of MMP-9. Endogenous NO from NOS2 stably expressing HBEI cells, or exogenously-generated NO from the NOdonor, diethylenetriamine NONOate (DETA NONOate) (10 [mu]M), demonstrate stimulatory effects on epithelial wound repair, and MMP-9 expression was found to be dependent on a cGMP-mediated pathway. Moreover, pharmacological MMP inhibition and siRNA knockdown of MMP-9 expression demonstrate the significant contribution of MMP-9 to NOmediated wound repair.
In addition to NO promoting epithelial repair, we also reveal a novel participation of the NADPH oxidase homolog, Dual Oxidase (Duox)-I, which may be involved in cellular redox signaling. We demonstrate that ATP-mediated activation of Duox-1 promotes epithelial cell migration and wound repair also through regulation of MMP-9. Collectively, we reveal a strong correlation for NO-mediated epithelial cell migration and wound repair depending on the local environment within the airway, through its regulation of MMP-9 activation. This data suggests a potential important role for NO-mediated epithelial remodeling and inflammatory processes in vivo.