Oxidative and nitrative stresses are thought to be critical mediators of lung injury and subsequent induction of inflammatory responses. Proteins constitute a major target of superoxide (O) and nitric oxide (NO) reactivity. Under exposure to these reactive stresses, protein modifications such as S-nitrosylation (SNO), carbonylation and 3-nitrotyrosine (3NT) occur and have been shown to act both physiologically and pathologically by altering protein function. The pulmonary collectins, Surfactant Proteins (SP)-A and SP-D modulate immune functions in the lung and nitration of SP-A abrogates its functions. Preliminary data indicate differential localization and expression of SNO and 3NT after bleomycin-induced lung injury in rodents. In addition, we have documented dramatic increases in lavage hyaluronic acid (HA) in injured lungs and HA-binding peptide treatment limits the inflammatory and fibrotic response to injury. Interestingly, reactive oxygen and nitrogen species (RONS) fragment high molecular weight (HMVV) HA into low molecular weight (LMW) forms that promote macrophage activation, cytokine gene expression and chemotaxis. The HA receptors CD44 and RHAMM have been implicated in these inflammatory responses both in vitro and in vivo. Preliminary data indicate that anti-RHAMM antibody blocks SP-A-mediated macrophage chemotaxis and that membrane lipid rafts are required for SP and HA signaling. Using the rodent intratracheal bleomycin model of lung injury, we will test the hypothesis that RONS, occurring as a result of lung injury, generate both GAG adducts and the post.translational modification of specific protein targets that collectively regulate macrophage activation, inflammatory cytokine gene expression and chemotaxis so as to promote pulmonary inflammation. Using both pharmacologic (chemical blockers) and transgenic (SP-ND knockouts) approaches, Aim 1 will determine the contribution of RONS to post-translational modifications of SPND, formation of LMW HA and inflammation after bleomycin injury. In addition, using specific antibody and peptide blockers, Aim 2 will focus on defining the roles of HA, CD44 and RHAMM in the regulation of macrophage functions by native and nitrated SP-ND in vitro.
In Aim 3, the mechanisms of altered macrophage function will be further examined by determining the formation of a lipid raft-associated signaling unit that regulates SP-ND and LMW HA-mediated macrophage behavior. Therapeutic intervention with specific blockers of lipid rafts will then be tested for their efficacy in blocking inflammation after lung injury. The experiments described in this proposal will define the molecular mechanisms of pulmonary collectin and LMW HA regulation of macrophage function and will explore novel targets for therapeutic intervention to limit the inflammatory response to lung injury.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL073896-04
Application #
7279188
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Harabin, Andrea L
Project Start
2004-04-01
Project End
2009-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
4
Fiscal Year
2007
Total Cost
$319,974
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pediatrics
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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