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Substance P Affects Growth Factors in Pseudomonas aeruginosa–Infected Mouse Cornea

Foldenauer, Megan E. B. MA; McClellan, Sharon A. BS; Barrett, Ronald P. BS; Zhang, Yunfan MD; Hazlett, Linda D. PhD

doi: 10.1097/ICO.0b013e31824d6ffd
Basic Investigation

Purpose: This study analyzed the influence of substance P (SP) on growth factors related to wound healing in mice in the presence of infectious keratitis.

Methods: Naturally resistant mice were injected intraperitoneally with SP or phosphate-buffered saline and infected with Pseudomonas aeruginosa, and corneal messenger RNA (mRNA) levels of growth factors and apoptosis genes were tested. Enzyme-linked immunosorbent assay determined the protein levels, whereas immunohistochemistry tested the distribution, macrophage phenotype, and cell quantitation. In vitro, macrophages were stimulated with lipopolysaccharide (LPS; with or without SP) and mRNA levels of proinflammatory and antiinflammatory cytokines and apoptosis genes were tested.

Results: After SP, epidermal growth factor mRNA and protein levels were disparately regulated early, with no differences later in the disease. Hepatocyte growth factor and fibroblast growth factor-7 mRNA and protein levels were increased after SP treatment. Enumerating dual-labeled stromal cells revealed no difference between SP-treated versus phosphate-buffered saline–treated groups in the percentage of epidermal growth factor–labeled fibroblasts or macrophages, but there were significant increases in both hepatocyte growth factor– and fibroblast growth factor-7–labeled cells. Type 2 (M2) macrophages and caspase-3 mRNA levels were decreased, whereas B-cell lymphoma-2 mRNA expression was increased after SP treatment. In vitro, mRNA levels of several proinflammatory cytokines and B-cell lymphoma-2 were elevated, whereas transforming growth factor β was decreased after macrophage stimulation with SP (with LPS) over LPS alone. (Mice: n = 105 control; 105 experimental.)

Conclusions: These data show that treatment with SP in infectious keratitis elevates growth factors but also adversely affects the disease by enhancing the inflammatory response and its sequelae.

Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI.

Reprints: Linda D. Hazlett, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201 (e-mail:

Supported by National Institutes of Health Grants R01EY02986 and P30EY04068.

The authors declare no conflicts of interest.

Received August 3, 2011

Accepted January 27, 2012

Copyright © 2012 Wolters Kluwer Health, Inc. All rights reserved.