Beta2-adrenergic receptor signaling mediates corneal epithelial wound repair

Abstract

Purpose: Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting

Results: With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing.

Methods: Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein).

Conclusions: The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair.

Figure 1

Superficial corneal wounding is confined to the epithelium. A 2-mm disposable biopsy punch was blotted with methylene blue and was gently depressed against the murine cornea, outlining the zone for deepithelialization. Two-millimeter circular, axial corneal defects were created using a 2-mm crescent blade and a LASEK micro hoe. Dissected corneas (n = 4) were embedded in methacrylate resin, cut at a thickness of 1.5 μm, and stained with toluidine blue.

Figure 2

β2-AR is required for β-adrenergic agent-mediated modulation of corneal epithelial cell migration. Passage 1 adult murine corneal epithelial cells from β2-AR^+/+ or β2-AR^−/− mice were plated on collagen-coated, glass-bottom culture dishes at a density of 26 cells/mm² in mCGM and were incubated for 2 hours at 37°C. The migration of single cells in the microscopic field of view was measured for a 1-hour period while the cells were in mCGM alone, 1 μM isoproterenol, or 20 μM timolol. Data displayed are the mean ± SEM of the speed (μm/min) of each individual cell for each treatment group. *P < 0.001 compared with control; Student's t-test.

Figure 3

β2-AR is required for the delay of in vivo corneal epithelial wound healing by a β-AR agonist and the acceleration of healing by a β-AR antagonist. Using male β2-AR^+/+ (n = 25) or β2-AR^−/− mice (n = 25), 2-mm diameter circular, axial corneal epithelial wounds were created using a crescent blade and a LASEK micro hoe. Corneas were treated topically with BSS, BSS with 1% isoproterenol every 4 hours, or BSS with 0.5% timolol twice daily, and wound healing was monitored stereomicroscopically using fluorescein staining, as shown (A). The polygonal area (A_p) of the wound was determined using NIH ImageJ, as described (B, C). Values plotted are the rates of wound healing, with standard error of the coefficient illustrated by error bars (D). *P < 0.05 using the F-test, compared with congenic, control group β2-AR^+/+ mice.

Figure 4

Deletion of the β2-AR in corneal epithelial cells interrupts the modulation of ERK signaling by β-adrenergic agents. Lysates of confluent cultured AMCE cells from male β2-AR^+/+ and β2-AR^−/− mice that had been treated with mCGM alone (control), 1 μM isoproterenol (β-AR agonist), or 20 μM timolol (β-AR antagonist) were electrophoresed and immunoblotted with an anti-ERK antibody and an anti-phospho-ERK (P-ERK) antibody (A). Blots from separate experiments were scanned, and densitometry was performed (NIH Image 1.62). Data were normalized so that the average of the mean phosphorylated ERK signal (42 kDa P-ERK) divided by the mean ERK signal for control was set at 1. All data were averaged, statistically analyzed, and represented graphically as a ratio of the control (B). Values plotted are mean ± SEM. *P < 0.01 compared with control. Data shown are representative of two independent experiments in duplicate from murine corneal epithelial cells isolated from multiple corneas.

Figure 5

Murine corneal epithelial cells expressed the catecholamine synthetic enzyme tyrosine hydoxylase and contained epinephrine. Lysates of confluent cultured AMCE cells from β2-AR^+/+ and β2-AR^−/− mice were electrophoresed and immunoblotted with an anti-tyrosine hydroxylase antibody (A). Lysates of corneal epithelial sheets from β2-AR^+/+ and β2-AR^−/− mice obtained by mechanical deepithelialization were tested in triplicate in an epinephrine (EPI) enzyme immunoassay. The amount of epinephrine (pg) per milligram protein in the extract was graphically represented ± SEM (B). *P < 0.05 compared with HCGS levels of epinephrine.