Low levels of hydrogen peroxide stimulate corneal epithelial cell adhesion, migration, and wound healing

Abstract

Purpose: Intracellular reactive oxygen species have been reported to associate with growth factor and integrin signalings in promoting cell adhesion in many cell types. This study is to explore if exogenous H(2)O(2) at low levels can be beneficial to cell adhesion, migration, and wound healing.

Methods: Primary rabbit corneal epithelial cells treated with 0-70 μM H(2)O(2) were tested for viability by MTT assay, adhesion by centrifugation assay, focal contacts of vinculin and F-actin by immunofluorescence, activated Src(pY416), EGF receptor (pY845), vinculin(pY1065), FAK(pY397), and FAK(pY576) by immunoblotting. Cell migration was examined with 0-50 μM H(2)O(2) using the scratch wound technique. Corneal wound healing of ex vivo pig model and in vivo mouse model was examined using H(2)O(2) with and without antioxidant N-acetylcysteine (NAC).

Results: Compared with the untreated control, H(2)O(2) at 10-50 μM stimulated cell viability and facilitated adhesion and migration with clear induction of vinculin-rich focal adhesions and F-actin-containing stress fibers by increasing activated Src, FAK(pY576), and vinculin(pY1065). H(2)O(2) also increased phosphorylation of EGFR(Y845) parallel to that of activated Src, but both were eliminated by NAC and PP1 (Src inhibitor). Finally, H(2)O(2) induced faster wound healing in cornea both in vitro and in vivo, but the healing was diminished by NAC.

Conclusions: These findings suggest that H(2)O(2) at low levels promotes cell adhesion, migration, and wound healing in cornea cells or tissue, and the interaction of H(2)O(2) with Src plays a major role.

Figure 1.

Effect of low concentrations of H₂O₂ on the viability of RCE cells. Cultured RCE cells (2 × 10⁵cells /mL) were serum starved (with 0.5% FBS) overnight and then cultured in EGF and serum-free medium for 30 minutes before experiment. Cells were thereby exposed to between (control) and 70 μM H₂O₂ diluted with serum and EGF-free medium for 24 hours. The viability of RCE cells was measured using an MTT assay and data expressed as percentage cell viability compared with control. Results are shown as mean ± SE (n = 6). *P < 0.05 and **P < 0.01 compared with control.

Figure 2.

Effect of low concentration of H₂O₂ on RCE cell-to-matrix adhesion by centrifugation assay. RCE cells were gradually serum starved as in Figure 1. Cells were then pretreated with between 0 (control) and 60 μM H₂O₂, 100 μM NAC, and 100 μM NAC + 20 μM H₂O₂, respectively, in suspension (2 × 10⁵cells /mL). (A) Cells were seeded on Fn-coated dishes in the absence of serum and growth factor with H₂O₂ concentrations of 0, 1, 5, 10, 20, 50, and 60 μM H₂O₂. After 30 minutes, nonadherent cells were removed with centrifugation force at 200g. Adherent cells were fixed and stained with formaldehyde/crystal violet, quantified by measuring the absorbance at OD₅₇₀. (B) Same experiment as (A), except that the cells were allowed 60 minutes of adhesion before centrifugation at 300g. (C) Comparison of cells treated with NAC alone, NAC + H₂O₂, H₂O₂ alone, and the untreated control in conditions the same as in (B). The adhered cells were plotted as the percentage of control (0 μM H₂O₂) and shown as mean ± SE (*P < 0.05 and **P < 0.01 vs. control; n = 6).

Figure 3.

Low-concentration H₂O₂ accelerates RCE cell attachment to ECM. RCE cells were serum starved and pretreated with 0 μM H₂O_2, 20 μM H₂O₂, 100 μM NAC, and 100 μM NAC + 20 μM H₂O₂ as described in Figure 2. Cells in each group were plated onto an Fn-coated dish for 15 minutes, 30 minutes, and 60 minutes, respectively. Cells were then fixed, and individual fields from multiple experiments were photographed by phase-contrast microscopy. The result is representative of three independent experiments. Arrow indicates cells with polarity shape.

Figure 4.

The effect of low concentration of H₂O₂ on focal adhesion and stress fiber formation. RCE cells, which grew to confluence in normal medium overnight, were taken as the positive control (panel 1). Cells pretreated with either 0 μM H₂O₂, 20 μM H₂O₂, 100 μM NAC, or 100 μM NAC + 20 μM H₂O₂ were plated onto an Fn-coated dish for 30 minutes, fixed, and stained for F-actin (red) and vinculin (green). Cells pretreated with 20 μM H₂O₂ (panel 4) revealed filament-like actin (arrow) and dotlike focal adhesions containing vinculin (arrowhead), which were not present in the 0 μM H₂O₂-treated cells (panel 2). NAC (100 μM) antagonized 20 μM H₂O₂–facilitated formation of the focal adhesion of vinculin and F-actin (panel 5), whereas NAC alone (panel 3) showed the same results as the cells without H₂O₂ treatment in panel 2.

Figure 5.

Low concentrations of H₂O₂ increases Src activity on integrin engagement. (A) Cells were resuspended in serum and EGF-free medium containing either 0 μM H₂O₂, 20 μM H₂O₂, 100 μM NAC, 100 μM NAC + 20 μM H₂O₂, 10 μM PP1, or 10 μM PP1 + 20 μM H₂O₂ and kept in suspension for 30 minutes. Thereby, cells were plated onto an Fn-coated dish for 30 minutes, then lysed and immunoblotted with antibodies against Src(pY419) and total Src. (B) The ratio of Src(pY419)/Src was quantified by densitometric scanning of immunoblots and plotted as a percentage of control (0 μM H₂O₂) and shown as mean ± SE (*P < 0.05 vs. control; n = 3). (C) Cells were processed as in (A) and then lysed and immunoblotted with antibodies against EGFR(pY 845) and EGFR. (D) The ratio of EGFR(pY 845)/EGFR was quantified as in (B). (E) Cells pretreated with 0 μM H₂O₂ or 20 μM H₂O₂ were plated onto an Fn-coated dish for 30 minutes, lysed, and immunoblotted with antibodies against vinculin(pY1065) and total vinculin, as well as (F) FAK(pY397), FAK(pY576), and FAK. (G) The ratios of vinculin(pY 1065)/vinculin, FAK(pY 397)/FAK, and FAK(pY 576)/FAK were quantified by densitometric scanning of immunoblots and plotted as percentage of control (0 μM H₂O₂) and shown as mean ± SE (*P < 0.05 vs. control; n = 3). Con., control.

Figure 6.

Effect of low H₂O₂ on scratch wound closure in vitro. (A) Cells pretreated with 0 (control), 5, 10, 20, 50 μM H₂O₂, 100 μM NAC, or 100 μM NAC + 20 μM H₂O₂ were grown to confluence on Fn-coated 12-well plates, and a scratch wound on the monolayer was created by a sterile plastic pipette tip. Cells were then allowed to migrate into the wound area in the absence of serum and growth factor. Micrographs of cell migration into the scratch-wound region were recorded at 0, 8, and 16 hours under phase-contrast microscope. (B) Confluent monolayer of cells pretreated with 0 (control), 20 μM H₂O_2, 10 μM PP1 (Src inhibitor), and 10 μM PP1 + 20 μM H₂O₂ were each made a scratch wound as in (A), and the wound closure (migration) was observed and recorded over 0, 8, and 16 hours (C). Images of wound closure were analyzed quantitatively at 16 hours. In total, four different areas were sampled for each group. Data are expressed as percentage rate of wound closure in comparison with that of the control and shown as mean ± SE (*P < 0.01, #P < 0.05, n = 3).

Figure 7.

The effect of low level of H₂O₂ on wound healing in cornea organ culture. (A) The cornea wound on fresh pig eyeball was made using a hollow plastic column (9 mm in diameter) in the center of the cornea. Epithelium inside the circle was removed. The wounded corneas were allowed to heal in the medium containing 20 μM H₂O₂ with or without 40 mM NAC in a humidified CO₂ incubator. After 24 or 48 hours of incubation, the corneas were stained with Richardson's dye for taking photos. A wounded cornea without treatment was used as a negative control. In addition one cornea without wounding and another cornea with wound were stained with Richardson's dye at 0 hour, and the photos were taken and included for comparison. (B) Graph represents the percentage of the wound area healed in 24 and 48 hours, shown as mean ± SE. The data at 24 and 48 hours in H₂O₂ and H₂O₂ + NAC group were compared with the control group (*P < 0.01, n = 3).

Figure 8.

Effect of low H₂O₂ on mouse corneal epithelial wound healing in vivo. (A) C57BL/6 mice were used for this in vivo study. Each cornea was scraped at the center area with a spatula to form an epithelial wound. These animals were divided into four groups, and the eyes were treated every 6 hours up to 48 hours with the following eye drop formulations: The first group was treated with an eye drop formulation containing 400 μM H₂O₂; the second group was treated with an eye drop containing 400 μM H₂O₂ + 40 mM NAC; the third group was treated with an eye drop containing 40 mM NAC; and the fourth group (control) was treated with PBS without any additive. Photos were taken at time intervals of 0, 12, 24, 36, and 48 hours after using fluorescein staining of the corneas to allow clear demarcation of the wounding surface (green). White dotted outline indicates the fluorescein-stained wound region. (B) Quantification of the wound healing in the in vivo mouse study. The area of the healed region in each corneal wound was quantified, expressed as percentage rate of wound healing as a function of time (hours) and shown as mean ± SE. The data at 36 hours in H₂O₂, NAC, and H₂O₂ + NAC group was each compared with control group (*P < 0.01, n = 4).