Purchase this article with an account.
JooHee Park, Dong Ju Kim, Roy S Chuck, Choul Yong Park; The Effect of Nitric Oxide on Human Corneal Epithelial Wound healing. Invest. Ophthalmol. Vis. Sci. 2017;58(8):145.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the effect of exogenous nitric oxide (NO) on corneal epithelial wound healing
Human corneal epithelial cells (HCEC) were cultured and exposed to various concentration of NaNO2 as NO donor. Cell viability and intracellular ROS generation were measured. The activation of autophagy and mTOR pathway was investigated by measured using western blot analysis. The effect of NO on HCEC migration was assayed using in vitro scratch model. LPS was used to induce cytotoxicity in HCECs. The rescuing effect of NO from LPS induced cytotoxicity was measured. The effect of NO on MAPKs (ERK, p38 and JNK) activation of HCECs was also investigated using western blot analysis. Corneal alkali burn model was made using Balb/c mice (20 males, 7 weeks old). The effect of NO on in vivo corneal wound healing from alkali burn was monitored. Inflammatory cytokine expression from alkali injured murine cornea was analyzed using quantitative PCR.
NaNO2 (0.1μM to 10μM) increased HCEC viability measured at 6, 24 and 48 hour exposure and HCEC migration in scratch assay for 5 days. NO induced dose dependent increase of intracellular ROS generation from HCEC at 20 min and 1 hr, however, this effect disappeared at 24 hr. Mild increase of LC3A/B II form was observed with 1 to 100 μM of NaNO2 with 24 hr incubation but statistically insignificant. No significant mTOR activation was found with NO up to 24 hr. NaNO2 (0.1μM to 10μM) rescued HCECs from LPS induced cytotoxicity. However, 1mM of NaNO2 increased autophagy and enhanced cytotoxicity induced LPS. LPS increased ERK activation of HCECs. Addition of NaNO2 (0.1μM to 10μM) in LPS stimulated HCECs further increased ERK phosphorylation. Other MAPKs such as p38 and JNK showed no significant change with addition of either LPS or NO. Topical treatment of 10 μM of NaNO2 solution for one week after alkani burn of cornea significantly enhanced corneal epithelial healing and decreased corneal opacity induced by chemical burn. The expression of key inflammatory cytokines such as MMP2, VEGF and IL-6 showed significant decrease with NaNO2 treatment.
Low concentration of NO increased HCEC proliferation and enhanced corneal epithelial wound healing both in vitro and in vivo. NO also rescued LPS induced HCEC cytotoxicity in dose dependent manner. ERK pathway is involved in HCEC wound healing promotion by NO.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only