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Cristina M. Kenney, Marilyn Chwa, James V. Jester, Donald J. Brown; Mechanical Strain Generates ROS/RNS Production by Human Corneal Fibroblasts in vitro. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2049.
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Using non-linear optical microscopy to image second harmonic generated signals from collagen, we have previously shown that keratoconus (KC) corneas have a structural defect in the macroscopic organization of the stroma that may destabilize the mechanical integrity and increase the susceptibility to shear stress. The present study was designed to determine if mechanical strain, such as would be found in KC corneas, can induce production of reactive oxygen/nitrogen species (ROS/RNS) in human corneal fibroblasts in vitro.
Normal human corneal fibroblasts were plated on Collagen I BioFlex Plates. Cells were exposed to repeated 15% strain for 45 repetitions and then allowed to recover for 60 minutes. The ROS/RNS levels were measured with the H2DCFDA assay. Cells were also pretreated for 1 hour with antioxidant inhibitors: (1) N-Acetyl-L cysteine (L-NAC, 10µM), a precursor of the antioxidant glutathione and a ROS scavenger; (2) Nω-nitro-L-arginine (Nitroarginine, L-NNA, 10µM), a nitro derivative of the amino acid arginine and an irreversible inhibitor of constitutive nitric oxide synthase (eNOS) and a reversible inhibitor of iNOS; and (3) Apocynin (30µM), a NAD(P)H oxidase inhibitor for superoxides. Control cells were not stretched.
In corneal fibroblasts the ROS/RNS levels increased 78% with the mechanical stretch compared to untreated control cells (p<0.001). When pretreated with L-NAC prior to stretching, the ROS/RNS levels decreased by 15% compared to the stretched cells (p=0.026). Pretreatment with Apocynin prior to stretching resulted in 57% decrease in ROS/RNS levels compared to the stretched cells (p=0.0025). Cells pretreated with L-NNA had a 29% decrease in ROS/RNS production compared to the stretch alone (p=0.042). Cultures treated with inhibitors alone showed ROS/RNS levels similar to untreated controls.
This work shows that mechanical strain which could occur on an unstable matrix, can lead to higher ROS/RNS levels involving both the NAD(P)H oxidase and the nitric oxide pathways. We propose that mechanical instability can influence the corneal cells to respond with increased oxidative stress, a feature common in human ectatic corneal diseases.
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