Increased Stress-Induced Generation of Reactive Oxygen Species and Apoptosis in Human Keratoconus Fibroblasts

Abstract

PURPOSE.To determine whether keratoconus (KC) corneal fibroblast cultures have increased reactive oxygen species (ROS) production and are more susceptible to stress-related challenges. METHODS. Normal (n ϭ 9) and KC (n ϭ 10) stromal fibroblast cultures were incubated in either neutral-or low-pH conditions, with or without hydrogen peroxide. Catalase activities were measured with a fluorescent substrate assay. Superoxide and ROS/reactive nitrogen species (RNS) productions were determined with an amine-reactive green-dye assay and 2Ј,7Ј-dichlorodihydrofluorescein diacetate (H 2 DCFDA) dye assay, respectively. Cell viability was analyzed by a dye-exclusion assay. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. A cationic (green) dye was used to measure the mitochondrial membrane potential (⌬⌿m). RESULTS. KC fibroblasts had increased superoxide and ROS/RNS production (6.2-fold, P Ͻ 0.001 and 1.8-fold, P Ͻ 0.001, respectively) and catalase activity (P Ͻ 0.01) with higher concentrations of H 2 O 2 compared with normal cultures (P ϭ 0.16). After a low-pH stress challenge, KC fibroblasts maintained higher ROS/RNS levels (3.3-fold, P Ͻ 0.02), showed higher caspase-3 activity (7.5-fold, P Ͻ 0.02) and decreased ⌬⌿m (2.6-fold, P Ͻ 0.04), and had decreased cell viability (37%, P Ͻ 0.005 vs. 20%, P Ͻ 0.27) compared with normal fibroblasts. CONCLUSIONS. Under identical conditions, KC fibroblasts had increased basal generation of ROS/RNS and were more susceptible to stressful challenges (low-pH and/or H 2 O 2 conditions) than were normal fibroblasts. In addition, the stressed KC fibroblasts possessed characteristics similar to those found in the intact KC corneas (increased catalase activity, ROS production, and apoptosis). These properties may play a role in the pathogenesis of KC. (Invest Ophthalmol Vis Sci.

Keratoconus: an inflammatory disorder?

“…19,20,106,[108][109][110]144 An important decrease in the level of protease inhibitors such as cystatins (inhibitors of cysteine proteases) and TIMP-1 (inhibitor of MMPs) have also been reported. 95,96,101,[129][130][131] The increased activity of several proteolytic enzymes results in higher concentrations of ROS, RNS, cytotoxic aldehydes (CAs) and peroxynitrates (Ps) (which decreases the activity of TIMP-1 and increase MMP-2), 63,143,144,146,149 and given the lower production of SOD 143 possibly related to IL-1α, 158 an environment with high oxidative stress and low pH is formed, causing an increase in the activation of the caspases (caspase-9 and -12), mitochondrial dysfunction (MD), and DNA damage, 156 which eventually lead to increased apoptosis. All of these could probably be the result of a complex interaction of both genetic predisposition and environmental triggering factors, such as eye rubbing and contact lenses wear (the 'two-hit hypothesis') in keratoconus.…”

“…Recent studies have shown that keratoconic corneas have increased levels of mtDNA mutations compared with controls. [63][64][65][66] In addition, it has been also established that the mean relative mtDNA content is higher in patients with the disease. 67 Genetics of keratoconus has been recently extensively reviewed by Abu-Amero et al 52 …”

“…63,143,144,146 In addition, in vitro studies have shown that IL-1α, which is high in keratoconus, has the ability to decrease the synthesis of the SOD, whereby antioxidant defenses become insufficient. 147 NO is an endogenous substance, which regulates complex processes in ocular tissues.…”

Apoptosis in the initiation, modulation and termination of the corneal wound healing response

“…Thus, in studies that included unwounded control corneas in rabbits (Helena, et al, 1998, Mohan, et al, 2003, Szentmary, 2005, mice , or humans (Kim, et al, 1999), almost no apoptotic keratocytes or other stromal cells were noted in control corneas, even when dozens of tissue sections were examined. Apoptotic keratocytes may, however, be noted away from sites of epithelial injury in keratoconus corneas (Kim, et al, 1999), leading to the hypothesis that abnormally high levels of ongoing keratocyte apoptosis could play a role in the pathophysiology of this ectatic corneal disease (Kim, et al, 1999;Chwa, et al, 2006). Abnormally high levels of keratocyte apoptosis have also been associated with the pathophysiology of aniridia (Ramaesh, et al, 2006).…”

VSX1 gene variants are associated with keratoconus in unrelated Korean patients

“…Based on studies reporting significant linkage of keratoconus, several pathogenic mutations in VSX1 (MIM 605020; visual system homeobox gene 1, zebrafish, homolog of), located on 20p11.21, have been detected in multigenerational pedigrees of keratoconus, with a strong association and genetic variants of VSX1 that has been validated by multiple studies (Aldave et al 2006;Bawazeer et al 2000;Bisceglia et al 2005;Chwa et al 2006;Heon et al 2002;Kenney and Brown 2003;Kuming and Joffe 1977;Liskova et al 2007;Tang et al 2008). Although several variants of VSX1, e.g., L17P, L159 M, and R166 W, have been reported to be pathogenic in multigenerational pedigrees of keratoconus with various associations, the identified pathogenic mutations has shown various associations in familial keratoconus patients with different ethnicities (Aldave et al 2006;Bisceglia et al 2005;Heon et al 2002;Liskova et al 2007;Tang et al 2008).…”