Bernard F. Godley

Oxidative stress-induced mitochondrial DNA damage in human retinal pigment epithelial cells: a possible mechanism for RPE aging and age-related macular degeneration

Oxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD). Although the vision loss of AMD results from photoreceptor damage in the central retina, the initial pathogenesis involves degeneration of RPE cells. Evidence from a variety of studies suggests that RPE cells are susceptible to oxidative damage. Mitochondrial DNA (mtDNA) is particularly prone to oxidative damage compared to nuclear DNA (nDNA). Using the quantitative PCR assay, a powerful tool to measure oxidative DNA damage and repair, we have shown that human RPE cells treated with H(2)O(2) or rod outer segments resulted in preferential damage to mtDNA, but not nDNA; and damaged mtDNA is not efficiently repaired, leading to compromised mitochondrial redox function as indicated by the MTT assay. Thus, the susceptibility of mtDNA to oxidative damage in human RPE cells, together with the age-related decrease of cellular anti-oxidant system, provides the rationale for a mitochondria-based model of AMD.

Blue light induces mitochondrial DNA damage and free radical production in epithelial cells

Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390–550 nm at 2.8 milliwatts/cm2) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis.

Mitochondrial DNA damage and its potential role in retinal degeneration.

Mitochondria are central to retinal cell function and survival. There is increasing evidence to support an association between mitochondrial dysfunction and a number of retinal pathologies including age-related macular degeneration (AMD), diabetic retinopathy and glaucoma. The past decade has highlighted mitochondrial genomic instability as an important factor in mitochondrial impairment culminating in age-related changes and age-related pathology. This represents a combination of the susceptibility of mitochondrial DNA (mtDNA) to oxidative damage and a limited base excision repair pathway. This random cumulative mtDNA damage leads to cellular heteroplasmy and, if the damage affects a sufficient proportion of mitochondria within a given cell, results in loss of cell function and greater susceptibility to stress. mtDNA damage is increased in the neural retina and RPE with ageing and appears to be greatest in AMD. It thus appears that the mitochondrial genome is a weak link in the antioxidant defenses of retinal cells and that deficits in mitochondrial DNA (mtDNA) repair pathways are important contributors to the pathogenesis of retinal degeneration. Specifically targeting mitochondria with pharmacological agents able to protect against oxidative stress or promote repair of mtDNA damage may offer potential alternatives for the treatment of retinal degenerations such as AMD.

Dietary curcumin prevents ocular toxicity of naphthalene in rats

Administration of naphthalene is known to cause cataract formation in rats and rabbits and naphthalene-initiated cataract is frequently used as a model for studies on senile cataract in humans. Oxidative stress has been implicated in the mechanism of naphthalene-induced cataract. Curcumin, a constituent of turmeric, a spice used in Indian curry dishes, is an effective antioxidant and is known to induce the enzymes of glutathione-linked detoxification pathways in rats. During the present studies, we have examined whether low levels of dietary curcumin could prevent naphthalene-induced opacification of rat lens. The presence of apoptotic cells in lens epithelial cells was also examined by catalytically incorporating labeled nucleotide to DNA with either Klenow fragment of DNA polymerase or by terminal deoxynucleotidyl transferase (TdT), which forms polymeric tail using the principle of TUNEL assay. The results of these studies demonstrated that the rats treated with naphthalene and kept on a diet supplemented with only 0.005% (w/w) curcumin had significantly less opacification of lenses as compared to that observed in rats treated only with naphthalene. Our studies also demonstrate, for the first time, that naphthalene-initiated cataract in lens is accompanied and perhaps preceded by apoptosis of lens epithelial cells and that curcumin attenuates this apoptotic effect of naphthalene.

Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells.

Purpose. To determine whether hydrogen peroxide (H 2 O 2) , a physiological mediator of oxidative stress induces apoptosis in retinal pigment epithelial (RPE) cells. Methods. To demonstrate that oxidatively stressed retinal pigment epithelial cells undergo apoptosis consequential to mitochondrial dysfunction, biochemical parameters of apoptosis were determined in cultured cells after treatment with 50–200 µM H 2 O 2 for different times. Caspase-3 protease activity was determined from hydrolysis of DEVD-?-nitroanilide. Expression of the anti-apoptotic protein, bcl-2 and the pro-apoptotic proteins p53 and p21 were analyzed by western blotting. Results. Caspase-3 activity significantly increased in cells exposed to H 2 O 2. Also, the expression of bcl-2 in cells treated with 200 µM H 2 O 2 was diminished, whereas expression of p53 and p21 waf-1 was increased compared to the controls. Conclusions. Exposure of retinal pigment epithelial cells to concentrations of H 2 O 2 that cause in vitro mitochondrial DNA damage also promotes apoptosis.

Mitochondrial DNA Damage and Repair in RPE Associated with Aging and Age-Related Macular Degeneration

PURPOSE Mitochondrial DNA (mtDNA) damage may be associated with age-related diseases, such as age-related macular degeneration (AMD). The present study was designed to test whether the frequency of mtDNA damage, heteroplasmic mtDNA mutations, and repair capacity correlate with progression of AMD. METHODS Macular and peripheral RPE cells were isolated and cultured from human donor eyes with and without AMD. The stages of AMD were graded according to the Minnesota Grading System. Confluent primary RPE cells were used to test the frequency of endogenous mtDNA damage by quantitative PCR. Mutation detection kits were used to detect heteroplasmic mtDNA mutation. To test the mtDNA repair capacity, cultured RPE cells were allowed to recover for 3 and 6 hours after exposure to H(2)O(2), and repair was assessed by quantitative PCR. The levels of human OGG1 protein, which is associated with mtDNA repair, were analyzed by Western blot. RESULTS This study showed that mtDNA damage increased with aging and that more lesions occurred in RPE cells from the macular region than the periphery. Furthermore, mtDNA repair capacity decreased with aging, with less mtDNA repair capacity in the macular region compared with the periphery in samples from aged subjects. Most interestingly, the mtDNA damage was positively correlated with the grading level of AMD, whereas repair capacity was negatively correlated. In addition, more mitochondrial heteroplasmic mutations were detected in eyes with AMD. CONCLUSIONS These data show macula-specific increases in mtDNA damage, heteroplasmic mutations, and diminished repair that are associated with aging and AMD severity.

Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium

Purpose. To investigate the interrelationships between DNA damage, mitochondrial activity, and apoptosis in retinal pigment epithelial cells (RPE) after exposure to rod outer segments (ROS). Methods. After incubation of cultured human RPE with ROS, mitochondrial redox function was evaluated from MTT reduction. Mitochondrial (mt) and nuclear (n) DNA damage were determined by quantitative polymerase chain reactions (QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosphatidyl serine (PS) using fluorescence microscopy. The expression of the pro-apoptotic proteins, p53 and p21 waf-1, and DNA repair enzymes, apurinic/apyrimidinic endonuclease (APE ref-1) and DNA polymerase ß (ß-pol) were quantitatively determined by Western blotting analysis. Results. Mitochondrial function decreased by 20 ± 5% and annexin V immunofluorscent binding was enhanced after exposure of cells to physiological levels of ROS (3.8 × 10 6 cm -2) for 4h. MtDNA was preferentially damaged after exposure to ROS with increased lesion frequencies of 1.49 ± 0.37 and 2.2 ± 0.14 per 10 kb base pairs (bp), respectively after 5 and 7h contact, compared to untreated controls (zero class damage). APE ref-1 expression increased more than 340% above controls after exposure to ROS for 7 and 24h. The expression of ß-pol in cultures increased 110% above controls after 24h contact with the ROS. The expression of p53 and p21 in cells increased 100 and 38% above controls after 24h exposure to the ROS. Conclusions. Exposure of ROS to ROS induced mtDNA damage and dysfunction and activated nDNA repair pathways, which did not prevent apoptosis.

The Effect of Microgravity on Ocular Structures and Visual Function: A Review

Ocular structural and functional changes, including optic disk edema and reduction of near visual acuity, have been recently described in some astronauts returning from long-duration space travels. It is hypothesized that ocular changes related to spaceflight may occur, in predisposed individuals, as a result of cephalad shift of body fluids, possibly leading to elevated intracranial pressure (ICP). Results from head-down bed-rest studies (used to simulate the effects of microgravity) and from parabolic flight experiments (used to produce transient periods of microgravity) indicate that ocular blood flow and intraocular pressure (IOP) may undergo changes in a low-gravity environment. Recent studies suggest that changes in translaminar pressure (i.e., IOP minus ICP) may be implicated in the pathophysiology of optic disk neuropathies. Because postural changes exert an effect on both IOP and ICP, the head-down bed-rest analog may also be used as a platform to characterize the relationship between IOP and ICP, and their reciprocal influence in the pathophysiology of conditions such as optic disk edema or glaucoma.

Factors affecting visual outcomes after small-incision phacoemulsification in diabetic patients.

Purpose: To examine the factors affecting visual outcome after phacoemulsification and evaluate the use of preoperative visual potential in assessing the visual prognosis in diabetic patients. Setting: Department of Ophthalmology, University of Texas Medical Branch, Galveston, Texas, USA. Methods: In a retrospective chart review of 1345 consecutive patients who had uneventful small‐incision phacoemulsification, operated eyes from 106 diabetic and 55 nondiabetic control patients were selected. Data on demographics, level of retinopathy, perioperative glycosylated hemoglobin (HbAIc), surgical duration, preoperative best corrected visual acuity (BCVA), and visual potential were collected. Results: The age, sex, preoperative BCVA, and visual potential in the diabetic and control eyes were comparable. Throughout the postoperative period, BCVA was worse of the diabetic group. At 1 year, BCVA was 20/40 in 82.1% of the diabetic group and 94.7% of the control group (P = .01). The most important factors affecting postoperative BCVA included coexisting diabetes and preoperative level of retinopathy. No correlation was found between perioperative and postoperative BCVA. Diabetic patients were less likely than control patients to achieve a BCVA better than or equal to the preoperative visual potential at 4 years (hazard ratio 0.6; 95% confidence interval, 0.4‐0.9; P = .011). Patients with nonproliferative diabetic retinopathy were nearly 5 times less likely (P = .023) and patients with proliferative diabetic retinopathy 30 times less likely (P < .0001) to achieve a postoperative BCVA of 20/40 than diabetic patients without retinopathy. Conclusions: Although uneventful small‐incision phacoemulsification improved visual acuity in diabetic patients, this group had an overall worse visual outcome than nondiabetic patients. The most important predictors of visual outcome were coexisting diabetes and the extent of preoperative retinopathy. Methods used to assess preoperative visual potential provided a reasonable estimate of postoperative BCVA in diabetic patients. Given the inverse association between the level of retinopathy and visual outcome, it may be better to perform cataract extraction in diabetic patients during earlier stages of retinopathy.

Lipopolysaccharide Increases the Incidence of Collagen‐Induced Arthritis in Mice Through Induction of Protease HTRA‐1 Expression

OBJECTIVE The protease HTRA-1 is closely associated with rheumatoid arthritis (RA). The molecular mechanisms that control HTRA-1 expression are currently unknown. This study was undertaken to determine the regulatory role of Toll-like receptors (TLRs) on HTRA-1 expression in mice with collagen-induced arthritis (CIA) and in synovial cells from RA patients. METHODS HTRA-1 messenger RNA and protein production in mouse fibroblasts, mouse macrophages, and freshly isolated RA patient synovial cells treated with TLR ligands were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Arthritis incidence and severity were determined using clinical scores and histopathologic analysis. Involvement of HTRA-1 in lipopolysaccharide (LPS)-increased arthritis incidence and severity in mice was determined using anti-HTRA-1 monoclonal antibody. The signal pathways involved in HTRA-1 expression were accessed by specific inhibitors, RNA interference, dual-luciferase reporter, and chromatin immunoprecipitation methods. RESULTS LPS and tenascin-C, but not the other TLR ligands tested, strongly induced HTRA-1 expression. LPS significantly increased HTRA-1 expression in the joint tissue as well as arthritis incidence and severity in mice with CIA. Blocking HTRA-1 by antibody significantly decreased LPS-promoted CIA severity. Inhibiting NF-κB significantly decreased LPS-induced HTRA-1 expression in mouse and human cells. Dual-luciferase reporter assay and ChIP analysis showed that p65 directly binds to HTRA-1 promoter (amino acid 347). CONCLUSION Our findings indicate that TLR-4 activation increases HTRA-1 expression through the NF-κB pathway in fibroblasts and macrophages. HTRA-1 expression is involved in the enhancing effects of LPS on CIA. This study offers new insights into the regulation of HTRA-1 expression via LPS/TLR-4 and the role of HTRA-1 in RA pathogenesis.