MicroRNA expression profiling in tears and blood as predictive biomarkers for anti-VEGF treatment response in wet age-related macular degeneration.

PURPOSE: This study aimed to investigate the potential of microRNAs (miRNAs) in tears, blood, and aqueous humor as biomarkers for predicting treatment response in wet age-related macular degeneration (AMD) patients undergoing anti-vascular endothelial growth factor (anti-VEGF) therapy. METHODS: In a single-center prospective cohort study, treatment-naïve wet AMD patients and age-matched controls were enrolled. Clinical data and miRNA levels (miR-199a-3p, miR-365-3p, miR-200b-3p, miR-195-5p, miR-335-5p, and miR-185-5p) in tears, blood, and aqueous humor were collected. Treatment response was categorized into responders and non-responders based on visual acuity and central subfield thickness. MiRNA levels were quantified using reverse-transcription PCR. Statistical analyses were performed, including ROC analysis, to evaluate predictive accuracy. RESULTS: Dysregulated miRNA profiles were observed in wet AMD tears and blood compared to controls. Specifically, miR-199a-3p, miR-195-5p, and miR-185-5p were upregulated, while miR-200b-3p was downregulated in tears. All six miRNAs were elevated in wet AMD blood samples. Notably, responders showed higher tear expression of miR-195-5p and miR-185-5p. Combining these miRNAs yielded the highest predictive power (AUC = 0.878, p = 0.006) for anti-VEGF responders. CONCLUSIONS: Dysregulated miRNA profiles in tears and blood suggest their potential as biomarkers for wet AMD. MiR-195-5p and miR-185-5p in tears demonstrate predictive value for anti-VEGF treatment responders. This study underscores the non-invasive prediction potential of miRNA tear analysis in wet AMD treatment responses.

Unilateral acute idiopathic maculopathy related to hand-foot-mouth disease: Case report and literature review.

Unilateral acute idiopathic maculopathy (UAIM) is a rare disease that may cause unilateral vision loss in young adults after a flu-like illness. Occasionally, it is associated with hand-foot-mouth disease (HFMD) and is often underdiagnosed. Herein, we report a case with characteristics of UAIM associated with HFMD with acute enteroviral infection. On the basis of the clinical findings using multimodality diagnostic imaging, including fundus image, optical coherence tomography, fluorescence angiography, and serological test for pan-enterovirus RNA polymerase chain reaction, we detailed the clinical course and postulated the pathogenesis of UAIM with choroiditis associated with HFMD. This study could remind every doctor of the potential visual loss caused by UAIM in HFMD, and referring patients to the ophthalmologic survey is important to eliminate potential visual impairment.

Microforceps-Assisted Transscleral Fixation of Intraocular Lens.

PURPOSE: We present a novel technique for intraocular lens (IOL) fixation. The technique can be used on single-piece acrylic IOLs and can manage the patients who are either aphakia or with a dislocated IOL. METHODS: One end of Gore-Tex suture is tied into the optic-haptic junction of the IOL. Another end is fixated in the scleral wall. The single sclerotomy and double sclerotomies settings can be applied to different situations. RESULTS: Twelve eyes received this procedure. After a follow-up period of up to 20 months, the IOLs were well centered. CONCLUSION: The technique is a reliable method for scleral fixation of IOLs, which can be applied on the widely used single-piece acrylic IOLs. In our experience, it is reproducible and rarely cause complications.

Association of Visual Impairment with Psychological Distress in Older Adults: A Survey of 105,092 Older People in Taiwan.

This study aimed to evaluate the association between visual impairment (VI) and psychological distress (PD) among older adults in Taiwan. The present cohort study included participants aged >65 years who participated in a physical examination program. Participants were divided into two groups on the basis of whether they had PD at baseline. The association between PD and VI with other variables was compared using the two-sample t-test for continuous variables and chi-squared test for discrete variables. Cox regression analyses were used to calculate the hazard ratio (HR). Cumulative incidence of PD was analyzed using the Kaplan-Meier method, and differences among participants with different severities of VI were analyzed using the two-tailed log-rank test. Subgroup analyses were performed to calculate the HR for PD among participants with different severities of VI. The PD group showed a significantly high percentage of VI. In addition, participants with VI showed a significantly higher HR and seven-year cumulative incidence rate of PD than those without VI. VI was independently and significantly associated with a higher incidence of PD among older Asian people. Therefore, identifying and treating correctible VI is important to prevent PD and improve the overall quality of life.

COVID-19 pandemic decreased the ophthalmic outpatient numbers and altered the diagnosis distribution in a community hospital in Taiwan: An observational study.

The aim of this study was to determine the effect of Coronavirus disease 2019 (COVID-19) pandemic on ophthalmic outpatient numbers and ophthalmic diagnosis distribution in a community hospital (Taipei City Hospital Zhongxiao Branch) in Taiwan. The COVID-19 pandemic period in Taiwan was defined as May 1 to July 31, 2021. Demographic data, including age, gender, and top 10 diagnoses from ophthalmic outpatients during this period, were collected. A corresponding control group from the same time in 2020 was also collected. The distribution of different diagnoses was analyzed, and the data of 10 most prominent diagnoses with decreased percentage of case numbers during the COVID-19 pandemic period were obtained. The number of cases during the COVID-19 pandemic decreased by 46.9% compared to the control group. The top three most common diagnoses were dry eye syndrome, glaucoma, and macular diseases. The 10 most prominent diagnoses with decreased number of cases during the COVID-19 pandemic were cataract, refraction & accommodation, macular degeneration, conjunctivitis, retinal detachment, vitreous body disorders, ophthalmic complications of diabetes mellitus, glaucoma, dry eye, and retinal vein occlusion. Identifying and treating these patients as scheduled may yield the highest cost-benefit effect in preventing visual loss during the COVID-19 pandemic.

Cardiac autonomic neuropathy predicts diabetic retinopathy progression in Asian population with type 2 diabetes mellitus.

PURPOSE: To investigate the role of cardiac autonomic neuropathy (CAN), vascular condition, and sensory function in diabetic retinopathy (DR) progression. METHODS: This 3-year cohort study conducted in a community hospital included 4850 patients over 20 with type 2 diabetes mellitus. Participants were assessed in 2017 at baseline and were followed up in 2020. Patients were divided into two groups based on whether they had DR progression or not and were compared using the chi-square test or two-sample t-test. Beta coefficient and odds ratio (OR) with 95% confidence intervals were calculated using binary logistic regression. The receiver operating characteristic (ROC) curve of various independent variables for DR progression was provided with C-statistics. RESULTS: Abnormal hemoglobin A1c (HbA1c) level/variation, estimated glomerular filtration rate, urine albumin-to-creatinine ratio, R-R interval variation, standard deviation of the average NN intervals, autonomic nervous system function, power of high-frequency (HF) bands, balance, cardio-ankle vascular index (CAVI), and warm stimulation (WS) were associated with DR progression. Average HbA1c, HF, and proliferative diabetic retinopathy were independent factors for patients developing DR progression. The top three areas under the curve of ROCs were HF + baseline DR grading, WS + baseline DR grading, and CAVI + baseline DR grading. These variable combinations were the most reliable predictors of DR progression. CONCLUSION: CAN, abnormal vascular condition, and sensory function are associated with DR progression. The combination of HF, WS, and CAVI with baseline DR grading provides the most accurate predictive model for DR progression. Early detection of these factors is important to prevent DR progression.

Association of visual impairment with cognitive decline among older adults in Taiwan.

This study investigated the association between visual impairment and cognitive decline among the elderly in Taiwan. The data were obtained from a government-sponsored, annual physical examination program for elderly citizens ≥ 65 years in Taipei City during 2005-2012. Distance presenting visual acuity was measured using the Snellen chart. Visual impairment was classified into low vision and blindness. The Short Portable Mental Status Questionnaire (SPMSQ) was selected to measure cognitive decline. The confounding factors including age, sex, sociodemographic factors: living status, marital status, education level, health behaviors: smoking, alcohol consumption, betel nut chewing, and physical comorbidities: BMI, hypertension, diabetes, cholesterol and triglyceride were collected for analysis. We recruited 105,208 participants and 4542 (4.3%) have abnormal SPMSQ. The abnormal SPMSQ had significantly higher prevalence of low vision (44.52% vs 18.79%) and blindness (8.89% vs 0.93%) compared with normal SPMSQ. The hazard ratios of abnormal SPMSQ in low vision and blindness were 2.34 (95% CI 2.17-2.52), and 5.13 (95% CI 4.50-5.85), after adjustment for confounders. In conclusion, visual impairment was independently and significantly associated with greater incident cognitive decline among elderly Asian people. Prevention of visual impairment may help to reduce the incidence of cognitive decline in the aged Asian population.

Correlation between Visual Acuity and Optical Coherence Tomography Angiography Parameters in Unilateral Idiopathic Epiretinal Membrane.

BACKGROUND: The tangential traction by idiopathic epiretinal membrane (iERM) may alter the hemodynamics of the macula. We investigated the correlation between visual acuity and the optical coherence tomography angiography (OCTA) parameters in unilateral iERM. METHODS: We included 61 eyes of 61 consecutive patients with unilateral iERM between January 2018 and December 2018. The flow area of the retinal superficial capillary plexus (SCP), deep capillary plexus (DCP), and choroidal capillary plexus (CCP) were measured using OCTA. The normal fellow eyes were used for comparison. The iERM patients were divided into those with a presence of foveal concavity and those with a loss of foveal concavity. RESULTS: When compared with fellow eyes, the flow areas showed a statistically significant decrease in the SCP and CCP of those with iERM (p = 0.037 and p = 0.011, respectively). In the DCP, no significant reduction in flow area was found in iERM (p = 0.054). The flow area of the CCP was the only factor significantly associated with best vision (p = 0.012). No significant differences in the flow areas of the SCP, DCP, and CCP were found between the presence and loss of foveal concavity. CONCLUSIONS: The flow area of the CCP is an important determinant of vision, emphasizing the crucial role of choroidal circulation in iERM. Moreover, mechanical stretch by iERM is not the only mechanism affecting the flow area.

Association of macular choroidal thickness with optical coherent tomography morphology in patients with idiopathic epiretinal membrane.

PURPOSE: To compare macular choroidal thickness of idiopathic epiretinal membrane (ERM) and fellow eyes, and before and after vitrectomy in terms of the morphological features on spectral-domain optical coherent tomography (SD-OCT). METHODS: Eighty-four patients with unilateral idiopathic ERM were involved. Patients were categorized into: Group 1, ERM without membrane contraction; Group 2, ERM with membrane contraction and retinal folding; and Group 3, ERM with membrane contraction and macular edema. Twenty-two patients received surgical removal of ERM. Choroidal thickness was compared between ERM and fellow eyes, and before and after treatment. RESULTS: Mean choroidal thickness was significantly greater in ERM eyes than in fellow eyes (234.4 ± 22.5 vs 220.6 ± 20.8 µm; P<0.01). Group 1 (n = 20) showed no significant difference in choroidal thickness between ERM and fellow eyes. Eyes in Group 2 (n = 27) and Group 3 (n = 37) showed statistically significant differences in mean choroidal thickness between ERM and fellow eyes (229.6 ± 23.8 vs 220.8 ± 19.6 µm; 242.6 ± 27.8 vs 221.0 ± 21.8 µm, respectively; P<0.05). In Group 2 (n = 8) and Group 3 (n = 16), choroidal thickness in ERM eyes decreased significantly at 1 month and 6 months after surgery, compared with that before surgery (P<0.05 for all comparisons). CONCLUSIONS: Membrane contraction contributed to the increase in choroidal thickness in idiopathic ERM patients. This finding may help to elucidate the pathophysiologic features of idiopathic ERM as well as the response to treatment in these patients.

Correlation between visual acuity changes and optical coherence tomography morphological findings in idiopathic epiretinal membranes.

PURPOSE: To analyze the influence of spectral-domain optical coherence tomography (SD-OCT) features on visual acuity changes in patients with idiopathic epiretinal membranes (ERMs). METHODS: Seventy-nine eyes of 71 patients were included in this study. SD-OCT was performed for all patients; data were collected upon ERM diagnosis and at the final visit. The patients were divided into subgroups based on their SD-OCT features. The initial best corrected visual acuity (BCVA) and changes in BCVA for each subgroup were compared. A multivariate analysis was performed to assess the factors associated with changes in BCVA. RESULTS: During a mean follow-up period of 20.78 months, the mean change in logMAR visual acuity was 0.052 ± 0.089. Eyes with inner segment/outer segment (IS/OS) junction disruption and cystoid macular edema (CME) had a significantly lower mean initial BCVA than those without disruption and CME (P = 0.036 and P = 0.012, respectively). However, only eyes with CME had significant changes in BCVA (P = .034). Multivariate analysis revealed the presence of CME as the only factor that had a significant correlation with VA changes. CONCLUSIONS: In patients with idiopathic ERMs, the presence of CME and IS/OS disruption detected by OCT correlated with a poorer initial BCVA. Most patients' visual acuity remained stable during follow-up. The presence of CME with OCT represented a predictor of the progression of visual acuity. These results may provide valuable clinical information regarding the management of patients with idiopathic ERMs. We demonstrated that the presence of CME and IS/OS disruption detected with OCT correlated with a poorer BCVA in idiopathic ERMs. The visual acuity of most patients was stable during the follow-up period. The presence of CME in OCT represented a predictor of vision deterioration for patients with idiopathic ERMs.

Chitosan oligosaccharides prevented retinal ischemia and reperfusion injury via reduced oxidative stress and inflammation in rats.

The purpose of the present study was to investigate the protective effect and mechanism of chitosan oligonucleotides (COS) on retinal ischemia and reperfusion (I/R) injury. Rats pretreated with PBS, low-dose COS (5 mg/kg), or high-dose COS (10 mg/kg) were subjected to retinal ischemia by increasing their intraocular pressure to 130 mm Hg for 60 min. The protective effect of COS was evaluated by determining the electroretinograms (ERGs), morphology of the retina, and survival of retinal ganglion cells (RGCs). The oxidative damage was determined by imuunohistochemistry and ELISA, respectively. The expressions of inflammatory mediators (TNF-α, IL-1ß, MCP-1, iNOS, ICAM-1) and apoptotic-related proteins (p53, Bax, Bcl-2) were quantified by PCR and Western blots. The detection of NF-κB p65 in the retina was performed by immunofluorescence. The protein levels of IκB and phosphorylated mitogen-activated protein kinases [MAPK; viz. extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK) and p38] and the NF-κB/DNA binding ability were assessed by Western blot analysis and EMSA. We found that pretreatment with COS, especially a high dosage, effectively ameliorated the I/R-induced reduction of the b-wave ratio in ERGs and the retinal thickness and the survival of RGCs at 24 h. COS decreased the expression of inflammatory mediators, p53 and Bax, increasing Bcl-2 expression and thereby reducing retinal oxidative damage and the number of apoptotic cells. More importantly, COS attenuated IκB degradation and p65 presence in the retina, thus decreasing NF-κB/DNA binding activity after I/R. In addition, COS decreased the phosphorylation levels of JNK and ERK but increased the phosphorylation level of p38. Pretreatment with p38 inhibitor (SB203580) abolished the protective effect of COS on retinal oxidative damage, as indicated by increased retinal 8-OHdG stains, and significantly increased the expression of inflammatory mediators (TNF-α, MCP-1, iNOS, ICAM-1) in I/R-injured rats. In conclusion, COS prevented retinal I/R injury through its inhibition of oxidative stress and inflammation. These effects were achieved by blocking the activation of NF-κB, JNK, and ERK but promoting the activation of p38 activation.

Induced pluripotent stem cells without c-Myc ameliorate retinal oxidative damage via paracrine effects and reduced oxidative stress in rats.

PURPOSE: To investigate the efficacy and mechanisms of non-c-Myc induced pluripotent stem cell (iPSC) transplantation in a rat model of retinal oxidative damage. METHODS: Paraquat was intravitreously injected into Sprague-Dawley rats. After non-c-Myc iPSC transplantation, retinal function was evaluated by electroretinograms (ERGs). The generation of reactive oxygen species (ROS) was determined by lucigenin- and luminol-enhanced chemiluminescence. The expression of brain-derived neurotrophic factor, ciliary neurotrophic factor, basic fibroblast growth factor (bFGF), stromal cell-derived factor (SDF)-1α, and CXCR4 was measured by immunohistochemistry and ELISA. An in vitro study using SH-SY5Y cells was performed to verify the protective effects of SDF-1α. RESULTS: Transplantation of non-c-Myc iPSCs effectively promoted the recovery of the b-wave ratio in ERGs and significantly ameliorated retinal damage. Non-c-Myc iPSC transplantation decreased ROS production and increased the activities of superoxide dismutase and catalase, thereby reducing retinal oxidative damage and apoptotic cells. Moreover, non-c-Myc iPSC transplantation resulted in significant upregulation of SDF-1α, followed by bFGF, accompanied by a significant improvement in the ERG. In vitro studies confirmed that treatment with SDF-1α significantly reduced apoptosis in a dose-dependent manner in SH-SY5Y cells. Most transplanted cells remained in the subretinal space, with spare cells expressing neurofilament M markers at day 28. Six months after transplantation, no tumor formation was seen in animals with non-c-Myc iPSC grafts. CONCLUSIONS: We demonstrated the potential benefits of non-c-Myc iPSC transplantation for treating oxidative-damage-induced retinal diseases. SDF-1α and bFGF play important roles in facilitating the amelioration of retinal oxidative damage after non-c-Myc iPSC transplantation.

Chitosan oligosaccharides attenuate ocular inflammation in rats with experimental autoimmune anterior uveitis.

We investigated the protective effects and mechanisms of chitosan oligosaccharides (COS) on experimental autoimmune anterior uveitis (EAAU) in rats. EAAU was induced in Lewis rats by footpad and intraperitoneal injections of melanin-associated antigen. The rats received intraperitoneal injections of low-dose (5 mg/kg) or high-dose (10 mg/kg) COS or PBS daily after the immunization. The effects of COS were evaluated by determining the clinical scores and the morphology of the iris/ciliary body (ICB). The expression of inflammatory mediators was evaluated using western blot, immunofluorescence, and ELISA. Treatment with COS significantly attenuated the clinical scores and the leukocyte infiltration in the ICB in a dose-dependent manner. COS effectively reduced the expression of inflammatory mediators (TNF-α, iNOS, MCP-1, RANTES, fractalkine, and ICAM-1). Moreover, COS decreased the IκB degradation and p65 presence in the ICB, which resulted in the inhibition of NF-κB/DNA binding activity. In an in vitro study, sensitized spleen-derived lymphocytes of the COS-treated group showed less chemotaxis toward their aqueous humor and decreased secretion of the above inflammatory mediators in the culture media. COS treated EAAU by inhibiting the activation of NF-κB and reducing the expression of inflammatory mediators. COS might be a potential treatment for acute anterior uveitis.

Docosahexaenoic acid reduces linoleic acid induced monocyte chemoattractant protein-1 expression via PPARγ and nuclear factor-κB pathway in retinal pigment epithelial cells.

SCOPE: To investigate whether docosahexaenoic acid (DHA) could inhibit linoleic acid (LA) induced monocyte chemoattractant protein (MCP)-1 expression in human retinal pigment epithelial (RPE) cells. METHODS AND RESULTS: ARPE-19 cells were pretreated with DHA and then exposed to LA. The expression of MCP-1 and PPARγ was examined using RT-PCR and Western blot analysis. LA at 10, 25, or 50 µM induced expression of MCP ARPE-19 cells in a dose-dependent manner (p < 0.05). DHA at 50 and 100 µM effectively inhibited LA-induced MCP-1 expression and production (p < 0.05) and NF-κB activation. In addition, the culture medium from LA-stimulated ARPE-19 cells could induce tube formation in choroidal endothelial cells (RF6A), whereas 100 µM DHA inhibited tube formation. DHA at 100 µM increased the expression and activity of PPARγ (p < 0.05). Pretreatment with PPARγ inhibitor (GW9662) abolished the inhibitory effect of DHA (100 µM) on LA-induced IκB degradation, p65 translocation, and MCP-1 expression in ARPE-19 cells (p < 0.05), as well as tube formation in RF6A. CONCLUSION: DHA reduced LA-induced MCP-1 expression via a PPARγ- and NF-κB-dependent pathway in ARPE-19 cells. These results suggest the molecular mechanisms underlying the beneficial effects of increased consumption of DHA and reduced consumption of LA on age-related macular degeneration.

Chitosan oligosaccharides attenuates oxidative-stress related retinal degeneration in rats.

This study investigated the therapeutic potential and mechanisms of chitosan oligosaccharides (COS) for oxidative stress-induced retinal diseases. Retinal oxidative damage was induced in Sprague-Dawley rats by intravitreal injection of paraquat (PQ). Low-dose (5 mg/kg) or high-dose (10 mg/kg) COS or PBS was intragastrically given for 14 days after PQ injection. Electroretinograms were performed to determine the functionality of the retinas. The surviving neurons in the retinal ganglion cell layer and retinal apoptosis were determined by counting Neu N-positive cells in whole-mounted retinas and TUNEL staining, respectively. The generation of reactive oxygen species (ROS) was determined by lucigenin- and luminol-enhanced chemiluminescence. Retinal oxidative damages were assessed by staining with nitrotyrosine, acrolein, and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Immunohistochemical studies were used to demonstrate the expression of nuclear factor-kappa B (NF-κB) p65 in retinas. An in vitro study using RGC-5 cells was performed to verify the results. We demonstrated COS significantly enhanced the recovery of retinal function, preserved inner retinal thickness, and decreased retinal neurons loss in a dose-dependent manner. COS administration demonstrated anti-oxidative effects by reducing luminol- and lucigenin-dependent chemiluminenscense levels and activating superoxide dismutase and catalase, leading to decreased retinal apoptosis. COS markedly reduced retinal NF-κB p65. An in vitro study demonstrated COS increased IκB expression, attenuated the increase of p65 and thus decreased NF-κB/DNA binding activity in PQ-stimulated RGC-5 cells. In conclusion, COS attenuates oxidative stress-induced retinal damages, probably by decreasing free radicals, maintaining the activities of anti-oxidative enzymes, and inhibiting the activation of NF-κB.

Transplantation of induced pluripotent stem cells without C-Myc attenuates retinal ischemia and reperfusion injury in rats.

Induced pluripotent stem cells (iPSC) are novel stem cell populations, but the role of iPSC in retinal ischemia and reperfusion (I/R) injury remains unknown. Since oncogene c-Myc is substantially contributed to tumor formation, in this study, we investigated the effects, mechanisms and safety of subretinal transplantation of iPSC without c-Myc (non-c-Myc iPSC) in a rat model of retinal I/R injury. Retinal I/R injury was induced by raising the intraocular pressure of Sprague-Dawley rats to 110 mmHg for 60 min. A subretinal injection of non-c-Myc iPSC or murine epidermal fibroblast was given 2 h after I/R injury. Electroretinograms (ERG) were performed to determine the functionality of the retinas. The surviving retinal ganglion cells (RGCs) and retinal apoptosis following I/R injury were determined by counting NeuN-positive cells in whole-mounted retinas and TUNEL staining, respectively. The generation of reactive oxygen species (ROS) and the activities of superoxide dismutase (SOD) and catalase (CAT) in the retinal tissues were determined by lucigenin- and luminol-enhanced chemiluminescence and enzyme-linked immunosorbent assay (ELISA). The degree of retinal oxidative damage was assessed by nitrotyrosine, acrolein, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. The expression of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF) in retinas was measured by immunohistochemistry and ELISA. We found that subretinal transplantation of non-c-Myc iPSC significantly suppressed the I/R-induced reduction in the ERG a- and b-wave ratio, attenuated I/R-induced loss of RGCs and remarkably ameliorated retinal morphological changes. Non-c-Myc iPSC potentially increased the activities of SOD and CAT, decreased the levels of ROS, which may account for preventing retinal cells from apoptotic cell death. In addition, the levels of BDNF and CNTF in retina were significantly elevated in non-c-Myc iPSC-treated eyes. Track the non-c-Myc iPSC after transplantation, most transplanted cells are remained in the subretinal space, with spare cells express neurofilament M markers at day 28. Six months after transplantation in I/R injured rats, no tumor formation was seen in non-c-Myc iPSC graft. In conclusion, non-c-Myc iPSC effectively rescued I/R-induced retinal damages and diminished tumorigenicity. Non-c-Myc iPSC transplantation attenuated retinal I/R injury, possibly via a mechanism involving the regulation of oxidative parameters and paracrinal secretion of trophic factors.

Comparative effects of fatty acids on proinflammatory gene cyclooxygenase 2 and inducible nitric oxide synthase expression in retinal pigment epithelial cells.

Dietary fat modification is a promising approach to prevent age-related macular degeneration (AMD). However, which types of fatty acids carry a greater risk for AMD remains unclear. In this study, we compared the effects of 18-carbon fatty acids with different degrees of unsaturation on the expression of the proinflammatory genes cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in human retinal pigment epithelium (RPE). Additionally, we investigated whether lutein could modulate these genes induced by fatty acids in RPE. Treatment with oleic acid, linoleic acid (LA), or linolenic acid increased the expression of iNOS and COX-2 genes and the production of prostaglandin E(2 )and nitric oxide (NO) in RPE, whereas the saturated fatty acid stearic acid had little effect on these genes. Of the fatty acids studied, LA had the greatest effects on the induction of these genes. Furthermore, LA also induced NF-kappaB transcriptional activation the most. Lutein inhibited LA-induced expression of COX-2 and iNOS in a dose-dependent manner. These data suggested that specific unsaturated fatty acids, particularly LA, can stimulate RPE cells to express proinflammatory genes, which may contribute to the pathogenesis of AMD. Lutein inhibited the expression of these genes induced by LA through blockade of NF-kappaB activation.

Overexpression of integrin alpha6 and beta4 enhances adhesion and proliferation of human retinal pigment epithelial cells on layers of porcine Bruch's membrane.

Transplantation of retinal pigment epithelium (RPE) following removal of choroidal neovascular membranes has been attempted in patients with age-related macular degeneration (AMD). However, inability of transplanted RPE to initially attach and subsequently proliferate on Bruch's membrane may lead to failure of RPE transplants and poor visual outcomes. Integrin alpha(6)beta(4) functions as a receptor for laminin, the major component of Bruch's membrane, and mediates the stable attachment of most epithelial cells to the underlying basement membrane. To improve adhesion and proliferation of transplanted RPE on Bruch's membrane, we elucidated the roles of integrin alpha(6)beta(4) in RPE adhesion to extracellular matrix and investigated whether ex vivo gene transfer of integrin alpha(6) and beta(4) in RPE could promote adhesion and proliferation of transplanted RPE on Bruch's membrane. The expression of integrin alpha(6) and beta(4) mRNA and surface protein in ARPE-19 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. We generated point mutation in the ligand binding domain of integrin alpha(6) and beta(4) by using site-directed mutagenesis and transfected these mutated constructs into ARPE-19 cells. Adhesion assay was used to determine the roles of integrin alpha(6) and beta(4) in RPE adhesion to extracellular matrix. In addition, we transfected full-length alpha(6) cDNA or beta(4) cDNA into ARPE-19 cells. The reattachment and proliferation ratios of alpha(6)-cDNA- or beta(4)-cDNA-transfected ARPE-19 cells on different layers of Bruch's membrane were determined by cell adhesion and proliferation assays. Cell morphology and surface coverage were evaluated by scanning electron microscopy 7 days after plating on various layers of Bruch's membrane. We found that integrin alpha(6) and beta(4) mRNA and proteins were constitutively expressed in ARPE-19 cells. Decreased endogenous integrin alpha(6) and beta(4) expression by selective mutation of amino acid residues caused a significant reduction in adhesion of ARPE-19 cells to laminin 5. Modification of integrin expression by transfection of alpha(6) cDNA into ARPE-19 cells induced a significant increase in cell adhesion to laminin 5, fibronectin, whereas transfection with beta(4) cDNA caused increased adhesion only to laminin 5. alpha(6)-cDNA-transfectants increased cell attachment and proliferation on all layers of Bruch's membrane, whereas beta(4)-cDNA-transfectants enhanced adhesion and proliferation on basal lamina and inner collagenous layers. These data indicate that integrin alpha(6) and beta(4) play a role in adhesion of ARPE-19 cells to extracellular matrix. Modification of integrin expression by ex vivo genetic manipulation in RPE might be an alternative strategy to increase the success of RPE transplantation.

Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-kappaB pathway in retinal pigment epithelial cells.

High linoleic acid (LA) intake is known to correlate with age-related macular degeneration (AMD), but the molecular mechanisms remain unclear. This study was conducted to investigate the effects of LA on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) and their associated signaling pathways in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with different concentrations of LA. Expressions of iNOS and COX-2 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Concentrations of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in the culture medium were determined by enzyme-link immunosorbent assay (ELISA). Activation of p42/44, p38, JNK mitogen-activated protein kinase (MAPK) and nuclear factors (NF)-kappaB were evaluated by Western blot analysis and electrophoretic mobility shift assay (EMSA). We found that LA induced expression of iNOS and COX-2 in RPE cells at the mRNA and protein levels in a time-and dose-dependent manner. Upregulation of iNOS and COX-2 resulted in increased production of NO and PGE(2). Moreover, LA caused degradation of IkappaB and increased NF-kappaB DNA binding activity. Effects of LA-induced iNOS and COX-2 expression were inhibited by a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). LA activated p42/44, but not p38 or JNK MAPK. Inhibition of p42/44 activity by PD98059 significantly reduced LA-induced activation of NF-kappaB. Linoleic acid-induced expression of iNOS and COX-2 as well as PGE(2) and NO release in RPE cells were sequentially mediated through activation of p42/p44, MAPK, then NF-kappaB. These results may provide new insights into both mechanisms of LA action on RPE cells and pathogenesis of age-related macular degeneration.