CHANGES IN SYSTEMIC LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AFTER INTRAVITREAL INJECTION OF AFLIBERCEPT OR BROLUCIZUMAB FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To analyze and compare the effects of intravitreal brolucizumab versus aflibercept on systemic vascular endothelial growth factor (VEGF)-A levels in patients with neovascular age-related macular degeneration. METHODS: In this prospective interventional case series study, brolucizumab (6.0 mg/50 µL) or aflibercept (2.0 mg/50 µL) was injected intravitreally in 30 patients each. Blood samples were drawn at baseline and 7 days and 28 days after the first injection. Systemic VEGF-A levels were measured using enzyme-linked immunosorbent assay. Thirty healthy individuals served as controls. RESULTS: The median baseline systemic VEGF-A levels in the brolucizumab, aflibercept, and control groups were 10.8 (8.0-13.2), 12.0 (8.0-18.5), and 10.0 (8.0-15.1) pg/mL, respectively (P = 0.315). In the brolucizumab group, VEGF-A levels significantly decreased to 8.0 (8.0-11.5) pg/mL on Day 7 (P = 0.0254) and to 8.0 (8.0-8.0) pg/mL on Day 28 (P < 0.001). In the aflibercept group, VEGF-A levels significantly decreased to 8.0 (8.0-8.0) pg/mL on Day 7 (P < 0.001) but returned to the baseline level, 12.5 (8.5-14.6) pg/mL, on Day 28 (P = 0.120). Vascular endothelial growth factor-A levels were significantly different between the treatment groups after 28 days (P < 0.001). CONCLUSION: Intravitreal brolucizumab resulted in a sustained reduction of systemic VEGF-A levels until 28 days posttreatment, which raises concerns regarding its safety and long-term effects.

Differential Circulating MicroRNA Expression in Age-Related Macular Degeneration.

This study explored the expression of several miRNAs reported to be deregulated in age-related macular degeneration (AMD). Total RNA was isolated from sera from patients with dry AMD (n = 12), wet AMD (n = 14), and controls (n = 10). Forty-two previously investigated miRNAs were selected based on published data and their role in AMD pathogenesis, such as angiogenic and inflammatory effects, and were co-analysed using a miRCURY LNA miRNA SYBR® Green PCR kit via quantitative real-time polymerase chain reaction (qRT-PCR) to validate their presence. Unsupervised hierarchical clustering indicated that AMD serum specimens have a different miRNA profile to healthy controls. We successfully validated the differentially regulated miRNAs in serum from AMD patients versus controls. Eight miRNAs (hsa-let-7a-5p, hsa-let-7d-5p, hsa-miR-23a-3p, hsa-miR-301a-3p, hsa-miR-361-5p, hsa-miR-27b-3p, hsa-miR-874-3p, hsa-miR-19b-1-5p) showed higher expression in the serum of dry AMD patients than wet AMD patients and compared with healthy controls. Increased quantities of certain miRNAs in the serum of AMD patients indicate that these miRNAs could potentially serve as diagnostic AMD biomarkers and might be used as future AMD treatment targets. The discovery of significant serum miRNA biomarkers in AMD patients would provide an easy screening tool for at-risk populations.

Altered serum levels of autophagy proteins Beclin-1 and mTOR in patients with exudative age-related macular degeneration.

Autophagy is a key process in the maintenance of cellular survival and homeostasis. Inhibition of autophagy results in degenerative changes resembling ageing. We wondered if autophagy can contribute to the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate the serum concentrations of two key autophagy regulators, Beclin-1 and mechanistic target of rapamycin (mTOR), in patients with exudative AMD. This retrospective case-control study included 38 patients with exudative AMD and 36 sex- and age-matched controls selected among senile cataract patients. Circulating Beclin-1 and mTOR were assessed using an enzyme-linked immunosorbent assay. The proteins levels were correlated with age, sex, duration of ocular symptoms, as well as angiographic and optical coherence tomography findings. Serum Beclin-1 levels were much lower in patients with AMD than in controls (median, 0.100 ng/ml versus 1.123 ng/ml; p = 0.0033), while mTOR levels did not differ (median, 4.377 ng/ml versus 3.608 ng/ml; p = 0.4522). Participants of the study older than 70 years had lower Beclin-1 levels than younger ones (p = 0.0444). However, this difference was the most evident in patients with AMD (p = 0.0024). Serum mTOR levels increased with age. In patients with AMD, lower mTOR levels were associated with drusen, while higher levels were observed in those with a fibrous scar in the contralateral eye (p = 0.0212). Our findings suggest that circulating Beclin-1 decreases with age and that is downregulated in patients with AMD.

Systemic counterregulatory response of angiopoietin-2 after aflibercept therapy for nAMD: a potential escape mechanism.

PURPOSE: To analyse the effect of intravitreal aflibercept injections on systemic angiopoietin-2 (Ang2) and vascular endothelial growth factor (VEGF)-A levels in patients with neovascular age-related macular degeneration (nAMD). METHODS: In a prospective, randomized study, aflibercept (2.0 mg/50 µl) or ranibizumab (0.5 mg/50 µl) was administered intravitreally to 38 treatment-naive patients. Blood samples were taken before, 7 days after, and 28 days after the first intravitreal therapy. Cytokine levels were measured by enzyme-linked immunosorbent assay. Twenty-two age- and sex-matched individuals served as controls. RESULTS: At baseline, there were no significant differences of systemic Ang2 and VEGF-A levels among the treatment and control groups. After intravitreal aflibercept administration, median (interquartile range: IQR) systemic Ang2 was significantly upregulated from 1819 pg/ml (1262-3099) to 2123 pg/ml (1441-3769; p = 0.011) 7 days after the drug injection and remained non-significantly elevated at 1944 pg/ml (1431-2546 pg/ml; p = 0.653) 28 days after the drug injection. Median (IQR) systemic VEGF-A levels were significantly reduced from 43 pg/ml (30-57) to 8 pg/ml (8-8; p < 0.0001) 7 days and 16 pg/ml (8-26; p = 0.001) 28 days after the injection in the aflibercept group. There were no significant effects on systemic VEGF-A and Ang2 levels in the ranibizumab group at any time point following the first injection. CONCLUSION: In this study, we report significant systemic upregulation of Ang2 after intravitreal aflibercept administration. This counterregulatory response may represent a potential escape mechanism from antiangiogenic therapy.

Differential effects of risuteganib and bevacizumab on AMD cybrid cells.

PURPOSE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) treatments are currently used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Chronic, repetitive treatments with anti-VEGF may have unintended consequences beyond the inhibition of angiogenesis. Most recently, clinical trials have been conducted with risuteganib (RSG, Luminate®), which is anti-angiogenic and has neuroprotective and anti-inflammatory properties. Mitochondrial damage and dysfunction play a major role in development of AMD. Transmitochondrial cybrids are cell lines established by fusing human retinal pigment epithelial (RPE) cells that are Rho0 (lacking mtDNA) with platelets isolated from AMD subjects or age-matched normal subjects. Cybrid cell lines have identical nuclei but mitochondria from different subjects, enabling investigation of the functional consequences of damaged AMD mitochondria. The present study compares the responses of AMD cybrids treated with bevacizumab (Bmab, Avastin®) versus risuteganib (RSG, Luminate®). METHODS: Cybrids were created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 3) cybrids were treated for 48 h with or without 1x clinical dose of 1.25 mg/50 µl (25,000 µg/ml) of Bmab or 1.0 mg/50 µl (20,000 µg/ml) of RSG. Cultures were analyzed for levels of cleaved caspase 3/7 and NucLight Rapid Red staining (IncuCyte® Live Cell Imager), mitochondrial membrane potential (ΔΨm, JC1 assay) or reactive oxygen species (ROS, H2DCFDA assay). Expression levels of genes related to the following pathways were analyzed with qRT-PCR: Apoptosis (BAX, BCL2L13, CASP-3, -7, -9); angiogenesis (VEGFA, HIF1α, PDGF); integrins (ITGB-1, -3, -5, ITGA-3, -5, -V); mitochondrial biogenesis (PGC1α, POLG); oxidative stress (SOD2, GPX3, NOX4); inflammation (IL-6, -18, -1ß, IFN-ß1); and signaling (P3KCA, PI3KR1). Statistical analyses were performed using GraphPad Prism software. RESULTS: The untreated AMD cybrids had significantly higher levels of cleaved caspase 3/7 compared to the untreated normal cybrids. The Bmab-treated AMD cybrids showed elevated levels of cleaved caspase 3/7 compared to untreated AMD or RSG-treated AMD cybrids. The Bmab-treated cybrids had lower ΔΨm compared to untreated AMD or RSG-treated AMD cybrids. The ROS levels were not changed with Bmab or RSG treatment. Results showed that Bmab-treated cybrids had higher expression levels of inflammatory (IL-6, IL1-ß), oxidative stress (NOX4) and angiogenesis (VEGFA) genes compared to untreated AMD, while RSG-treated cybrids had lower expression levels of apoptosis (BAX), angiogenesis (VEGFA) and integrin (ITGB1) genes. CONCLUSIONS: These data suggest that the mechanism(s) of action of RSG, an integrin regulator, and Bmab, a recombinant monoclonal antibody, affect the AMD RPE cybrid cells differently, with the former having more anti-apoptosis properties, which may be desirable in treating degenerative ocular diseases.

Association of plasma trace element levels with neovascular age-related macular degeneration.

Although the triggers causing angiogenesis in the context of neovascular age-related macular degeneration (nAMD) are not fully understood, oxidative stress is likely involved. Oxidative stress in the eye can occur through exposure of macular tissues to sunlight and local or systemic exposure to oxidative stressors associated with environmental or lifestyle factors. Because trace elements have been implicated as regulators of oxidative stress and cellular antioxidant defense mechanisms, we hypothesized that they may play a role as a risk factor, modifying the progression toward nAMD. Herein, we determined whether levels of human plasma trace elements are different in 236 individuals with nAMD compared to 236 age-matched controls without AMD. Plasma levels of 16 trace elements including arsenic, barium, calcium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, lead, antimony, selenium, vanadium and zinc were measured using inductively coupled plasma mass spectrometry. Associations of trace elements with demographic, environmental and lifestyle factors and AMD-associated genetic variants were assessed. Elevated levels of barium and cadmium and reduced levels of chromium were observed in nAMD patients compared to controls. Mean plasma concentrations of barium were 1.35 µg/L (standard deviation [SD] 0.71) in nAMD and 1.15 µg/L (SD 0.63) in controls (P = 0.001). Mean levels of chromium were 0.37 µg/L (SD 0.22) in nAMD and 0.46 µg/L (SD 0.34) in controls (P = 0.001). Median levels for cadmium, which were not normally distributed, were 0.016 µg/L (interquartile range [IQR] 0.001-0.026) in nAMD and 0.012 µg/L (IQR 0.001-0.022) in controls (P = 0.002). Comparison of the Spearman's correlation coefficients between nAMD patients and controls identified a difference in correlations for 8 trace elements. Cadmium levels were associated with the smoking status (P < 0.001), while barium levels showed a trend of association with the usage of antihypertensive drugs. None of the AMD-associated genetic variants were associated with any trace element levels. In conclusion, in this case-control study we detected elevated plasma levels of barium and cadmium and reduced plasma levels of chromium in nAMD patients. An imbalance in plasma trace elements, which is most likely driven by environmental and lifestyle factors, might have a role in the pathogenesis of AMD. These trace elements may be incorporated as biomarkers into models for prediction of disease risk and progression. Additionally, population-based preventive strategies to decrease Cd exposure, especially by the cessation of smoking, could potentially reduce the burden of nAMD. Future studies are warranted to investigate whether supplementation of Cr would have a beneficial effect on nAMD.

Peripheral blood CD163(+) monocytes and soluble CD163 in dry and neovascular age-related macular degeneration.

Macrophages are the main infiltrating immune cells in choroidal neovascularization (CNV), a hallmark of the human wet, or neovascular age-related macular degeneration (AMD). Due to their plasticity and ability to adapt to the local microenvironment in a tissue-dependent manner, macrophages display polar functional phenotypes characterized by their cell surface markers and their cytokine profiles. We found accumulation of hemoglobin-scavenging cluster of differentiation 163 (CD163)(+) macrophages in laser-induced CNV lesions and higher expression of CD163(+) monocytes in the peripheral blood on day 7 post injury in mice. In comparison, CD80(+) macrophages did not differ with laser-injury in young or aged mice and did not significantly change in the peripheral blood of CNV mice. We examined the percentages of CD163(+), CD206(+), and CD80(+) monocytes in the peripheral blood of patients with wet AMD, patients with dry AMD, and in age-matched individuals without AMD as controls. Percentages of peripheral blood CD163(+) monocytes in both dry AMD (P < .001) and wet AMD (P < .05) were higher than in age-matched non-AMD controls, while there was no difference between the groups in the percentages of peripheral CD206(+) and CD80(+) monocytes. Further, serum level of soluble CD163 (sCD163) was elevated only in patients with wet AMD (P < .05). An examination of 40 cytokine levels across the study groups revealed that anti-VEGF treated patients with wet AMD, who showed no exudative signs on the day of blood drawing had a cytokine profile that was similar to that of non-AMD individuals. These results indicate that CD163 could be further evaluated for its potential as a useful marker of disease activity in patients with neovascular AMD. Future studies will address the origin and potential mechanistic role of CD163(+) macrophages in wet AMD pathologies of angiogenesis and leakage of blood components.

Diacron reactive oxygen metabolites and biological antioxidant potential tests for patients with age-related macular degeneration.

BACKGROUND: Previously, we showed that serum malondialdehyde (MDA) was significantly higher in patients with neovascular age-related macular degeneration (nAMD) than in those without AMD. The Diacron reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) tests are known markers of oxidative stress. The aim of this study was to use d-ROMs and BAP tests to evaluate changes in systemic oxidative stress in patients with nAMD. METHODS: Blood serum samples were collected from 34 patients with nAMD (mean age: 76.5 ± 7.7 years; 22 men) and 20 control subjects (mean age: 62.9 ± 14.0 years; 10 men), and d-ROMs and BAP tests were examined. RESULTS: In men, the mean level of d-ROMs for the nAMD patients was significantly higher than that for the controls (312.0 ± 52.4 vs. 275.1 ± 45.5 U.CARR, respectively; P < .05). There was a significant correlation between d-ROM level and CNV lesion area in the male nAMD group (r = .42, P = .05). There were no significant differences in mean BAP test results between the nAMD patients and controls for either sex (men: 2241 ± 549 vs. 2136 ± 246 µmol/L; women: 2263 ± 292 vs. 2335 ± 161 µmol/L). CONCLUSION: The d-ROMs test may provide a useful indicator of nAMD in men but not in women.

MALONDIALDEHYDE LEVELS IN PATIENTS WITH AGE-RELATED MACULAR DEGENERATION: A Systematic Review and Meta-analysis.

BACKGROUND/PURPOSE: The role of blood malondialdehyde (MDA) in age-related macular degeneration (AMD), the leading cause of new blindness in industrialized countries, is still matter of debate. We performed a systematic review and meta-analysis of the published data on the MDA levels in AMD patients. METHODS: PubMed, ISI Web of Sciences, and Scopus searches were performed according to MOOSE guidelines. Case-control studies were eligible for inclusion. Participants and controls were AMD patients and subjects without AMD, respectively. The main outcome measures were wet AMD and dry AMD. MDA level was the main exposure variable. Data were pooled using a random-effects model. RESULTS: Twelve case-control studies were identified. A total of 634 AMD patients (mean age 66.7 years) and 656 controls without AMD (mean age 67.8 years) were evaluated. Extreme between-study heterogeneity was observed (I = 96.8%, P < 0.001). Pooled standardized mean difference showed that MDA values were significantly higher in patients with AMD (standardized mean difference = 1.91 µmol/L, 95% confidence interval = 1.08-2.74; P < 0.001). In a model including five studies, homogenous for age, sample matrix, and laboratory testing for MDA, heterogeneity decreased from extreme to moderate (I = 46.4%, P = 0.113), and pooled standardized mean difference, though attenuated, remained significantly higher in AMD patients (standardized mean difference = 1.07 µmol/L, 95% confidence interval = 0.82-1.31; P < 0.001). CONCLUSION: There is some evidence of higher levels of MDA in AMD patients compared with healthy controls; however, this result should be interpreted with caution because of extreme between-study heterogeneity and the possible effect of publication bias. Future studies, preferably well age-matched and of cohort design, are necessary before any firm conclusions on the putative role of elevated MDA on AMD can be drawn.

Change in cardiac troponin T level after intravitreal anti-vascular endothelial growth factor treatment: Prospective pilot study.

BACKGROUND: Evaluate subclinical myocardial injury associated with intravitreal anti-vascular endothelial growth factor therapy by measuring serum high-sensitivity cardiac troponin T. METHODS: This is a prospective pilot comparative study conducted at American University of Beirut Medical Center, Beirut, Lebanon. In total, 40 consecutive patients were randomized to receive either intravitreal bevacizumab or ranibizumab. Patients received three consecutive monthly injections of the assigned drug, then continued treatment as needed. Systemic concentrations of high-sensitivity cardiac troponin T and vascular endothelial growth factor were obtained at baseline, week 9, and week 24. Primary endpoint measure was change in high-sensitivity cardiac troponin T levels compared to baseline. Secondary endpoint measure was change in systemic vascular endothelial growth factor levels. RESULTS: There was no significant difference in high-sensitivity cardiac troponin T levels over time (p = 0.227) within each treatment group and no significant difference between treatments at any time point (p = 0.276). There was a significant decrease in plasma vascular endothelial growth factor levels at week 9 (p = 0.001) and week 24 (p < 0.001) compared to baseline. In the ranibizumab group, vascular endothelial growth factor levels were not significantly different at weeks 9 and 24 compared to baseline (p = 0.708 and p = 0.117, respectively). There was a significant association between the number of bevacizumab injections from weeks 8 to 24 and the decrease in vascular endothelial growth factor levels at week 24 (R = -0.67, p = 0.032). This correlation was not observed in the ranibizumab group (R = -0.341, p = 0.141). CONCLUSION: Repeated intravitreal bevacizumab or ranibizumab did not influence serum high-sensitivity cardiac troponin levels. Intravitreal bevacizumab but not ranibizumab lowered free-systemic vascular endothelial growth factor levels, which was observed in this study to be inversely related to the number of bevacizumab injections.

Systemic activation of the complement system in patients with advanced age-related macular degeneration.

PURPOSE: To examine the role of systemic activation of the complement system (assessed by levels of circulating C3a, Ba, and sC5b-9) in patients (n = 122) with advanced age-related macular degeneration, geographic atrophy, and neovascular age-related macular degeneration, compared with cataract controls (n = 27). METHODS: Plasma complement factors were measured using enzyme-linked immunosorbent assays. Statistical analysis included univariate and multivariate logistic regression (p < 0.05). RESULTS: Adjusted for age, the odds ratios of C3a and sC5b-9 for any advanced age-related macular degeneration were 1.78 (95% confidence interval = 1.16-2.73, p < 0.01) and 1.20 (95% confidence interval = 1.04-1.39, p = 0.01), respectively. We found a significantly elevated adjusted odds ratio of C3a (adjusted odds ratio = 1.71, 95% confidence interval = 1.12-2.60, p = 0.01) and sC5b-9 (adjusted odds ratio = 1.22, 95% confidence interval = 1.04-1.43, p = 0.01) for neovascular age-related macular degeneration. Adjusted for age, neither C3a, sC5b-9, nor Ba were associated with geographic atrophy. CONCLUSION: We suggest a role for elevated plasma levels of C3a and sC5b-9 in patients with neovascular age-related macular degeneration. The study's results reinforce the need for more investigation to assess the impact of therapeutic interventions targeted at the complement signaling pathways in age-related macular degeneration.

Circulating endothelial and progenitor cells in age-related macular degeneration.

PURPOSE: To evaluate circulating endothelial and circulating progenitor cells as biomarkers in age-related macular degeneration patients (both exudative and atrophic forms) in order to establish the possible clinical implication of their assessment. METHODS: We have enrolled 44 age-related macular degeneration patients: 22 patients with a recently diagnosed exudative (neovascular) form (Group A) and 22 patients with an atrophic (dry) form (Group B). The control group consisted of 22 age and sex-matched healthy subjects (Group C). The number of circulating endothelial progenitor cells (CD34+/KDR+, CD133+/KDR+, and CD34+/KDR+/CD133+), circulating progenitor cells (CD34+, CD133+, and CD34+/CD133+), and circulating endothelial cells were determined in the peripheral venous blood samples by flow cytometry. Neovascular age-related macular degeneration patients were evaluated at baseline and 4 weeks after a loading phase of three consequent intravitreal injections of ranibizumab. RESULTS: Comparing age-related macular degeneration patients with the control group, endothelial progenitor cell and circulating progenitor cell levels were not significantly different, while age-related macular degeneration patients showed significantly higher levels of circulating endothelial cells (p = 0.001). Anti-vascular endothelial growth factor treatment with intravitreal ranibizumab was associated with a significant reduction of endothelial progenitor cell levels, with no significant influence on circulating progenitor cells and circulating endothelial cells. CONCLUSION: We reported higher levels of circulating endothelial cells in age-related macular degeneration patients in comparison with the control group, thereby supporting the hypothesis of an involvement of endothelial dysregulation in the age-related macular degeneration and a reduction of the endothelial progenitor cell level in neovascular age-related macular degeneration patients after three intravitreal injections of ranibizumab.

Chitinase-3-Like-1 Promotes M2 Macrophage Differentiation and Induces Choroidal Neovascularization in Neovascular Age-Related Macular Degeneration.

Purpose: Choroidal neovascularization (CNV) is the principal pathological factor contributing to blindness in neovascular age-related macular degeneration (nAMD). Infiltration of M2 macrophage is thought to contribute to CNV progress, although the way that regulates its differentiation remains unclear. Here, we investigate the role of CHI3L1 in M2 differentiation and angiogenesis in CNV. Methods: Serums from nAMD patients were tested for CHI3L1 expression. Mice were subjected to laser injury to induce CNV, and lesion expansion were tracked using fundus fluorescence angiography (FFA) and immunofluorescence analysis. Several strategies were taken to verify the contribution of M2 macrophage and CHI3L1: macrophage depletion by clodrosome, local CHI3L1 inhibition using intravitreally injection neutralize antibody (mAY), and depletion of CHI3L1 receptor (IL13-Ra2) by small-interfering RNA (siRNA). Tuber analysis was used to further determine angiogenetic effect of CHI3L1. Anti-VEGFA was used as positive control for mAY. Results: Serum levels of CHI3L1 were highly elevated in nAMD patients. CHI3L1 was expressed by infiltrating M2 macrophages and was elevated as CNV progress in a mice model. System macrophage depletion and local suppression of CHI3L1 alleviated CNV formation while enhancing anti-VEGFA therapeutic effect. Stimulation of macrophage with recombinant CHI3L1 activated MAPK signaling cascade and induced transition to M2, while siRNA knockdown of IL13-Ra2 abolished it. In an in vitro coculture system, supernatants from CHI3L1-stimulated M2 macrophages and promoted tube vascularization. Conclusions: These results unveil novel angiogenic regulation of CHI3L1 and M2 polarized macrophages in CNV development. These mechanistic insights may point to CHI3L1 as a new therapeutic target for treatment for nAMD.

Neutrophil-to-lymphocyte ratio in age-related macular degeneration: a systematic review and meta-analysis.

Age-related macular degeneration (AMD) is aetiologically linked to immunological ageing and dysfunction. One aspect of this is the altered neutrophil-to-lymphocyte ratio (NLR), which in other domains have been associated with inflammation and angiogenesis, and therefore investigated in patients with AMD in several papers. In this systematic review and meta-analysis, we summarize findings in patients with AMD in relation to NLR, both qualitatively and quantitatively. We searched PubMed/MEDLINE, EMBASE, Web of Science, and the Cochrane Central and identified six studies from where we extracted data on 1178 individuals (777 patients with AMD and 401 healthy controls). Patients with AMD had a higher NLR (weighted mean difference: 0.37, CI 95% 0.08 to 0.66, p = 0.013) when compared to healthy controls. In subgroup analyses, we did not find a significant difference between patients with dry AMD and healthy controls (weighted mean difference: 0.34, CI 95% -0.03 to 0.69, p = 0.068), but did find a strong significant difference between patients with neovascular AMD and healthy controls (weighted mean difference: 0.54, CI 95% 0.23 to 0.86, p = 0.00068). Hence, we find that the association between AMD and elevated NLR may have stronger relevance to the neovascular subtype of AMD. However, the clinical value of measuring the NLR remains unclear.

Cytokine Profiling in Patients With Exudative Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy.

Purpose: The purpose of this study was to investigate the cytokine profiles in plasma and aqueous humor of patients with choroidal neovascularization (CNV) due to exudative AMD and polypoidal choroidal vasculopathy (PCV). Methods: In this cross-sectional study, 16 patients clinically diagnosed with AMD, 18 patients with PCV, and 50 age- and sex-matched cataract patients without AMD/PCV (controls) were enrolled. Study subjects were treatment naïve, and 200 µL undiluted aqueous humor and 5 mL peripheral venous blood were collected from the study subjects. Clinical samples were analyzed for 41 different cytokines by Luminex bead-based multiplex assay. Cytokines concentrations with detection rates of 50% or more were included for the analysis, and the differences in plasma and aqueous humor cytokines levels between each group were analyzed. Results: The age of the patients with AMD and PCV was 70.62 ± 10.15 (mean ± SD) and 71.48 ± 9.08 years, respectively, and that in the control group was 62.8 ± 10.67 years. Aqueous humor cytokines growth-regulated oncogene (GRO), macrophage-derived chemokine (MDC), and macrophage inflammatory protein (MIP)-1α were significantly higher in AMD patients than controls (all P < 0.04), and GRO, MDC, MIP-1α, IL-8, IFN-γ-inducible protein 10, and monocyte chemotactic protein levels were significantly higher in PCV patients than controls (all P < 0.03). Soluble CD40 ligand and platelet-derived growth factor-AA levels were higher in plasma of healthy controls compared with AMD subjects. No significant differences in cytokine levels were observed between AMD and PCV patients for both plasma and aqueous humor. Conclusions: In AMD and PCV patients, our data suggest that the pathologic changes are primarily driven by dysregulation of local immune factors in the eye, whereas the plasma cytokine levels are not elevated.

Increased soluble urokinase plasminogen activator receptor (suPAR) levels in neovascular age-related macular degeneration: a role for inflammation in the pathogenesis of the disease?

PURPOSE: To evaluate the plasma concentration of the soluble form of the urokinase-type plasminogen activator receptor ((s)uPAR), an established biomarker of chronic inflammation, in patients affected by neovascular age-related macular degeneration. METHODS: Forty consecutive patients affected by age-related macular degeneration and 52 subjects with no history of the disease were included in this case-control study. The two groups of individuals considered for the study were matched for age, sex, and class of medications taken. Plasma concentration of suPAR was measured using a specific ELISA assay (suPARnostic, Birkeroed, Denmark). RESULTS: The case and control groups were similar for age, gender distribution, weight, height, and systolic and diastolic blood pressure, as well as for dyslipidemia and high blood pressure medication (P > 0.28). The plasma concentrations of suPAR were significantly increased in patients with neovascular age-related macular degeneration when compared to controls (6.19 ± 2.2 ng/ml, vs 5.21 ± 1.5, respectively, mean ± SD P = 0.01). CONCLUSIONS: Patients with neovascular age-related macular degeneration display increased plasma levels of suPAR, suggesting that chronic inflammation may be involved in the pathogenesis of the disease.

Plasma markers of chronic low-grade inflammation in polypoidal choroidal vasculopathy and neovascular age-related macular degeneration.

PURPOSE: Ageing is the strongest predictor of neovascular age-related macular degeneration (AMD), where neuroinflammation is known to play a major role. Less is known about polypoidal choroidal vasculopathy (PCV), which is an important differential diagnosis to neovascular AMD. Here, we report plasma markers of inflammation with age (inflammaging) in patients with PCV, patients with neovascular AMD and a healthy age-matched control group. METHODS: We isolated plasma from fresh venous blood obtained from participants (n = 90) with either PCV, neovascular AMD, or healthy maculae. Interleukin(IL)-1ß, IL-6, IL-8, IL-10 and tumour necrosis factor receptor 2 (TNF-R2) were measured using U-PLEX Human Assays. Routine plasma C-reactive protein (CRP) was measured using Dimension Vista 1500. RESULTS: Patients with PCV had plasma levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-R2 similar to that in healthy controls. Patients with neovascular AMD had significantly higher plasma IL-1ß, IL-6 and IL-10 than healthy controls, whereas no significant differences were observed for plasma IL-8 and TNF-R2. Differences between plasma IL-1ß, IL-6 and IL-10 possessed a positive but weak ability in discriminating neovascular AMD from PCV. Both patients with PCV and patients with neovascular AMD had significantly higher levels of routine plasma CRP. CONCLUSION: Patients with PCV differ from patients with neovascular AMD in terms of plasma inflammaging profile. Apart from increased CRP, no signs of inflammaging were observed in patients with PCV. In patients with neovascular AMD, we find a specific angiogenesis-twisted inflammaging profile.

Cytokine profiles in the aqueous humor and serum of patients with dry and treated wet age-related macular degeneration.

PURPOSE: To identify disease-specific cytokine profile differences in the aqueous humor (AH) (other than the vascular endothelial growth factor) between patients with dry and treated wet age-related macular degeneration (AMD) and healthy controls. METHODS: This retrospective study drew on a case-series of patients diagnosed with dry AMD (n = 25) and treated wet AMD (n = 19), as well as on healthy controls (no systemic therapy; n = 20) undergoing phacoemulsification or vitrectomy. Samples of AH and serum were collected in parallel at the beginning of surgery. The levels of 43 cytokines were simultaneously determined using the Bio-Plex® multiplex beads system. Differences between the three groups were statistically compared using the Kruskal-Wallis H-Test after applying the Bonferroni correction for multiple comparisons (p<0.0012). RESULTS: The concentrations of three cytokines were elevated in the AH of patients with dry AMD (CXCL6; p = 0.00067) and treated wet AMD (CXCL5, CXCL6, MIG/XCXL; all p<0.001) relative to those in the healthy controls. No other differences between the three groups were identified. The AH levels of seven cytokines (16%), including CXCL6, ranged below the lower limit of quantitation of the assay. Without the correction for multiple comparisons (p<0.05), the levels of 31 of the 43 cytokines in the AH of patients with AMD would have differed significantly from those in the control. The systemic cytokine profiles (serum) were similar in all three groups. CONCLUSIONS: No systematic differences in the AH cytokine environment were identified between patients with dry AMD and those with treated wet AMD. This finding might indicate that AMD is either the result of a persistent imbalance in the physiological tissue milieu, or that the localized process induces no significant change in the cytokine environment of the anterior ocular segment.

The association of matrix metalloproteinases polymorphisms and interleukins in advanced age-related macular degeneration.

PURPOSE: To assess the impact of matrix metalloproteinase (MMP)1-1607 1G/2G (rs1799750), MMP7-181 A/G (rs11568818) single-nucleotide polymorphism and systemic cytokins interleukin-1 beta (IL-1ß), IL-6 levels on the development of exudative age-related macular degeneration (eAMD) Methodology: The study group comprised 282 patients with eAMD, and the control group enrolled 379 randomly selected persons. The genotyping of MMP1-1607 (rs1799750) and MMP7-181 (rs11568818) was performed by using the polymerase chain reaction-based restriction fragment length polymorphism method. To determine IL-1ß and IL-6 serum levels, the immunoenzymatic method with monoclonal antibodies coated plates was performed. RESULTS: MMP1 rs1799750 1G/2G genotype was more frequently found in the development of eAMD. It was associated with a 4.3-fold increased risk for eAMD under the codominant model and a 4.9-fold increased risk for eAMD under the overdominant model. The effect was more pronounced at the age of less than 65 years. IL-1ß concentration was significantly higher for MMP1 rs1799750 1G/1G genotype and MMP7 rs11568818 A/G genotype in eAMD patients compared with control group subjects. CONCLUSIONS: MMP1 rs1799750 1G/2G genotype was found to play a significant role in the development of eAMD at the age of less than 65 years. IL-1ß concentration was significantly higher in eAMD patients for MMP1 rs1799750 1G/1G genotype and MMP7 rs11568818 A/G genotype compared with control group subjects.