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.

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.

A profile of circulating vascular progenitor cells in human neovascular age-related macular degeneration.

BACKGROUND/OBJECTIVE: A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS: Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion, the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS: There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media, and markers such as NG2 and αSMA in mesenchymal media. However, analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS: CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However, nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease.

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.

T-cell differentiation and CD56+ levels in polypoidal choroidal vasculopathy and neovascular age-related macular degeneration.

Polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (AMD) are prevalent age-related diseases characterized by exudative changes in the macula. Although they share anatomical and clinical similarities, they are also distinctly characterized by their own features, e.g. vascular abnormalities in PCV and drusen-mediated progression in neovascular AMD. PCV remains etiologically uncharacterized, and ongoing discussion is whether PCV and neovascular AMD share the same etiology or constitute two substantially different diseases. In this study, we investigated T-cell differentiation and aging profile in human patients with PCV, patients with neovascular AMD, and age-matched healthy control individuals. Fresh venous blood was prepared for flow cytometry to investigate CD4+ and CD8+ T-cell differentiation (naïve, central memory, effector memory, effector memory CD45ra+), loss of differentiation markers CD27 and CD28, and expression of aging marker CD56. Patients with PCV were similar to the healthy controls in all aspects. In patients with neovascular AMD we found significantly accelerated T-cell differentiation (more CD28-CD27- cells) and aging (more CD56+ cells) in the CD8+ T-cell compartment. These findings suggest that PCV and neovascular AMD are etiologically different in terms of T cell immunity, and that neovascular AMD is associated with T-cell immunosenescence.

CD11b and CD200 on Circulating Monocytes Differentiate Two Angiographic Subtypes of Polypoidal Choroidal Vasculopathy.

Purpose: To investigate surface expression of CD11b and CD200 on circulating monocytes in patients with polypoidal choroidal vasculopathy (PCV). Methods: This was a prospective case-control study of patients with PCV (n = 27), age-matched healthy controls (n = 27), and patients with neovascular AMD (n = 49). All participants underwent a comprehensive ocular examination. Fluorescein and indocyanine green angiography were performed in patients suspected of neovascular AMD or PCV. Polypoidal choroidal vasculopathy was angiographically categorized into those with a strong presence of a branching vascular network (BVN) (type 1) or with a faint/no clear presence of a BVN (type 2). Fresh venous blood was stained with fluorescent antibodies for flow cytometric analyses. We compared the percentages of CD11b+, CD200+, and CD11b+CD200+ monocytes between groups of diagnosis and between different angiographic subtypes of PCV. Results: Overall, CD11b+ monocytes were both increased in patients with PCV and neovascular AMD. CD200+ and CD11b+CD200+ monocytes were increased in patients with neovascular AMD. An age-related increase in CD11b+CD200+ monocytes was absent in patients with PCV and neovascular AMD. Patients with PCV type 1 had significantly higher CD11b+, CD200+, and CD11b+CD200+ monocytes, whereas patients with PCV type 2 had levels similar to that in healthy controls. Conclusions: We found that PCV is immunologically heterogeneous with significant differences between angiographic subtypes. Increased CD11b+ and CD200+ monocytes in those with a strong presence of BVN indicate that BVN development may be associated with retinal injury and a VEGF-mediated process that is either reflected or propelled by systemic changes in monocytes.

Associations between Serum Vitamin D and Genetic Variants in Vitamin D Pathways and Age-Related Macular Degeneration in the European Eye Study.

PURPOSE: To study associations between early and late age-related macular degeneration (AMD) and neovascular AMD (nvAMD) with serum 25-hydroxy vitamin D (25(OH)D) and genetic variants in vitamin D pathway genes. DESIGN: Population-based, cross-sectional study in a random sample aged 65 years or older from 7 European countries. PARTICIPANTS: Of 4753 participants, 4496 (2028 men and 2468 women), with a mean age of 73 years, provided a blood sample; 2137 had no signs of AMD, 2209 had early AMD, and 150 had late AMD, of whom 104 had nvAMD. METHODS: Participants were interviewed to determine smoking and alcohol use, sunlight exposure, and diet; underwent fundus photography. Fundus images were graded using the International Classification System for Age-Related Maculopathy. The 25(OH)D was measured by liquid chromatography-tandem mass spectrometry and categorized as deficient (<30 nmol/l), insufficient (30-50 nmol/l), or adequate (≥50 nmol/l). Genotyping was performed on a subsample of 1284 AMD cases and controls for 93 single nucleotide polymorphisms (SNPs) from 7 genes. Associations were investigated by linear or logistic regression adjusted for potential confounders. MAIN OUTCOME MEASURES: Adjusted odds ratio (OR) for 3 outcomes (early AMD, late AMD, nvAMD). RESULTS: No linear association was found with 25(OH)D and early or late AMD or nvAMD. There was no association between insufficient or deficient status with early or late AMD. Deficient status was associated with nvAMD (adjusted OR, 1.27; 95% confidence interval, 1.1-1.45; P < 0.0001). Significant (P < 0.05) associations with 25(OH)D were found for SNPs in genes GC, VDR, CYP2R1, and CYP27B1. Two SNPs (VDR) were associated with early AMD, 4 SNPs (RXRA) and 1 SNP (VDR) were associated with nvAMD, and 1 SNP (RXRA), 2 SNPs (VDR), and 1 SNP (CYP2R1) were associated with late AMD. After Bonferroni correction, no SNPs were associated with early AMD, late AMD, or nvAMD. CONCLUSIONS: Deficiency in 25(OH)D was associated with nvAMD, but the adjusted OR was small, and we cannot exclude residual confounding. The hypothesis of a causal association of vitamin D with AMD is not supported by clear evidence for an association of vitamin D SNPs with early AMD, late AMD, or nvAMD.

SERUM LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR BEFORE AND AFTER INTRAVITREAL INJECTION OF RANIBIZUMAB OR CONBERCEPT FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

OBJECTIVE: To investigate the serum levels of vascular endothelial growth factor (VEGF) before and after intravitreal injection of conbercept or ranibizumab for neovascular age-related macular degeneration and polypoidal choroidal vasculopathy patients. METHODS: This study is a prospective, interventional case series and involved 28 patients, 18 treated with 0.5 mg of conbercept and 10 treated with 0.5 mg of ranibizumab. Serum concentrations of VEGF were determined by enzyme-linked immunosorbent assay before the injection and at 1 day, 1 week, and 1 month after anti-VEGF treatments. RESULTS: The baseline serum VEGF level of the ranibizumab group was 367.11 ± 311.87 pg/mL, whereas that of the conbercept group was 315.06 ± 170.88 pg/mL (P = 0.653). In the conbercept group, VEGF level significantly decreased to 36.32 ± 72.11 pg/mL at 1 day (P = 0.03) and returned to 136.55 ± 144.62 pg/mL at 1 week (P = 0.03). At 1 month, the concentration increased to 334.48 ± 197.41 pg/mL and showed no significant difference compared with the baseline. In the ranibizumab group, the serum VEGF levels were 292.42 ± 239.80 pg/mL, 282.60 ± 201.36 pg/mL, and 308.83 ± 266.89 pg/mL at 1 day, 1 week, and 1 month after intravitreal injection, respectively. There was no significant difference in the ranibizumab group at each detection time point (P = 0.45). CONCLUSION: Conbercept significantly decreased serum VEGF level 1 day and 1 week after injection, but this effect was not sustained for 1 month. In contrast, ranibizumab had no significant effect on serum VEGF concentration changes. The reduction in serum VEGF by conbercept may affect its systemic safety profile.

Analysis of the Serum Lipid Profile in Polypoidal Choroidal Vasculopathy.

Polypoidal choroidal vasculopathy (PCV), the predominant subtype of neovascular age-related macular degeneration in the Asian population, is associated with genetic polymorphism of lipid metabolism. In this study, we performed the untargeted lipidomics approach of ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) to reveal the potential discriminating lipid profile of PCV patients in serum (21 PCV patients and 19 age-matched controls). Unsupervised principal component, supervised orthogonal partial least squares analysis, correlation analysis, and heatmap analysis were performed with the data obtained by UPLC-MS. Forty-one discriminating metabolites were identified. Receiver operating characteristic curve analysis, pathway analysis and functional analysis were performed subsequently, and platelet-activating factor (PAF) was further selected as the key indicator of the distinct lipid metabolism in PCV patients. Finally, the serum level of PAF was validated by enzyme-linked immunosorbent assay, which is significantly higher in PCV patients compared to controls (65 PCV patients and 63 age-matched controls, p < 0.0001), consistent with the UPLC-MS analysis. Our results suggested that PAF is considered as the major indicator of the distinct lipid metabolism in PCV patients.

[Hypoxia and hyperglycemia promote the proliferation and migration of human marrow mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the cell proliferation, apoptosis and migration of human marrow mesenchymal stem cells (hMSCs) under hypoxia and hyperglycemia. METHODS: The hMSCs from healthy adults were isolated and harvested by density gradient centrifugation followed by adherent cultures. The cells was divided into four groups: control group (210 mL/L O2 with 5.56 mmol/L glucose), high glucose and normoxia group (210 mL/L O2 with 30 mmol/L glucose), normal glucose and hypoxia group (50 mL/L O2 with 5.56 mmol/L glucose) and high glucose and hypoxia group (50 mL/L O2 with 30 mmol/L glucose). The cells at the third passage were cultured in different groups. Then, we used cell counting kit-8 (CCK-8) to detect the proliferation of hMSCs, Transwell(TM) assay to examine the migration of hMSCs, and flow cytometry to observe the apoptosis of hMSCs at 24 hours. RESULTS: Compared with the control group, the proliferation rate of hMSCs increased in the other groups at 24 and 48 hours, and the maximum proliferation effect was found in high glucose and hypoxia group. Compared with the control group, the migration ability of hMSCs in the other groups was enhanced dramatically at 24 hours, whereas there was no significant difference in the apoptosis rate among the four groups. CONCLUSION: Both high glucose and hypoxia could improve the migration and proliferation of hMSCs, but they have no effect on apoptosis.

Interleukin-1ß Level Is Increased in Vitreous of Patients with Neovascular Age-Related Macular Degeneration (nAMD) and Polypoidal Choroidal Vasculopathy (PCV).

PURPOSE: To examine the expression of pro-interleukin-1ß (pro-IL-1ß) and interleukin-1ß (IL-1ß) in the vitreous body of patients with neovascular age-related macular degeneration(nAMD), polypoidal choroidal vasculopathy (PCV), proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO) or Eales' disease to further elucidate the role of IL-1ß and inflammation in the pathogenesis of neovascular retinal disease. DESIGN: Prospective clinical laboratory investigation study. METHODS: All patients enrolled had vitreous hemorrhage due to nAMD, PCV, PDR, RVO or Eales' disease that required vitrectomy. Patients were excluded for any history of active intraocular inflammation, or other ophthalmic surgery besides vitrectomy. Control samples were obtained from patients with idiopathic macular epiretinal membrane. A total of fifty vitreous samples were collected from patient during vitrectomy. Pro-IL-1ß and IL-1ß expression were measured by enzyme-linked immunosorbent assay (ELISA). Results were analyzed statistically using nonparametric tests. RESULTS: Expression of pro-IL-1ß protein was increased by 2.83-fold and 9.19-fold in PCV and nAMD vitreous samples relative to control, respectively. Expression of IL-ß protein was increased by 10-fold and 4.83-fold in PCV and nAMD vitreous samples relative to control, respectively. CONCLUSIONS: Our results demonstrate that expression of pro-IL-1ß and IL-1ß proteins is higher in PCV and nAMD. The roles of pro-IL-1ß and IL-1ß as inflammatory mediators in the development of PCV and nAMD may be associated with photoreceptor degeneration and neovascularization which necessitates further study.

Multiphasic changes in systemic VEGF following intravitreal injections of ranibizumab in a child.

PURPOSE: To investigate whether intravitreal ranibizumab injections administered to a child alter systemic plasma levels of total and free VEGF 165. METHODS: A 9-year-old child sustained a choroidal rupture from blunt trauma. He subsequently developed a secondary choroidal neovascular membrane, which was treated with five ranibizumab injections over a period of 8 months. Peripheral venous blood samples were taken at each visit over a period of 12 months and plasma was extracted. Plasma VEGF 165 levels were determined using enzyme-linked immunosorbent assay and were assayed both pre- and post-immunodepletion to remove complexed VEGF. RESULTS: Plasma VEGF 165 levels proved labile following intravitreal injection of ranibizumab. Levels increased by 30% above baseline following the first intravitreal ranibizumab injection, but then returned to baseline despite two subsequent injections. There was then a rebound increase of 67% in total plasma VEGF levels following a further injection, which remained above baseline for 12 weeks despite two further intravitreal ranibizumab injections. Baseline levels were re-attained 26 weeks after the final injection. CONCLUSIONS: These results suggest intravitreal ranibizumab injections can cause significant, multiphasic changes in systemic VEGF levels. This may be of particular clinical significance in children as VEGF is known to be vital in the development of major organs, in addition to its role in the maintenance of normal organ function in adults.

The reduction of serum soluble Flt-1 in patients with neovascular age-related macular degeneration.

PURPOSE: To evaluate serum soluble Flt-1 (sFlt-1) in age-related macular degeneration (AMD) patients. DESIGN: Case-control study. METHODS: Study involved 56 non-AMD participants, 53 early AMD patients, and 97 neovascular AMD patients from Belfast in Northern Ireland. Serum samples were collected from each patient. Serum sFlt-1 was measured by human sVEGFR1/sFlt-1 ELISA kit. The results were analyzed by Excel and SPSS. RESULTS: Serum sFlt-1 concentration of non-AMD, early AMD, and neovascular AMD were 90.8 ± 2.9 pg/mL (± standard error of the mean), 88.2 ± 2.6 pg/mL, and 79.9 ± 2.2 pg/mL. sFlt-1 from neovascular AMD patients was significantly decreased compared to non-AMD and early AMD patients (ANOVA, P < .01). For each 10-point increase in sFlt-1, the odds for having neovascular AMD compared with non-AMD and neovascular AMD decrease by 27.8%, odds ratio (OR) = 0.722 (95% confidence interval [CI]: 0.588-0.888, P = .002) and 27.0%, OR = 0.730 (95% CI: 0.594-0.898, P = .003), respectively. In patients over 73 years of age, serum sFlt-1 <80 pg/mL was associated with a >6-fold higher risk of neovascular AMD. CONCLUSIONS: Reduced serum sFlt-1 differentiates those patients with neovascular AMD from both early AMD and non-AMD participants. In those aged over 73, serum sFlt <80 pg/mL seems to indicate a particularly high risk of neovascular AMD. Our results indicate serum sFlt-1 could be a biomarker for development of neovascular AMD.

Changes in Clotting Time, Plasma Fibrinogen Levels, and Blood Viscosity After Administration of Ranibizumab for Treatment of Choroidal Neovascularization.

PURPOSE: To observe changes in clotting time, plasma fibrinogen levels, and blood viscosity after intravitreal ranibizumab (IVR) injection in patients with macular choroidal neovascularization (CNV). METHODS: A total of 77 patients were enrolled in the study. Patients were divided into a study group (n = 42 CNV patients) and a control group (n = 35 age- and gender-matched healthy subjects). Study group patients received IVR injections; control group patients received none. Clotting times, plasma fibrinogen levels and blood viscosity were evaluated before, and 1 week and 1 month after the first IVR injection, and again 1 month after the second injection in the study group, but only once in the control group. A paired-sample t-test was used to analyze data at four time points in the study group. Study group patients were further categorized as those with neovascular age-related macular degeneration (AMD subgroup) or CNV secondary to pathological myopia (PM subgroup). Indicators were also analyzed for each subgroup. RESULTS: There were no significant differences between study and control group patients in baseline values. Results showed that 1 week after the first IVR injection, the mean activated partial thromboplastin time (APTT) of study group patients was significantly reduced compared with baseline values (27.88 ± 4.00 versus 30.70 ± 5.56 s), respectively. Low-, median- and high-shear viscosity rates were increased significantly compared with baseline values. No statistically significant changes in tested indicators were found at other time points. In AMD subgroup patients, changes in all indicators were similar to those found overall. In contrast, only changes in median- and high-shear viscosity rates were statistically significant in PM subgroup patients. CONCLUSION: IVR injection may cause short-term fluctuations in APTT and blood viscosity in AMD patients. Further studies are needed to establish the long-term safety of IVR treatment.

Association of plasma malondialdehyde with ARMS2 genetic variants and phenotypes in polypoidal choroidal vasculopathy and age-related macular degeneration.

PURPOSE: To evaluate the relationships between plasma malondialdehyde (MDA) level and ARMS2 variants and phenotypes in patients with polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (nAMD). METHODS: This study is a retrospective case-control study. Plasma MDA was measured in 84 controls, 62 patients with PCV, and 42 patients with nAMD. Participants were genotyped for ARMS2 polymorphism. Phenotypes including bilaterality and greatest linear dimension based on fluorescein angiography (FA-GLD) and indocyanine green angiography (ICGA-GLD), were evaluated. RESULTS: Plasma MDA in the PCV and nAMD groups was higher than in the control group (P < 0.001, respectively). For ARMS2 variants, plasma MDA of homozygous high-risk genotype (TT) was higher than that of homozygous low-risk genotype (GG) in all groups (P < 0.001, respectively). Plasma MDA was higher in homozygous high-risk genotype than in heterozygous genotype in the control, PCV, and nAMD groups (P = 0.021, 0.002, and 0.004, respectively). In the nAMD group, there was a correlation between plasma MDA and both FA-GLD (r = 0.418, P = 0.006) and ICGA-GLD (r = 0.329, P = 0.033). There was a difference in plasma MDA between patients with unilateral and bilateral lesions in both PCV and nAMD (P = 0.017 and 0.019, respectively). CONCLUSION: This study revealed significant relationships between the plasma MDA level and ARMS2 variants and phenotypes in PCV and nAMD.

Endothelial progenitor cells and plasma vascular endothelial growth factor and stromal cell-derived factor-1 during ranibizumab treatment for neovascular age-related macular degeneration.

PURPOSE: To evaluate endothelial progenitor cell [late outgrowth endothelial progenitor cells (OECs)], vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1α (SDF-1α) plasma levels as potential biomarkers before and during ranibizumab (Lucentis(®)) treatment for neovascular age-related macular degeneration (nvAMD). METHODS: Thirty-one patients with untreated nvAMD presenting for 3 consecutive intravitreal ranibizumab injections and a follow-up visit at 4 weeks intervals were enrolled. Peripheral blood was collected before each injection and at the follow-up visit and OEC clusters were cultured and evaluated according to previously published protocols. VEGF and SDF-1α plasma levels were measured by enzyme-linked immunosorbent assay and compared to values from healthy young and old control. RESULTS: Patients with a high OEC count before treatment presented significantly more often with a short symptom duration and a smaller choroidal neovascularization size. VEGF plasma levels were significantly higher in nvAMD (282.4±195.2 pg/mL) compared to young (45.5±6.8 pg/mL) and old control (46.1±8.5 pg/mL). OEC levels decreased nonsignificantly during ranibizumab treatment, returning to baseline levels after the third injection. VEGF and SDF-1α plasma levels decreased significantly during treatment toward control values. Patients needing retreatment after 3 ranibizumab injections had significantly higher VEGF plasma levels at pretreatment compared to patients not needing further treatment. CONCLUSIONS: The results presented here suggest that VEGF plasma levels may warrant further evaluation regarding biological, therapeutical, and predictive implications in nvAMD.

Different impact of high-density lipoprotein-related genetic variants on polypoidal choroidal vasculopathy and neovascular age-related macular degeneration in a Chinese Han population.

Neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) are both major serosanguinous maculopathies among the Asian elderly. They are similar in phenotype. Genetic variants in high-density lipoprotein (HDL) pathway were discovered to be associated with AMD in two genome-wide association studies. In this study with a Chinese Han cohort, we investigated the impacts of these genetic variants on nAMD and PCV separately. The missense coding variants and previously identified variants at LIPC, ABCA1, CETP, LPL and FADS1 loci were genotyped in 157 nAMD patients, 250 PCV patients and 204 controls without any macular abnormality. The known variants in CFH, ARMS2 and near HTRA1 were also genotyped. Fasting serum cholesterol levels were determined. The variants in CFH, ARMS2 and near HTRA1 were strongly associated with both PCV (P < 10(-6), 10(-7) and 10(-7) respectively) and nAMD (P < 10(-6), 10(-16) and 10(-17) respectively). None of the studied HDL-related variants were significantly associated with nAMD. A missense variant in CETP, rs5882, was significantly associated with PCV (P = 2.73 × 10(-4)). The rs5882 GG genotype had a 3.53-fold (95% CI: 1.93-6.45) increased risk for PCV, and conferred a significantly lower serum HDL-cholesterol level for PCV patients than the AA genotype (P = 0.048). These results suggest the need to separate PCV from nAMD in association studies especially with Asian cohorts, and that the HDL pathway may involve in the pathogenesis of PCV and nAMD differently.

New biomarker for neovascular age-related macular degeneration: eotaxin-2.

Recently, eotaxin-CCR3 was reported to play an important role in choroidal neovascularization (CNV) development and was documented to be superior than vascular endothelial growth factor-A treatment when tested in CNV animals. As eotaxin studies are lacking in the human age-related macular degeneration (AMD) patients, we sought to determine whether eotaxin-2 (CCL24) has any association with inflammatory processes that occur in CNV. CCL24 levels were determined by enzyme linked immunosorbant assay (ELISA) after normalization to total serum protein and levels of ELISA were correlated to various risk factors in about 133 AMD patients and 80 healthy controls. The CCL24 levels were significantly higher in wet AMD patients as compared with dry AMD and normal controls. There was a significant difference when compared among wet AMD patients (i.e., minimally classic, predominantly classic, and occult). We also report significant difference in the CCL24 levels of Avastin-treated and untreated AMD patients. This study shows that CCL24 levels were found to be significantly increased in AMD patients despite Avastin treatment as compared with normal controls and those without Avastin, indicating that CCL24 may have an association with CNV and may be an important target to validate future therapeutic approaches in AMD in tandem with Avastin treatment.