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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.

Purpose: To assess the long-term anatomical and functional findings in patients with symptomatic vitreomacular traction (VMT), with or without full thickness macular hole (FTMH), after eye treatment with intravitreal ocriplasmin injection (IOI). Methods: This longitudinal case series includes 51 eyes from 51 symptomatic patients with VMT (

Purpose To compare the effect of adding a corticosteroid or switching to another anti-VEGF treatment in patients with exudative age-related macular degeneration (eAMD) with persisting intraretinal fluid on anti-VEGF monotherapy. Methods This retrospective, interventional case series involved 43 pseudophakic eyes with eAMD and persistent intraretinal fluid on anti-VEGF treatment that switched treatment to a combination of Ozurdex® or Triamcinolone and anti-VEGF therapy (group 1) or to another anti-VEGF agent (group 2). The number of injections, time to re-injection, change in central retinal thickness (CRT), and best-corrected visual acuity (BCVA) from 12 months prior to 12 months after switch to third-line therapy were defined as primary outcomes. Results Whereas the treatment demand was reduced (from 8.8±2.2 to 4.6±2.9 injections; p=0.001) and the re-injection interval extended in group 1 (from 1.5±0.4 months to 4.4±3.8 months; p=0.001), these parameters did not change in group 2 (7.4±1.6 to 7.3±2.2; p=0.90 and 1.7±0.3 months to 1.9±0.8 months; p=0.75). Mean CRT decreased from 455.7±30.1 and 427.6±36.0µm (groups 1 and 2, respectively) to 359.1±38.2 and 303.1±44.4µm (intergroup p=0.03). The mean baseline BCVA of 62.6±3.8 letters (group 1) and 63.0±1.9 letters (group 2) remained stable under therapy in both groups (intergroup p=0.67). Conclusion In eyes with eAMD with persistent intraretinal fluid on anti-VEGF monotherapy despite frequent re-injections, corticosteroids achieved a similar functional and morphological outcome over 12 months as switching to another anti-VEGF therapy, but with a reduced injection burden. In selected cases, corticosteroid treatment may thus be an option for third-line therapy in refractory exudative AMD.

Controversy remains about how diet affects the vascular endothelial dysfunction associated with disordered insulin-glucose homeostasis. It is postulated that the type and level of certain macronutrients contribute to endothelial dysfunction in vascular diabetes complications. However, it is not well understood how specific macronutrients affect the molecular inflammatory response under conditions of hyperglycemia. Here, we examined retinal microvascular endothelial injury in streptozotocin (STZ)-diabetic rats fed a laboratory Western diet (WD). WD, characterized by its high content of saturated fat, cholesterol, and sugar, significantly increased retinal leukocyte accumulation and endothelial injury in the STZ-diabetic rats. Suppression of endothelial NF-κB signaling in the STZ model reduced the WD-induced increase in leukocyte accumulation. To isolate the effect of dietary fat, we generated high-fat diets with varying fatty acid balance and type. These diets contained moderate amounts of carbohydrates but no sugar. We found that neither high levels of saturated or unsaturated fats per se increased retinal leukocyte accumulation and endothelial injury in the STZ-diabetic rat model but that the combination of high levels of dietary cholesterol with specific saturated fatty acids that are abundant in WD exacerbated leukocyte accumulation and endothelial injury in the retinas of STZ-diabetic rats.—Barakat, A., Nakao, S., Zandi, S., Sun, D., Schmidt-Ullrich, R., Hayes, K. C., Hafezi-Moghadam, A. In contrast to Western diet, a plant-based, high-fat, low-sugar diet does not exacerbate retinal endothelial injury in streptozotocin-induced diabetes.

The aim of this paper is to summarise our own and to review published experience regarding the long-term outcome of intravitreal treatment for macular neovascularisation (MNV) secondary to Sorsby’s fundus dystrophy (SFD). A systematic literature search using the MeSH terms [Sorsby] and [anti-vascular endothelial growth factor (VEGF)] was conducted in NCBI/PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), ScienceDirect, Google Scholar and ClinicalTrials.gov to identify publications reporting anti-VEGF treatment outcomes in SFD. Treatment outcomes were extracted for this meta-analysis from 14 publications and an own patient reporting a total of 31 cases with a mean follow-up (FU) of 54 months. Both eyes were affected in ten (32.3%) instances. Heterogenous reporting limited the comparability of the outcomes. All papers in common, however, reported satisfied to excellent responses to anti-VEGF therapy if patients were diagnosed and treated immediately after onset of symptoms. Of 20 eyes, for which visual acuity was reported before and after treatment, five worsened and seven improved by more than 1 line, whereas eight eyes maintained their function by end of the follow up, and 11 eyes (55%) maintained a driving vision (Snellen VA ≥ 0.5). Of six eyes with a VA < 0.5, VA improved in one to VA ≥ 0.5, whereas of 14 eyes with an initial VA ≥ 0.5, this dropped to

Diabetic retinopathy (DR) is a sight-threatening late complication of diabetes mellitus (DM). Even though its pathophysiology has not been fully elucidated, several studies suggested a role for transforming growth factor- (TGF-) β, matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinase (TIMP) in the onset and progression of the disease. Consequently, the aim of this study was to analyze the concentrations of TGF-β1, TGF-β2, TGF-β3, MMP-3, MMP-9, and TIMP-1 in patients with different stages of DR in order to identify stage-specific changes in their concentrations during the progression of the disease. Serum and aqueous humor (AH) samples were collected during intraocular surgery, and eyes were classified into the following groups: healthy controls (n = 17), diabetic patients with non-apparent DR (n = 23), mild/moderate nonproliferative DR (NPDR) (n = 13), and advanced NPDR/proliferative DR (PDR) without vitreal hemorrhage (n = 14). None of the patients had been under anti-VEGF or laser treatment within six months prior to surgery. In the AH, TGF-β1 levels increased in advanced NPDR/PDR by a factor of 5.5 compared to the control group. Similarly, an increase in MMP-3 and TIMP-1 levels in the AH was evident in the later stages of DR, corresponding to a 7.7- and 2.4-fold increase compared to the control group, respectively, whereas serum levels of the studied proteins remained similar. In conclusion, increased concentrations of TGF-β1, MMP-3, and TIMP-1 in the AH, but not in the serum, in advanced NPDR/PDR indicate that the intraocular regulation for these cytokines is independent of the systemic one and suggest their involvement in the progression of DR.

Diabetic retinopathy (DR) is a sight-threatening late complication of diabetes mellitus (DM). Even though its pathophysiology has not been fully elucidated, several studies suggested a role for transforming growth factor- (TGF-) β, matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinase (TIMP) in the onset and progression of the disease. Consequently, the aim of this study was to analyze the concentrations of TGF-β1, TGF-β2, TGF-β3, MMP-3, MMP-9, and TIMP-1 in patients with different stages of DR in order to identify stage-specific changes in their concentrations during the progression of the disease. Serum and aqueous humor (AH) samples were collected during intraocular surgery, and eyes were classified into the following groups: healthy controls (n=17), diabetic patients with non-apparent DR (n=23), mild/moderate nonproliferative DR (NPDR) (n=13), and advanced NPDR/proliferative DR (PDR) without vitreal hemorrhage (n=14). None of the patients had been under anti-VEGF or laser treatment within six months prior to surgery. In the AH, TGF-β1 levels increased in advanced NPDR/PDR by a factor of 5.5 compared to the control group. Similarly, an increase in MMP-3 and TIMP-1 levels in the AH was evident in the later stages of DR, corresponding to a 7.7- and 2.4-fold increase compared to the control group, respectively, whereas serum levels of the studied proteins remained similar. In conclusion, increased concentrations of TGF-β1, MMP-3, and TIMP-1 in the AH, but not in the serum, in advanced NPDR/PDR indicate that the intraocular regulation for these cytokines is independent of the systemic one and suggest their involvement in the progression of DR.

To define a clinically tailored therapeutic strategy for the treatment of viral anterior uveitis (VAU).A PubMed search spanning the past 5 years was conducted using the MesH-terms "viral anterior uveitis" and "therapy."The herpes simplex virus (HSV), the varicella zoster virus (VZV), and the cytomegalovirus (CMV) are the predominant pathogens in VAU. Other viruses, including rubella, chikungunya, and zika, have been linked with distinct forms of the disease. Depending on the causative agent and the host immunocompetence, the mainstay treatment for suspected VAU is a combination of topical or systemic antivirals and topical corticosteroids, supplemented with cycloplegics and intraocular-pressure-lowering medication.Oral acyclovir, valacyclovir, and famciclovir are the mainstay of treatment for HSV- and VZV-induced infections. Brivudin serves as an alternative in insufficiently responsive cases. CMV-induced infections respond well to valganciclovir. A 3- to 12-month course of prophylactic treatment against recurrences is worth considering.

The aim of this study is to assess the effect of repeated injections of dexamethasone implants in patients with persistent diabetic macular edema (DME) despite prior therapies.This retrospective interventional study involved 47 DME-afflicted eyes, which were administered ≥2 intravitreal injections of dexamethasone. Group 1 (34 eyes) received a dexamethasone monotherapy, whereas group 2 (13 eyes) received a combination therapy with intravitreal antivascular endothelial growth factor as needed. The duration of dexamethasone effect until retreatment and the change in best-corrected visual acuity (BCVA) and central retinal thickness (CRT) were defined as outcome measures.A total of 197 injections of dexamethasone were administered in group 1 and 52 in group 2 during a mean follow-up of 23 ± 10 and 24 ± 13 months, respectively. Mean time to reinjection was 4.6 ± 0.5 (group 1) and 5.3 ± 1.0 months (group 2; P = 0.17). Reinjection intervals did not shorten over time for up to 10 dexamethasone injections per eye in group 1 and BCVA improved from before 1 month after the first implantation, 7.0 letters (P = 0.04). In group 2, there was no significant improvement in BCVA at any time point. CRT decreased from 534 ± 208 and 529 ± 215 μm to 287 ± 115 and 371 ± 78 μm at 3 months and increased to 460 ± 186 μm and 547 ± 175 μm before reinjection (groups1 and 2, respectively). The maximal CRT before each implantation remained stable over time.In eyes with chronic DME that respond incompletely to prior therapy or require frequent reinjections, dexamethasone shows promising long-term anatomic and functional improvement. The absence of a treatment effect reduction over time argues against a relevant rebound phenomenon.

Pseudoexfoliation syndrome (PEXS) may go along with capsular bag shrinkage and luxation. In the present study, we focus on an association of isoforms of TGF-β with capsular bag luxation.Aqueous humor was collected intraoperatively from 20 healthy controls and from 73 otherwise healthy patients with PEXS [PEXS without complications (PEX, n = 33), late PEXS with glaucoma (PEXG, n = 30) and with IOL and capsular bag luxation (PEXL, n = 10)]. The concentrations of TGF-β1, TGF-β2 and TGF-β3 were compared using the Bio-Plex® multiplex beads system based on the non-parametric Kruskal-Wallis H test (p 0.01).Concentrations of TGF-β 1, TGF-β 2 and TGF-β 3 were higher in the sub-groups PEX and PEXG than in controls (TGF-β 1; p = 0.009 and 0.0005; TGF-β 2; p = 0.002 and 0.005 and TGF-β 3; 0.0005 and 0.0005; respectively), whereas for TGF β2, no significant difference between controls and PEXL was revealed (p = 1.0). TGF-β2 concentrations were elevated in a similar degree in early PEX and PEXG, but not in PEXL compared to controls (p = 0.002). The concentrations of of TGF-β 1 and TGF-β 3 increased in parallel with the progression of disease. The levels of TGF-β 3, however, did not attain pathophysiological levels (100 pg/ml) in any group.A stage-dependent increase in the concentrations of TGF-β1 and TGF-β3, but not of TGF-β2, accords to the shrinkage of the capsular bag. This could increase the tension on the zonular fibers and contribute to luxation of the capsular bag.

Purpose To compare the effect of adding a corticosteroid or switching to another anti-VEGF treatment in patients with exudative age-related macular degeneration (eAMD) with persisting intraretinal fluid on anti-VEGF monotherapy. Methods This retrospective, interventional case series involved 43 pseudophakic eyes with eAMD and persistent intraretinal fluid on anti-VEGF treatment that switched treatment to a combination of Ozurdex® or Triamcinolone and anti-VEGF therapy (group 1) or to another anti-VEGF agent (group 2). The number of injections, time to re-injection, change in central retinal thickness (CRT), and best-corrected visual acuity (BCVA) from 12 months prior to 12 months after switch to third-line therapy were defined as primary outcomes. Results Whereas the treatment demand was reduced (from 8.8±2.2 to 4.6±2.9 injections; p=0.001) and the re-injection interval extended in group 1 (from 1.5±0.4 months to 4.4±3.8 months; p=0.001), these parameters did not change in group 2 (7.4±1.6 to 7.3±2.2; p=0.90 and 1.7±0.3 months to 1.9±0.8 months; p=0.75). Mean CRT decreased from 455.7±30.1 and 427.6±36.0µm (groups 1 and 2, respectively) to 359.1±38.2 and 303.1±44.4µm (intergroup p=0.03). The mean baseline BCVA of 62.6±3.8 letters (group 1) and 63.0±1.9 letters (group 2) remained stable under therapy in both groups (intergroup p=0.67). Conclusion In eyes with eAMD with persistent intraretinal fluid on anti-VEGF monotherapy despite frequent re-injections, corticosteroids achieved a similar functional and morphological outcome over 12 months as switching to another anti-VEGF therapy, but with a reduced injection burden. In selected cases, corticosteroid treatment may thus be an option for third-line therapy in refractory exudative AMD.

PURPOSE To compare the cytokine profiles of phakic (p) and pseudophakic (ps) eyes with primary rhegmatogenous retinal detachment (RD) to eyes with macular holes (MH) and to identify differences in the specific cytokine profiles. METHODS Aqueous humour (AH) and vitreous fluid (VF) were obtained from patients with primary RD without proliferative vitreoretinopathy undergoing vitrectomy. AH and VF of patients with macular holes (MH) served as controls. Forty-three different cytokines were quantified using multiplex cytokine analysis. Intergroup and intragroup comparisons were performed. To control for multiple comparisons, Holm's correction was applied. RESULTS VF and AH samples of 71 eyes with RD (pRD N = 38; psRD N = 33) and 26 eyes with MH were included. Cytokine levels in psRD and pRD were similar (none with >10-fold difference). The levels of 39 of 43 cytokines in the VF were significantly higher in eyes with RD than in those with MH (>10-fold: CXLC5, CCL26, CCL1, IL-6, CXCL11, CCL7, CCL13, MIG/CXCL9, CCL19 and TGF-β1). In the AH, 23 of 43 cytokines were significantly higher compared to MH (>10-fold: CXCL5, IL-4, IL-6, IL-8/CXCL8 and CCL7). CONCLUSION A complex, but nonspecific cytokine environmental response seems to initiate immunological and profibrotic processes following RD. Relevant differences in the cytokine profiles of eyes with pRD and psRD were not identified, whereas cytokine differences between AH and VF in RD could be explained by upregulation in the vitreous, a higher turn around in the anterior chamber, or differences in inflammatory cascades in both compartments.

PurposeVarious profibrotic and proinflammatory cytokines have been found upregulated in uncomplicated primary retinal detachment (pRD), but without providing a uniform picture. Here, we compare the cyto- and chemokine profiles in pRD with and without proliferative vitreoretinopathy (PVR) in an attempt to unravel relevant differences not in single cytokines, but in the cytokine profiles at diagnosis.MethodsUndiluted vitreous fluid (VF) was obtained at the beginning of surgery from 174 eyes with pRD without relevant PVR (maximally grade B; group 1; n = 81) and with moderate or advanced PVR requiring a gas tamponade (group 2; n = 49) or silicon oil filling (group 3; n = 44). VF of eyes undergoing macular hole (MH) surgery served as controls (group 4; n = 26). Forty-three cytokines were quantified in parallel using a multiplex cytokine analysis system (Bioplex). For all comparisons we applied Holm's correction to control for multiple comparisons.Results44.9% of group 2 eyes presented grade C1 and 55.1% C2-C3, whereas 86.4% of group 3 eyes exhibited a PVR grade of C2-D. CCL19 was the only cytokine that displayed higher concentrations in the vitreous of eyes with PVR C1 compared to lower PVR grades. Eyes with PVR C2-D showed higher levels of CCL27, CXCL6, IL4, IL16, CXCL10, CCL8, CCL22, MIG/CXCL9, CCL15, CCL19, CCL 23 and CXCL12 compared to controls. Interestingly, no difference of cytokine levels was detected between C1 and C2-D PVR.ConclusionsCCL19 may represent a potential biomarker for early PVR progression that holds promise for future diagnostic and therapeutic applications.

To survey the clinical responses to treatment of chronic postoperative and uveitic cystoid macular edema (CME) with a dexamethasone-based intravitreal implant (Ozurdex(®)).This retrospective, interventional case series reports on patients with chronic CME after uncomplicated vitrectomy for epiretinal gliosis or phacoemulsification (group 1: 12 eyes) or secondary to noninfectious endogenous uveitis (group 2: 36 eyes). Central retinal thickness (CRT), best-corrected visual acuity (BCVA, logMAR), and intraocular pressure (IOP) throughout follow-up were gleaned from the medical records.In group 1, CRT decreased, compared with baseline, from 519 ± 43 to 297 ± 23 and 356 ± 49 μm by the 1- and 3-month visit (P = 0.02) and to 429 ± 57 μm before reimplantation. In group 2, CRT decreased from 460 ± 31 to 300 ± 21 and 312 ± 26 μm by the 1- and 3-month follow-up, respectively (P = 0.001), and to 373 ± 32 μm before reimplantation. Complete resolution of CME was achieved in 67% and 94% (groups 1 and 2, respectively) by 1 month and in 42% and 80% by 3 months after injection. In group 1, BCVA improved from 0.46 ± 0.08 to 0.27 ± 0.09 and 0.20 ± 0.06 (P = 0.01) by the 1- and 3-month follow-up, respectively, and to 0.32 ± 0.08 before reimplantation. In group 2, BCVA improved from 0.47 ± 0.06 to 0.34 ± 0.09, 0.26 ± 0.07, and 0.29 ± 0.08 (P 0.05) at 1 and 3 months of follow-up and before reimplantation, respectively. A significant IOP increase was not observed in either group. Mean time to reimplantation of Ozurdex was 6.4 ± 5.7 and 6.6 ± 3.4 months for postoperative and uveitic CME, respectively.Ozurdex seems to achieve a sustained effect over up to 6 months in postsurgical and uveitic CME.

Purpose To determine the inherent risks of handling results below the lowest detectable value in the analysis of multiple cytokines in the aqueous humor of patients with retinal diseases by comparing possible statistical strategies to lower the risk of mis interpretation or over interpretation of results. Furthermore, in analyzing multiple cytokines simultaneously, the challenge of multiple comparison arises. Methods The analyses were based on parallel testing of 43 cytokines in 58 aqueous humor samples from patients with macular hole or epiretinal membrane. Substitution of values below the detection limit with 0.1 ×, 0.5 ×, or 1.0× of the lowest level of quantitation was compared with handling as missing value. The impact of correction for multiple comparisons was assessed using the Holm correction. Results When comparing macular hole with epiretinal membrane, not substituting the missing data revealed a difference (P < 0.05) for five compared with wight cytokines after their substitution, indicating an increased risk for under-estimating group differences (type II error). Correcting for multiple comparisons revealed a relevant risk of over estimating group differences (type I error). Conclusions Physiologic cytokine concentrations in ocular fluids typically range at or below the lowest level of quantitation. Handling of results below this cutoff as missing leads to increased type II errors. Not correcting for multiple comparisons increases the risk of a type I error. Taken together, both harbor a systematic inherent risk of misinterpretation of the results. Translational Relevance Ignoring the inherent risks of data misinterpretation in analyses of ocular fluid samples may result in mis leading conclusions regarding their biological relevance.

To report outcomes in patients with neovascular age-related macular degeneration (nAMD) after treatment with aflibercept for up to 4 years using a treat-and-extend (TE) regimen.Observational study.Patients with newly diagnosed nAMD treated with aflibercept in a TE protocol.Subjects received 3 injections of aflibercept at monthly intervals followed by a TE protocol for at least 12 months. At each clinical visit after the loading phase, OCT and best-corrected visual acuity (BCVA) testing were performed to monitor disease activity.Change in BCVA over time, number of injections and visits per year, and percentage of patients reaching a treatment interval of ≥12 weeks.Of 231 consecutive eyes (231 patients) with a mean follow-up time of 2.9 (1-5.5) years, 173 were followed up for ≥2 years, 112 were followed up for ≥3 years, and 62 were followed up for ≥4 years. Mean BCVA increased from 59.8 letters (20/60) at diagnosis to 65.8 letters (20/50) after the loading phase (+6.0 letters; standard deviation [SD], 11.1) and to 65.5 letters at 12 months (+5.7 letters; [SD], 17). After 4 years of treatment, mean BCVA was maintained insignificantly better than baseline (63.4 letters, +3.6 letters gain, SD, 20.6; P 0.05). To achieve this, a mean of 7.7 (±1.2) injections and 4.4 (±1.6) clinic visits in the first year and 4.4 (±1.9) injections and 4.3 (±1.3) clinical visits per year thereafter were required. By 2 years of follow-up, 46.9% of patients reached a treatment interval of ≥12 weeks.By using a TE regimen, patients with nAMD maintained stable visual function over 4 years in a real-world setting with a reasonable treatment burden.

Click here to listen to the PodcastBackground/aimsTo compare the efficacy of combined intravitreal injection of bevacizumab and a Rho-kinase inhibitor, fasudil (intravitreal bevacizumab (IVB)/intravitreal fasudil (IVF)), with IVB alone for centre-involving diabetic macular oedema (DME).MethodsIn this prospective randomised clinical trial, 44 eyes with centre-involving DME were randomised into two groups. The combined group received three consecutive injections of IVB (1.25 mg) and IVF (50 µM/L) monthly, while the monotherapy group received only one IVB (1.25 mg) injection per month for 3 months. Changes in best-corrected visual acuity (BCVA) and central macular thickness (CMT) were compared between the two groups at months 3 and 6. The primary outcome measure was the mean change in BCVA at month 6.ResultsMean BCVA was significantly improved in both groups at month 3 (PConclusionAdjunctive intravitreal injection of a Rho-kinase inhibitor may enhance and prolong the therapeutic effects of anti-vascular endothelial growth factor drugs for centre- involving DME.

This study evaluated the effects of intravitreal injection of fasudil (IVF), a Rho-kinase inhibitor, in cases of recent-onset nonarteritic anterior ischemic optic neuropathy (NAION). In this interventional case series, 13 eyes of 13 patients diagnosed with NAION within 14 days of onset were included. The affected eyes received a 0.025 mg/0.05 mL IVF. Functional and structural outcomes were assessed 1 and 3 months following treatment. Best corrected visual acuity (BCVA) was the main outcome measured, with mean deviation (MD) index of the VF test and peripapillary retinal nerve fiber layer thickness as secondary measures. There was a statistically significant improvement in the patients' BCVA 1 and 3 months following IVF; BCVA improved from 1.69 ± 0.55 logMAR at baseline to 0.98 ± 0.47 and 0.93 ± 0.51 logMAR at 1 and 3 months, respectively (P = .004). The change in BCVA was not significant between month 1 and month 3 (P = .22). Peripapillary retinal nerve fiber layer thickness decreased from 173.5 ± 29.28 µm in the baseline evaluation to 85.8 ± 8.8 µm at 1 month, and 62.9 ± 5.97 µm at 3 months (P = .003). MD values changed from 24.60 ± 3.80 to 21.0 ± 6.10 and 20.5 ± 6.50 at 1 and 3 months, respectively (P = .007 and .005, respectively). This pilot study suggests that IVF may be an effective treatment for patients with recent-onset NAION. Larger studies are required to establish the therapeutic role of fasudil for NAION.

SummaryAge is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

The Rho-kinase/ROCK (Rho-associated coiled-coil-containing protein kinase) pathway is involved in the pathogenesis of multiple ocular and systemic disorders. Recently, ROCK inhibitors have been suggested as novel treatments for various ocular diseases. Several in vitro, in vivo and clinical studies have demonstrated the safety and efficacy of ROCK inhibitors in the management of ocular disorders such as corneal epithelial and endothelial damage, glaucoma, retinal and choroidal neovascularisation, diabetic macular oedema and optic nerve disorders. In this review, these studies are explored with focus on the relevant clinical investigations.

The pathogenesis of diabetic retinopathy (DR) in metabolic syndrome (MetS) and type 2 diabetes (T2D) is not well studied, partly because an appropriate model has not been developed. Recently, we introduced a novel model of spontaneous T2D and MetS that replicates the relevant features of the human disease. In the current study, we investigated the retinal vascular changes in these animals. Experimental DR in streptozotocin (STZ)-injected rodents is described as an inflammatory disease, in which intercellular adhesion molecule 1 (ICAM-1) plays a key role. In comparison, advanced diabetes (HbA1c>10%) in the Nile grass rat (NGR) was associated with lower ICAM-1 protein expression when compared with that in normal or moderately diabetic animals. Vascular cell adhesion molecule 1 (VCAM-1) expression, however, was unaffected by the disease state. As opposed to the STZ-induced model of DR, in diabetic NGRs, most leukocytes accumulated in the retinal arteries. Consistent with the ICAM-1 reduction, leukocyte accumulation was significantly reduced in advanced disease. Similarly, leukocyte adhesions were significantly lower, with elevated plasma triglycerides (>200 mg/dl), and cholesterol (>240 mg/dl). However, these adhesions were significantly higher in animals with higher plasma insulin (>5 μIU/ml) and leptin (>20 ng/ml), suggesting a role for these hormones in diabetic retinal leukostasis. Diabetic NGRs showed substantial retinal endothelial injury, primarily in the microvessels, including vascular tortuosity, obliterated acellular capillaries, and pericyte ghosts. The NGR provides a convenient and realistic model for investigation of retinal changes in MetS/T2D with convincing advantages over the commonly used STZ-induced T1D.— Noda, K., Nakao, S., Zandi, S., Sun, D., Hayes, K. C., Hafezi-Moghadam, A. Retinopathy in a novel model of metabolic syndrome and type 2 diabetes: new insight on the inflammatory paradigm.

Purpose Cytokines play an important role in cell signaling in inflammatory and repair processes, also within the posterior segment of the eye. These molecules are thus implicated in the pathophysiology of several vitreoretinal diseases. In the present study, we compared vitreal cytokine profiles in patients with idiopathic epiretinal membranes (ERMs) and idiopathic full-thickness macular holes (MHs) without epiretinal membranes. Methods Native vitreal humor was collected during elective pars plana vitrectomy for the treatment of macular pathologies (group 1: ERM; group 2: MH) from patients without any other ocular or systemic disease. The concentrations of 43 chemokines and cytokines were measured in parallel by multiplex beads analysis. Intergroup comparisons were conducted using the Mann-Whitney U test and Bonferroni's correction, at a level of significance of P < 0.0012. Results Vitreal samples from 31 patients with ERMs (group 1) and from 30 with MHs (group 2) were analyzed. For 12 of the tested cytokines (GM-CSF, MCP-1, MIF, CCL15, CCL20, CCL17, CX3CL1, CXCL10, CXCL16, and TGF-β-1, -2, and -3), no intergroup differences were revealed; for the other 31, the concentrations were higher in the ERM than in the MH group (P < 0.0012 in each case). Conclusions The vitreal levels of 72% of the tested cytokines were higher in ERM than in MH. This indicates that even in the absence of clinical markers, activation of inflammatory and profibrotic mechanisms is implicated in the progression of ERMs. Although frequently used as such in the past, eyes with ERMs should be considered with caution as a healthy control group.

Cathepsin B (CtsB) contributes to atherosclerosis and cancer progression by processing the extracellular matrix and promoting angiogenesis. Although CtsB was reported to promote and reduce angiogenesis, there is no mechanistic explanation that reconciles this apparent discrepancy. CtsB cleaves CD18 from the surface of immune cells, but its contribution to angiogenesis has not been studied. We developed an in vivo technique for visualization of immune cell transmigration from corneal vessels toward implanted cytokines. Wild-type (WT) leukocytes extravasated from limbal vessels, angiogenic stalks, and growing tip vessels and migrated toward the cytokines, indicating immune competence of angiogenic vessels. Compared to WT leukocytes, CtsB-/- leukocytes accumulated in a higher number in angiogenic vessels, but extravasated less toward the implanted cytokine. The accumulated CtsB-/- leukocytes in angiogenic vessels expressed more CD18. CD18-/- leukocytes extravasated later than WT leukocytes. However, once extravasated, CD18-/- leukocytes transmigrated more rapidly than their WT counterparts. These results suggest that, although CD18 facilitates efficient extravasation, outside of the vessel CD18 interaction with the extracellular matrix, it reduced transmigration velocity. Our results reveal an unexpected role for CtsB in leukocyte extravasation and transmigration, which advances our understanding of the complex contribution of CtsB to angiogenesis.-Nakao, S., Zandi, S., Sun, D., Hafezi-Moghadam, A. Cathepsin B-mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels.

LYVE-1+ corneal lymphatics contribute to drainage and immunity. LYVE-1 is widely accepted as the most reliable lymphatic marker because of its continuous expression in lymphatic endothelium. LYVE-1 expression in corneal lymphatics has not been examined. In this study, we report intact CD31+ corneal lymphatic capillary endothelial cells that do not express LYVE-1. The number of LYVE-1− gaps initially increased until 8 wk of age but was significantly reduced in aged mice. C57BL/6 mice showed a notably higher number of the LYVE-1−/CD31+ lymphatic regions than BALB/c mice, which suggests a genetic predisposition for this histological feature. The LYVE-1− lymphatic gaps expressed podoplanin and VE-cadherin but not αSMA or FOXC2. Interestingly, the number of LYVE-1− gaps in FGF-2, but not VEGF-A, implanted corneas was significantly lower than in untreated corneas. Over 70% of the CD45+ leukocytes were found in the proximity of the LYVE-1− gaps. Using a novel in vivo imaging technique for visualization of leukocyte migration into and out of corneal stroma, we showed reentry of extravasated leukocytes from angiogenic vessels into newly grown corneal lymphatics. This process was inhibited by VE-cadherin blockade. To date, existence of lymphatic valves in cornea is unknown. Electron microscopy showed overlapping lymphatic endothelial ends, reminiscent of microvalves in corneal lymphatics. This work introduces a novel corneal endothelial lymphatic phenotype that lacks LYVE-1. LYVE-1− lymphatic endothelium could serve as microvalves, supporting unidirectional flow, as well as immunological hot spots that facilitate reentry of stromal macropahges.—Nakao, S., Zandi, S., Faez, S., Kohno, R., Hafezi-Moghadam, A. Discontinuous LYVE-1 expression in corneal limbal lymphatics: dual function as microvalves and immunological hot spots.

Vascular adhesion protein-1 (VAP-1) contributes to inflammatory and angiogenic diseases, including cancer and age-related macular degeneration. It is expressed in blood vessels and contributes to inflammatory leukocyte recruitment. The cytokines IL-1β and vascular endothelial growth factor A (VEGF-A) modulate angiogenesis, lymphangiogenesis, and leukocyte infiltration. The lymphatic endothelium expresses intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, which facilitate leukocyte transmigration into the lymphatic vessels. However, whether lymphatics express VAP-1 and whether they contribute to cytokine-dependent lymph- and angiogenesis are unknown. We investigated the role of VAP-1 in IL-1β– and VEGF-A–induced lymph- and angiogenesis using the established corneal micropocket assay. IL-1β increased VAP-1 expression in the inflamed cornea. Our in vivo molecular imaging revealed significantly higher VAP-1 expression in neovasculature than in the preexisting vessels. VAP-1 was expressed in blood but not lymphatic vessels in vivo. IL-1β–induced M2 macrophage infiltration and lymph- and angiogenesis were blocked by VAP-1 inhibition. In contrast, VEGF-A–induced lymph- and angiogenesis were unaffected by VAP-1 inhibition. Our results indicate a key role for VAP-1 in lymph- and angiogenesis-related macrophage recruitment. VAP-1 might become a new target for treatment of inflammatory lymph- and angiogenic diseases, including cancer.

Angio- and lymphangiogenesis are inherently related processes. However, how blood and lymphatic vessels regulate each other is unknown. This work introduces a novel mechanism explaining the temporal and spatial relation of blood and lymphatic vessels. Vascular endothelial growth factor-A (VEGF-A) surprisingly reduced VEGF-C in the supernatant of blood vessel endothelial cells, suggesting growth factor (GF) clearance by the growing endothelium. The orientation of lymphatic sprouting toward angiogenic vessels and away from exogenous GFs was VEGF-C dependent. In vivo molecular imaging revealed higher VEGF receptor (R)-2 in angiogenic tips compared with normal vessels. Consistently, lymphatic growth was impeded in the angiogenic front. VEGF-C/R-2 complex in the cytoplasm of VEGF-A–treated endothelium indicated that receptor-mediated internalization causes GF clearance from the extracellular matrix. GF clearance by receptor-mediated internalization is a new paradigm explaining various characteristics of lymphatics.

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

Metabolic syndrome (MetS) is a prevalent and complex disease, characterized by the variable coexistence of obesity, dyslipidemia, hyperinsulinaemia, and hypertension. The alarming rise in the prevalence of metabolic disorders makes it imperative to innovate preventive or therapeutic measures for MetS and its complications. However, the elucidation of the pathogenesis of MetS has been hampered by the lack of realistic models. For example, the existing animal models of MetS, i.e., genetically engineered rodents, imitate certain aspects of the disease, while lacking other important components. Defining the natural course of MetS in a spontaneous animal model of the disease would be desirable. Here, we introduce the Nile grass rat (NGR), Arvicanthis niloticus, as a novel model of MetS. Studies of over 1100 NGRs in captivity, fed normal chow, revealed that most of these animals spontaneously develop dyslipidemia (P

Atrial natriuretic peptide (ANP) is a hormone with diuretic, natriuretic, and vasodilatory properties. ANP blocks vascular endothelial growth factor (VEGF) production and signaling in vitro; however, its role in vascular leakage and angiogenesis is unknown. In vitro, retinal barrier permeability (transepithelial electrical resistance (TEER)) was measured in cultured retinal endothelial (HuREC) and retinal epithelial (ARPE-19) cells with VEGF (10 ng/ml), ANP (1 pM to 1 micromol/L), and/or isatin, an ANP receptor antagonist. In vivo, blood-retinal barrier (BRB) leakage was studied using the Evans Blue dye technique in rats treated with intravitreal injections of ANP, VEGF, or vehicle. Choroidal neovascularization was generated by laser injury, and 7 days later, lesion size and leakage was quantitated. ANP significantly reversed VEGF-induced BRB TEER reduction in both HuREC and ARPE-19 cells, modeling the inner and the outer BRB, respectively. Isatin, a specific ANP receptor antagonist, reversed ANP's effect. ANP reduced the response of ARPE-19 cells to VEGF apically but not basolaterally, suggesting polarized expression of the ANP receptors in these cells. ANP's TEER response was concentration but not time dependent. In vivo, ANP significantly reduced VEGF-induced BRB leakage and the size of laser-induced choroidal neovascularization lesions. In sum, ANP is an effective inhibitor of VEGF-induced vascular leakage and angiogenesis in vivo. These results may lead to new treatments for ocular diseases where VEGF plays a central role, such as age-related macular degeneration or diabetic retinopathy.

OBJECTIVE—Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy. Involvement of the Rho/Rho kinase (ROCK) pathway in diabetic microvasculopathy and therapeutic potential of fasudil, a selective ROCK inhibitor, are investigated. RESEARCH DESIGN AND METHODS—Localization of RhoA/ROCK and Rho activity were examined in retinal tissues of rats. Impact of intravitreal fasudil administration on retinal endothelial nitric oxide synthase (eNOS) and myosin phosphatase target protein (MYPT)-1 phosphorylation, intercellular adhesion molecule-1 (ICAM-1) expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from diabetic retinopathy patients or nondiabetic control subjects to cultured microvascular endothelial cells was quantified. The potential of fasudil for endothelial protection was investigated by measuring the number of adherent neutrophils and terminal transferase-mediated dUTP nick-end labeling–positive endothelial cells. RESULTS—RhoA and ROCK colocalized predominantly in retinal microvessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, whereas it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from diabetic retinopathy patients showed significantly higher adhesion to cultured endothelium and caused endothelial apoptosis, which was significantly reduced by fasudil. Blockade of the Fas-FasL interaction prevented endothelial apoptosis. The protective effect of fasudil on endothelial apoptosis was significantly reversed by Nω-nitro-l-arginine methyl ester, a NOS inhibitor, whereas neutrophil adhesion remained unaffected. CONCLUSIONS—The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy. Fasudil protects the vascular endothelium by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury. ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.

Vascular adhesion protein-1 (VAP-1) is an endothelial cell adhesion molecule involved in leukocyte recruitment. Leukocytes and, in particular, macrophages play an important role in the development of choroidal neovascularization (CNV), an integral component of age-related macular degeneration (AMD). Previously, we showed a role for VAP-1 in ocular inflammation. Here, we investigate the expression of VAP-1 in the choroid and its role in CNV development. VAP-1 was expressed in the choroid, exclusively in the vessels, and colocalized in the vessels of the CNV lesions. VAP-1 blockade with a novel and specific inhibitor significantly decreased CNV size, fluorescent angiographic leakage, and the accumulation of macrophages in the CNV lesions. Furthermore, VAP-1 blockade significantly reduced the expression of inflammation-associated molecules such as tumor necrosis factor (TNF) -α, monocyte chemoattractant protein (MCP) -1, and intercellular adhesion molecule (ICAM) -1. This work provides evidence for an important role of VAP-1 in the recruitment of macrophages to CNV lesions, establishing a novel link between VAP-1 and angiogenesis. Inhibition of VAP-1 may become a new therapeutic strategy in the treatment of AMD.—Noda, K., She, H., Nakazawa, T., Hisatomi, T., Nakao, S., Almulki, L., Zandi, S., Miyahara, S., Ito, Y., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Mashima, Y., Hafezi-Moghadam, A. Vascular adhesion protein-1 blockade suppresses choroidal neovascularization.

The role of lymphatics in atherosclerosis is not yet understood. Here, we investigate lymphatic growth dynamics and marker expression in atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. The prolymphangiogenic growth factor, VEGF-C, was elevated in atherosclerotic aortic walls. Despite increased VEGF-C, we found that adventitial lymphatics regress during the course of formation of atherosclerosis (P < 0.01). Similar to lymphatic regression, the number of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1(+)) macrophages decreased in the aortic adventitia of apoE(-/-) mice with atherosclerosis (P < 0.01). Intimal lymphatics in the atherosclerotic lesions exhibited an atypical phenotype, with the expression of podoplanin and VEGF receptor 3 (VEGFR-3) but not of LYVE-1 and prospero homeobox protein 1. In the aortas of atherosclerotic animals, we found markedly increased soluble VEGFR-2. We hypothesized that the elevated soluble VEGFR-2 that was found in the aortas of apoE(-/-) mice with atherosclerosis binds to and diminishes the activity of VEGF-C. This trapping mechanism explains, despite increased VEGF-C in the atherosclerotic aortas, how adventitial lymphatics regress. Lymphatic regression impedes the drainage of lipids, growth factors, inflammatory cytokines, and immune cells. Insufficient lymphatic drainage could thus exacerbate atherosclerosis formation. Our study contributes new insights to previously unknown dynamic changes of adventitial lymphatics. Targeting soluble VEGFR-2 in atherosclerosis may provide a new strategy for the liberation of endogenous VEGF-C and the prevention of lymphatic regression.-Taher, M., Nakao, S., Zandi, S., Melhorn, M. I., Hayes, K. C., Hafezi-Moghadam, A. Phenotypic transformation of intimal and adventitial lymphatics in atherosclerosis: a regulatory role for soluble VEGF receptor 2.

This study evaluated the effects of intravitreal injection of fasudil (IVF), a Rho-kinase inhibitor, in cases of recent-onset nonarteritic anterior ischemic optic neuropathy (NAION). In this interventional case series, 13 eyes of 13 patients diagnosed with NAION within 14 days of onset were included. The affected eyes received a 0.025 mg/0.05 mL IVF. Functional and structural outcomes were assessed 1 and 3 months following treatment. Best corrected visual acuity (BCVA) was the main outcome measured, with mean deviation (MD) index of the VF test and peripapillary retinal nerve fiber layer thickness as secondary measures. There was a statistically significant improvement in the patients' BCVA 1 and 3 months following IVF; BCVA improved from 1.69 ± 0.55 logMAR at baseline to 0.98 ± 0.47 and 0.93 ± 0.51 logMAR at 1 and 3 months, respectively (P = .004). The change in BCVA was not significant between month 1 and month 3 (P = .22). Peripapillary retinal nerve fiber layer thickness decreased from 173.5 ± 29.28 µm in the baseline evaluation to 85.8 ± 8.8 µm at 1 month, and 62.9 ± 5.97 µm at 3 months (P = .003). MD values changed from 24.60 ± 3.80 to 21.0 ± 6.10 and 20.5 ± 6.50 at 1 and 3 months, respectively (P = .007 and .005, respectively). This pilot study suggests that IVF may be an effective treatment for patients with recent-onset NAION. Larger studies are required to establish the therapeutic role of fasudil for NAION.

Diabetic retinopathy (DR) is a microvas- cular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumula- tion of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR-2) in retinal and choroidal vessels of diabetic animals (P

Purpose Inflammation and immune cells regulate choroidal neovascularization (CNV) and could become therapeutic targets in age-related macular degeneration (AMD). Lymphangiogenesis is a key component of various inflammatory diseases. Whether lymphangiogenesis and lymph node-mediated immunity are involved in the pathogenesis of AMD is not understood. Methods To investigate lymphangiogenesis in CNV, we generated CNV in animals by laser and studied surgically removed CNV membranes from uveitis and AMD patients. Immunohistochemistry was performed with lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1) and podoplanin antibodies. VEGF-C and VEGFR-3 expressions were examined with immunohistochemistry and Western blotting. To examine the role of lymph node in CNV, we lasered lymphotoxin alpha-deficient mice (LTα-/-) and measured the CNV volume. Results Immunohistochemistry showed that LYVE-1(+) macrophages infiltrated in acutely induced CNV, although lymphatic tubes did not form. CNV membranes from patients did not show LYVE-1(+)podoplanin(+) vessels, suggesting the lack of lymphangiogenesis in AMD and uveitis. Western blots and immunostaining revealed VEGF-C and VEGFR-3 expression in CNV lesions, mainly in macrophages and angiogenic endothelial cells. Using fluorescent microsphere tracers, we show a path for cellular migration from the eye to the cervical lymph nodes (LNs) during CNV. However, CNV injury did not cause LN swelling. CNV volume did not differ between wild-type and LN-deficient mice, suggesting that LN is not a key component of early CNV formation. Conclusions Laser-induced CNV is not primarily dependent on acquired immunity, nor does the fundus injury affect peripheral LNs. Our results reveal a previously unknown cellular connection between the ocular fundus and the cervical LNs. This connection that in function resembles lymphatics is actively utilized in CNV.