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This prospective, open-label, single-arm, non-randomized clinical trial, assessed the efficacy of a 2-year treat-and-extend (T&E) regimen involving intravitreal aflibercept injection (IAI), with the longest treatment interval set to 16 weeks, and adjunct focal/grid laser in diabetic macula edema (DME) patients. We examined 40 eyes (40 adults) with fovea-involving DME from 8 Japanese centers between April 2015 and February 2017. Participants received IAI with an induction period featuring monthly injections and a subsequent T&E period featuring 8–16-week injection interval, adjusted based on optical coherence tomography findings. The primary endpoints were mean changes in the best-corrected visual acuity (BCVA) and central subfield macular thickness (CST) from baseline. Thirty patients (75%) completed the 2-year follow-up. The mean BCVA and CST changed from 60.5 ± 15.6 letters and 499.2 ± 105.6 µm at baseline to 66.6 ± 17.1 letters (P = 0.217) and 315.2 ± 79.0 µm (P

To investigate the efficacy and safety of a treat-and-extend (T&E) regimen using aflibercept (Eylea) for diabetic macular edema (DME). Prospective, open-label, multicenter, single-arm, nonblinded clinical study. Forty eyes of 40 patients with DME received a T&E regimen of intravitreal aflibercept injection (IAI) with the longest treatment interval set to 16 weeks and adjunct focal/grid laser for 1 year. An intent-to-treat analysis was performed using the same last-observation-carried-forward method. A per-protocol analysis was also performed for patients who completed a 1-year T&E regimen. The primary endpoints were mean changes in best-corrected visual acuity (BCVA) and central subfield macular thickness (CST) from baseline. Secondary endpoints included IAI-interval extension and resultant IAI numbers and the association between an early response to IAI and final BCVA gain at 1 year. Thirty-one patients (77.5%) completed the 1-year aflibercept T&E regimen. In these per-protocol participants, the mean CST improvement/reduction was 187.3 ± 145.0 µm (P

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti–vascular endothelial growth factor drugs used to treat AMD carry the risk of inducing subretinal fibrosis. We investigated the use of adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. The therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with those in wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-β and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, transforming growth factor-β and C-X-C chemokine receptor type 4 (CXCR4) inhibitors eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.

AimsTo evaluate quantitative metrics of the retinal microvasculature in eyes with diabetic retinopathy (DR) using various en face swept-source optical coherence tomography angiography (SS-OCTA) image sizes.MethodsNon-segmented and segmented images were acquired using an SS-OCTA device (PLEX Elite 9000; Carl Zeiss Meditec, Dublin, California, USA). The scanning protocols included the 3×3 mm, 6×6 mm and 12×12 mm fields of view. Quantitative analysis of the perfusion density (PD), vessel length density (VLD) and fractal dimension (FD) was performed. The area under the receiver operating characteristic curve was estimated to assess the ability of each image size to predict DR.ResultsThis prospective, cross-sectional study included 60 eyes, (non-DR, 13 eyes; non-proliferative DR (NPDR), 24 eyes; proliferative DR (PDR), 23 eyes) of 46 patients with diabetes and 21 eyes of 16 healthy individuals. In the 12×12 mm images, the PD of healthy individuals was significantly greater than that of patients with NPDR or PDR for all layers (NPDR, pConclusionsVascular changes in DR can be monitored in detail through quantitative evaluations that combine different SS-OCTA scan sizes and parameters.

To evaluate the degree of ischemia in eyes with retinal vein occlusion (RVO) using swept-source optical coherence tomography angiography (SS-OCTA) with the extended field imaging (EFI) technique, which extends the area encompassed by SS-OCTA by scanning through trial frames fitted with a +20-diopter lens. Retrospective observational study. Twenty-three consecutive eyes of 22 patients with RVO underwent 12 × 12 mm SS-OCTA imaging both with and without EFI for determination of extension rate. Two graders blinded to the clinical data evaluated the degree of retinal ischemia in paired EFI-SS-OCTA and fluorescein angiography (FA) images, and the concordance rates between the grades were statistically examined. One EFI-SS-OCTA image was not successfully obtained due to motion artifacts caused by the patient’s poor central vision, while SS-OCTA images without EFI were captured in all 23 eyes. The average extension rate of EFI-SS-OCTA over SS-OCTA was 1.39 ± 0.06 and the average scanning area was enlarged by 76.4%. Two graders evaluated the degree of retinal ischemia by measuring nonperfusion areas as the sum of disc areas/diameters. Although their assessments of the EFI-SS-OCTA images were in complete agreement (Cohen’s Unweighted Kappa coefficient = 1.00), concordance using FA images was only moderate (Cohen’s Unweighted Kappa coefficient = 0.60). EFI-SS-OCTA noninvasively produces wider field images of retinal vasculature with one capture and provides resolution sufficient to accurately evaluate retinal capillary nonperfusion in RVO.

Intravitreal injection of bevacizumab or aflibercept significantly reduced plasma vascular endothelial growth factor level for up to 1 month, whereas intravitreal ranibizumab produced no such effect. Plasma vascular endothelial growth factor showed no correlation with the severity of diabetic macular edema or diabetic retinopathy., Purpose: The aim of this study was to investigate the changes in plasma vascular endothelial growth factor (VEGF) level depending on the severity of diabetic retinopathy (DR) or diabetic macular edema (DME) and after intravitreal injection of bevacizumab, aflibercept, or ranibizumab for treatment of DME. Methods: Plasma VEGF level was evaluated in 72 patients with DR and changes were measured in 42 patients with DME receiving intravitreal injections of bevacizumab, aflibercept, or ranibizumab at the initial injection. Results: There were no correlations between plasma VEGF level and the severity of DME or DR. Baseline plasma VEGF level (51.9 pg/mL) was significantly reduced using bevacizumab to 11.9 pg/mL after 1 week and 24.1 pg/mL after 4 weeks (P = 0.0130 and 0.0201, respectively). In aflibercept-treated eyes, plasma VEGF decreased from 52.2 pg/mL to 7.8 pg/mL and 12.6 pg/mL, respectively, at the same time points (both P < 0.001). No such reductions were observed in patients receiving ranibizumab. Conclusion: Baseline plasma VEGF level showed no correlations with DR or DME severity, whereas intravitreal injection of bevacizumab or aflibercept significantly reduced plasma VEGF for up to 4 weeks and ranibizumab produced no such effects. Changes in plasma VEGF level seemed not to be critical in progression or treatment of DME and DR.

Diabetic macular edema (DME) is caused by blood-retinal barrier breakdown associated with retinal vascular hyperpermeability and inflammation, and it is the major cause of visual dysfunction in diabetic retinopathy. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilator. ADM is expressed in the eyes and is up-regulated in various eye diseases, although the pathophysiological significance is largely unknown. We investigated the effect of ADM on DME. In Kimba mice, which overexpress human vascular endothelial growth factor in their retinas, the capillary dropout, vascular leakage, and vascular fragility characteristic of diabetic retinopathy were observed. Intravitreal or systemic administration of ADM to Kimba mice ameliorated both the capillary dropout and vascular leakage. Evaluation of the transendothelial electrical resistance and fluorescein isothiocyanate-dextran permeability of an endothelial cell monolayer using TR-iBRB retinal capillary endothelial cells revealed that vascular endothelial growth factor enhanced vascular permeability but that co-administration of ADM suppressed the effect, in part by enhancing tight junction formation between endothelial cells. In addition, a comprehensive PCR array analysis showed that ADM administration suppressed various molecules related to inflammation and NF-κB signaling within retinas. From these results, we suggest that by exerting inhibitory effects on retinal inflammation, vascular permeability, and blood-retinal barrier breakdown, ADM could serve as a novel therapeutic agent for the treatment of DME.

Purpose To compare neovascularization identified in proliferative diabetic retinopathy (PDR) eyes by widefield swept-source (SS) OCT angiography (OCTA) using vitreoretinal interface (VRI) slab images, composed by automated and manual segmentation, with that identified by fluorescein angiography (FA). Design Retrospective study. Participants Forty-two eyes of 30 treatment-naive PDR patients who visited the outpatient clinic of the Department of Ophthalmology, Shinshu University, from June 2018 through October 2019. Methods All patients underwent comprehensive ophthalmologic examinations, including SS-OCTA and FA. Main Outcome Measures Neovascularization detected by en face SS-OCTA 15 × 15-mm VRI slab images and by FA in the same 15 × 15-mm areas were compared in terms of number and structure. Results Among 100 neovascularizations detected by FA, 73 also were visualized as neovascularization in SS-OCTA VRI slab images using automated segmentation. The sensitivity of VRI slab images for detecting neovascularization was 73%. Among the remaining 27 neovascularizations detected only by FA, but not by VRI slab, 15 were intraretinal microvascular abnormalities with fluorescence leakage, 1 was a diabetic papillopathy, and 11 were flat neovascularizations on the internal limiting membrane surface that were missed because of segmentation error. Conversely, among the 98 neovascularizations detected on VRI slab images, 25 were not detected as neovascularizations by FA. They included 9 small neovascularizations that exhibited too little leakage on FA and 16 false-positive results that were the result of segmentation errors. After reconstruction of SS-OCTA VRI slab images by means of manual segmentation, the sensitivity of VRI slab images for detecting neovascularizations increased to 84%. Conclusions The efficacy of SS-OCTA VRI slab images for detecting neovascularizations in PDR was comparable with that of FA. Swept-source OCTA VRI slab images may be better than FA for identifying intraretinal microvascular abnormalities and diabetic papillopathy from neovascularizations. Notably, however, FA and SS-OCTA VRI slab images demonstrated differences in identification efficacy in cases of small and flat neovascularizations. Further exploration of SS-OCTA technology is warranted to address this issue.

The effect of combination therapy using intravitreal ranibizumab (IVR) injections and short pulse focal/grid laser photocoagulation was evaluated for the treatment of diabetic macular edema (DME). The current investigation was a preliminary single-arm, open-label, prospective clinical study conducted on 21 eyes at 4 sites in Japan. Treatment protocol consisted of two phases. The induction IVR phase included two monthly IVRs followed by PRN IVR phase in which additional IVR was administered if the central macular thickness (CMT) exceeded 300 μm. One week after each IVR in both phases, short pulse focal/grid laser was delivered to treat residual leakage outside of the fovea (>500 μm) and reduce edema fluid influx. At the 6-month endpoint, the effects of treatment were examined in terms of best corrected visual acuity (BCVA), CMT, and required number of IVR injections in eyes with or without perifoveal leaking microaneurysms (MAs). In eyes with initial BCVA ≤70 letters, mean BCVA was significantly ameliorated by 7.0 ± 7.4 letters (P = 0.0324) and mean CMT improved significantly by 174.8 ± 105.0 µm (P = 0.0005). Both BCVA improvement (P = 0.8693) and CMT reduction (P = 0.9336) were comparable between MA(−) and MA(+) groups. The MA(−) group required significantly fewer PRN-IVR injections than did the MA(+) group over the 6-month study period (mean 3.4 ± 1.6 vs. 5.3 ± 0.9, median 3.0 vs. 5.5; P = 0.0229). Short pulse focal/grid laser photocoagulation could reduce the number of IVR injections required to resolve macular edema and increase BCVA in a possible mechanism of reduced influx of edema fluid into the foveal area in eyes without apparent perifoveal microaneurysms.

To investigate the effect of leaking capillaries and microaneurysms (MAs) in the perifoveal capillary network (PCN) on the treatment of fovea-involving macular edema (ME) secondary to branch retinal vein occlusion (BRVO) by intravitreal ranibizumab (IVR) injections combined with focal, grid, and scatter laser photocoagulation. Retrospective comparative case series. The MA (+) group consisted of 12 patients with leaking MAs in the PCN and the MA (−) group contained 11 patients without. At 6 months following the initial IVR injection, best corrected visual acuity (BCVA) was evaluated as the primary outcome. Secondary outcomes included central macular thickness (CMT) and the number of IVR injections performed in a pro re nata (PRN) regimen when CMT was ≥300 µm and vision deteriorated by 0.1 logMAR or greater. Mean BCVA improved by 0.30 ± 0.25 logMAR in the MA (−) group and 0.28 ± 0.20 logMAR in the MA (+) group (both P

Background The ability to image wide fundus fields and to conduct swift, non-invasive examinations is increasingly important with the escalation in patients with diabetic retinopathy (DR). Methods Fifty eyes of 28 consecutive patients with DR were examined in this prospective observational study. A total of 46 eyes, 25 right and 21 left eyes, of 27 patients (male, 19; female, 8) were ultimately included in the analysis. All patients underwent comprehensive ophthalmological examination. A single image each was obtained using two ultra-wide-field (UWF) imaging systems: Optos® (Optos Carfornia®, Optos PLC, Dunfermline, United Kingdom) and Clarus™ (CLARUS 500™, Carl Zeiss Meditec Inc., Californea, USA), without mydriasis. The total retinal area captured and the obscured retinal area were compared between the two systems using nonparametric Wilcoxon matched-pairs signed-rank analysis. Early Treatment of Diabetic Retinopathy Study (ETDRS) and International Clinical DR severity were analyzed by κ statistics. Results The Optos® allowed capture of larger areas of the fundus than the Clarus™ (465 ± 117 vs. 243 ± 39 disc areas, P

Central retinal vein occlusion (CRVO) is an intractable disease that causes visual acuity loss with retinal ischemia, hemorrhage, and edema. In this study, we developed an experimental CRVO model in mice and evaluated the therapeutic potential of the pleiotropic peptide adrenomedullin (ADM) and its receptor activity-modifying protein 2 (RAMP2). The CRVO model, which had phenotypes resembling those seen in the clinic, was produced by combining i.p. injection of Rose bengal, a photoactivator dye enhancing thrombus formation, with laser photocoagulation. Retinal vascular area, analyzed using fluorescein angiography and fluorescein isothiocyanate-perfused retinal flat mounts, was decreased after induction of CRVO but gradually recovered from day 1 to 7. Measurements of retinal thickness using optical coherence tomography and histology revealed prominent edema early after CRVO, followed by gradual atrophy. Reperfusion after CRVO was diminished in Adm and Ramp2 knockout (KO) mice but was increased by exogenous ADM administration. CRVO also increased expression of a coagulation factor, oxidative stress markers, and a leukocyte adhesion molecule in both wild-type and Adm KO mice, and the effect was more pronounced in Adm KO mice. Using retinal capillary endothelial cells, ADM was found to directly suppress retinal endothelial injury. The retinoprotective effects of the Adm-Ramp2 system make it a novel therapeutic target for the treatment of CRVO.

Purpose: Evaluate the effect of foveal leaking microaneurysms (MAs) on the required number of intravitreal ranibiz­umab (IVR) injections in the treatment of center-involving diabetic macular edema (DME) when treated with focal/grid laser. Design: A pilot study of prospective, nonrandomized, multicenter clinical trial. Methods: This study enrolled 21 eyes with DME for which pro re nata IVR injections were combined with short-pulse focal/grid laser. At 12 months, best-corrected visual acuity (BCVA), central subfield macular thickness (CMT), and the required number of IVRs to maintain CMT < 300 µm were compared between eyes with or without foveal leaking MAs, termed the MA(+) and MA(–) groups, respectively. Results: Significant CMT improvements (p < 0.0001) and increased BCVA of 4.0 ± 8.5 letters were observed at 12 months. The MA(–) group required significantly fewer IVRs than did the MA(+) group (mean: 4.9 ± 3.0 vs. 8.6 ± 3.0; p = 0.0306). In the latter 6 months of the 1-year follow-up, 50% (4/8) of MA(–) eyes did not require any IVR administration to sustain CMT < 300 µm. Conclusions: A combination therapy of short-pulse focal/grid laser and reduced IVR injections appeared noninferior to previous reports of IVR monotherapy. Further large-scale investigations are warranted.

Calcitonin gene–related peptide (CGRP; official name CALCA) has a variety of functions and exhibits both angiogenic and anti-inflammatory properties. We previously reported the angiogenic effects of the CGRP family peptide adrenomedullin in oxygen-induced retinopathy; however, the effects of CGRP on ocular angiogenesis remain unknown. Herein, we used CGRP knockout (CGRP −/− ) mice to investigate the roles of CGRP in ocular vascular disease. Observation of pathological retinal angiogenesis in the oxygen-induced retinopathy model revealed no difference between CGRP −/− and wild-type mice. However, much higher levels of the CGRP receptor were present in the choroid than the retina. Laser-induced choroidal neovascularization (CNV), a model of exudative age-related macular degeneration, revealed more severe CNV lesions in CGRP −/− than wild-type mice, and fluorescein angiography showed greater leakage from CNV in CGRP −/− . In addition, macrophage infiltration and tumor necrosis factor (TNF)-α production were enhanced within the CNV lesions in CGRP −/− mice, and the TNF-α, in turn, suppressed the barrier formation of retinal pigment epithelial cells. In vivo , CGRP administration suppressed CNV formation, and CGRP also dose dependently suppressed TNF-α production by isolated macrophages . From these data, we conclude that CGRP suppresses the development of leaky CNV through negative regulation of inflammation. CGRP may thus be a promising therapeutic agent for the treatment of ocular vascular diseases associated with inflammation.

Background/Aims: To investigate the dynamics of macular hole (MH) closure in gas-filled eyes starting 20 min after vitrectomy using swept source optical coherence tomography (SS-OCT). Methods: Twenty consecutive eyes with MH underwent vitrectomy with internal limiting membrane peeling and gas tamponade. SS-OCT imaging was performed approximately 20 min after the operation, and then once a day, until MH closure was confirmed. The correlation between the base, top and minimum hole diameters and the duration required for MH closure was investigated. Results: MH closure in gas-filled eyes was confirmed in 1 eye on day 0, 10 eyes on day 1, 2 eyes on day 2, and 3 eyes on day 3, at which times face-down posturing was discontinued without MH recurrence. SS-OCT revealed a distinct closure pattern within the first 24 h postoperatively. MHs closing by day 1 had a significantly smaller minimum diameter (312.5 ± 105.2 µm) than holes closing on day 2 or later (510.8 ± 153.5 µm; p = 0.019). Conclusions: SS-OCT enables tomographic images of MH in gas-filled eyes immediately postoperatively, thus permitting early discontinuation of, or no necessity for, face-down positioning upon confirmation of MH closure.

Purpose To report en face swept-source optical coherence tomography (SS-OCT) as a noninvasive detection modality for regions of retinal layer thinning that are potential indicators of retinal nonperfusion areas (NPAs) in patients with branch retinal vein occlusion (BRVO). Methods Thirty-one eyes of 46 patients with BRVO showing a definite retinal NPA larger than 1 disc in diameter within the vascular arcades were included in this study. We calculated the overlapping correspondence ratio between presumed NPA, which appeared as a dark area in en face SS-OCT, and definite NPA in corresponding fluorescein angiography (FA) images. The correlation between brightness in en face SS-OCT images and corresponding ganglion cell complex (GCC) thickness determined by SD-OCT GCC maps was evaluated as well. Results Measured NPA size in FA and presumed NPA size in en face SS-OCT showed strong correlation (r = 0.746, pConclusions The results suggest that en face SS-OCT is a noninvasive and relatively reliable method for delineating retinal thinning as an indicator of NPAs in eyes with BRVO. Because SS-OCT can be performed without mydriasis, this procedure may be an option for evaluating NPAs on a screening basis and during follow-up to reduce the number of FA examinations that carry very rare, but potentially fatal, allergic side effects.

The accessory protein RAMP2 is a component of the CLR/RAMP2 dimeric adrenomedullin (AM) receptor and is the primary determinant of the vascular functionality of AM. RAMP2 is highly expressed in the brain; however, its function there remains unclear. We therefore used heterozygous RAMP2 knockout (RAMP2+/-) mice, in which RAMP2 expression was reduced by half, to examine the actions of the endogenous AM-RAMP2 system in cerebral ischemia. To induce acute or chronic ischemia, mice were subjected to middle cerebral artery occlusion (MCAO) or bilateral common carotid artery stenosis (BCAS), respectively. In RAMP2+/- mice subjected to MCAO, recovery of cerebral blood flow (CBF) was slower than in WT mice. AM gene expression was upregulated after infarction in both genotypes, but the increase was greater in RAMP2+/- mice. Pathological analysis revealed severe nerve cell death and demyelination, and a higher level of oxidative stress in RAMP2+/- mice. In RAMP2+/- mice subjected to BCAS, recovery of cerebral perfusion was slower and less complete than in WT mice. In an 8-arm radial maze test, RAMP2+/- mice required more time to solve the maze and showed poorer reference memory. They also showed greater reductions in nerve cells and less compensatory capillary growth than WT mice. These results indicate the AM-RAMP2 system works to protect nerve cells from both acute and chronic cerebral ischemia by maintaining CBF, suppressing oxidative stress, and in the case of chronic ischemia, enhancing capillary growth.

Adrenomedullin (AM) is a vasoactive peptide that possesses various bioactivities. AM receptors are dimers consisting of CLR with one of two accessory proteins, RAMP2 or RAMP3. The functional difference between CLR/RAMP2 and CLR/RAMP3 and the relationship between the two receptors remain unclear. To address these issues, we generated RAMP2 and RAMP3 knockout (-/-) mice and have been studying their physiological activities in the vascular system. AM-/- and RAMP2-/- mice die in utero due to blood vessel abnormalities, which is indicative of their essential roles in vascular development. In contrast, RAMP3-/- mice were born normally without any major abnormalities. In adult RAMP3-/- mice, postnatal angiogenesis was normal, but lymphangiography using indocyanine green (ICG) showed delayed drainage of subcutaneous lymphatic vessels. Moreover, chyle transport by intestinal lymphatics was delayed in RAMP3-/- mice, which also showed more severe interstitial edema than wild-type mice in a tail lymphedema model, with characteristic dilatation of lymphatic capillaries and accumulation of inflammatory cells. In scratch-wound assays, migration of isolated RAMP3-/- lymphatic endothelial cells was delayed as compared to wild-type cells, and AM administration failed to enhance the re-endothelialization. The delay in re-endothelialization was due to a primary migration defect rather than a decrease in proliferation. These results suggest that RAMP3 regulates drainage through lymphatic vessels, and that the AM-RAMP3 system could be a novel therapeutic target for controlling postoperative lymphedema.

Aims We evaluated skin autofluorescence (AF) as a marker of tissue advanced glycation end-product (AGE) accumulation and examined whether it was related to the prevalence and severity of diabetic retinopathy (DR) and of diabetic macular edema (DME) in patients with type 2 diabetes mellitus (DM). Methods This study included 138 type 2 DM patients consisting of 31 patients with proliferative DR, 71 patients with non-proliferative DR, and 36 patients without retinopathy, in addition to 111 non-DM control subjects. At the time of skin AF and HbA1c measurement, self-assessed duration of DM was also determined. DR and DME stages were classified according to international guidelines. Results Skin AF was significantly increased in patients with DM as compared with non-DM controls. Furthermore, skin AF was correlated with the severity of DR, whereas single measurement of HbA1c and self-assessed DM duration were not. None of these 3 factors showed a correlation with DME prevalence or severity. Conclusions Skin AF levels, which can be measured non-invasively on a screening basis without skin biopsy or blood sampling, have a greater predictive ability for the presence and severity of DR than single measurement of HbA1c or self-assessed DM duration in patients with type 2 DM.

Background/Aims: To establish a screening and treatment method for fovea-threatening diabetic macular edema (DME) using spectral-domain optical coherence tomography (SD-OCT). In order to maintain good visual acuity (VA), focal/grid laser treatment for screened fovea-threatening DME was evaluated based on macular thickness map images produced by SD-OCT. Methods: In 66 diabetic eyes with no visual deterioration, the sensitivity and the specificity of SD-OCT without the use of mydriatics for the detection of fovea-threatening DME were determined. A definite diagnosis of DME was made under mydriasis, using slitlamp biomicroscopy with a contact lens. Eyes with fovea-threatening DME then underwent prophylactic focal/grid laser treatment. The main outcome measures were corrected VA and central macular thickness (CMT). Results: A definitive diagnosis of DME was made in 5 of the 66 eyes, while macular thickening above the 99th percentile was detected in 11 (Cirrus®) or 13 (RS-3000®) eyes by SD-OCT. The focal/grid laser treatment of the 5 eyes with fovea-threatening DME successfully maintained good VA, which was 0.91 ± 0.76 (average: 20/22; 0.04 ± 0.12 logMAR) before treatment and 0.89 ± 0.70 (average: 20/22; 0.05 ± 0.15 logMAR; p = 0.88) 1 year afterwards. The average CMT was stable before and after focal/grid lasering at 302 ± 29 and 329 ± 55 µm, respectively (p = 0.99). Conclusions: The detection of fovea-threatening DME is feasible by SD-OCT without using mydriatics. Prophylactic treatment, such as with focal/grid lasers, was effective in maintaining good VA by avoiding an otherwise highly likely foveal involvement.

Purpose This study aimed to evaluate the consultation and referral pathway and benefit of urgent surgery within 24 hours of outpatient clinic presentation on increasing the ratio of eyes with fovea-on at surgery and visual outcomes in retinal detachment cases. Design This is a retrospective, interventional case series. Methods A total of 106 eyes underwent an operation for rhegmatogenous retinal detachment. Standard vitrectomy or explant scleral buckling was performed. The ratio of eyes with fovea-on at the time of operation, anatomical success rate, and postoperative best corrected visual acuity were measured. Results Of the106 eyes, 46 (43.4%) already were fovea-off at initial eye clinic visit, and 9 eyes became fovea-off during referral. Consequently, 55 patients (51.9%) were fovea-off when presenting to our outpatient clinic. Retinal detachment was within 1 disc diameter of the fovea in 9 of 51 eyes with fovea-on at outpatient clinic presentation, but surgery within 24 hours spared 6 eyes from foveal involvement. The anatomical success rate of primary surgery was 98.8% (81/82 eyes) by vitrectomy and 83.3% (20/24 eyes) by scleral buckling. Postoperative best corrected visual acuity of the fovea-on group was significantly higher (mean [SD], -0.019 [0.22] logarithm of the minimal angle of resolution) than that of the fovea-off group (mean [SD], 0.32 [0.45] logarithm of the minimal angle of resolution; P = 0.002). Conclusions More than half (51.9%) of our cohort had already been fovea-off by outpatient presentation. Therefore, efforts to urge patients to visit operating facilities promptly seem to be as important as the urgent surgeries themselves.

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第935号）・小山 晃英, Background-Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. Methods and Results-We generated endothelial cell-specific RAMP2 and AM knockout mice (E-RAMP2(-/-) and E-AM(-/-)). Most E-RAMP2(-/-) mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2(-/-) mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2(-/-) mice (DI-E-RAMP2(-/-)) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. Conclusions-Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage. (Circulation. 2013;127:842-853.), Article, CIRCULATION. 127(7):842-853 (2013)

Adrenomedullin (AM) was identified as a vasodilating and hypotensive peptide mainly produced by the cardiovascular system. The AM receptor calcitonin receptor-like receptor associates with receptor activity-modifying protein (RAMP), one of the subtypes of regulatory proteins. Among knockout mice ( −/− ) of RAMPs, only RAMP2 −/− is embryonically lethal with cardiovascular abnormalities that are the same as AM −/− . This suggests that the AM-RAMP2 system is particularly important for the cardiovascular system. Although AM and RAMP2 are highly expressed in the heart from embryo to adulthood, their analysis has been limited by the embryonic lethality of AM −/− and RAMP2 −/− . For this study, we generated inducible cardiac myocyte-specific RAMP2 −/− (C-RAMP2 −/− ). C-RAMP2 −/− exhibited dilated cardiomyopathy-like heart failure with cardiac dilatation and myofibril disruption. C-RAMP2 −/− hearts also showed changes in mitochondrial structure and downregulation of mitochondria-related genes involved in oxidative phosphorylation, β-oxidation, and reactive oxygen species regulation. Furthermore, the heart failure was preceded by changes in peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis. Metabolome and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) imaging analyses revealed early downregulation of cardiolipin, a mitochondrial membrane-specific lipid. Furthermore, primary-cultured cardiac myocytes from C-RAMP2 −/− showed reduced mitochondrial membrane potential and enhanced reactive oxygen species production in a RAMP2 deletion–dependent manner. C-RAMP2 −/− showed downregulated activation of cAMP response element binding protein (CREB), one of the main regulators of mitochondria-related genes. These data demonstrate that the AM-RAMP2 system is essential for cardiac metabolism and homeostasis. The AM-RAMP2 system is a promising therapeutic target of heart failure.

Background/Aims: We evaluated the effects of pattern scan laser (PSL) wavelength in delivering appropriate laser burns to the retina of eyes with an opaque lens. Methods: Sixteen shots of 2 × 2 square grids (64 laser spots) were delivered using green (532-nm), yellow (577-nm) and red (647-nm) lasers to the retinas of mice with mild cataract induced by chloral hydrate (400 mg/kg). Three eyes with clear lenses served as controls. One week after laser coagulation, the ratio of appropriate burns, defined as coagulation restricted to the outer half of the retina without retinal or choroidal hemorrhage, was investigated histologically. Results: With the green laser, we confirmed only 3.0 ± 2.0 appropriate burns in eyes with an opaque lens, in contrast to 13.7 ± 4.0 effective burns in eyes with a clear lens. On the other hand, the yellow and red lasers produced 18 ± 5.2 and 13 ± 1.5 appropriate burns, respectively, in eyes with an opaque lens. Conclusion: Although all three PSL wavelengths successfully delivered appropriate burns restricted to the outer half of the retina in eyes with an opaque lens, the longer-wavelength yellow and red lasers were significantly more effective than the green laser. PSL may be a treatment option to accompany anti-vascular endothelial growth factor drug therapy.

Various bioactive peptides have been implicated in the homeostasis of organs and tissues. Adrenomedullin (AM) is a peptide with various bioactivities. AM-receptor, calcitonin-receptor-like receptor (CLR) associates with one of the subtypes of the accessory proteins, RAMPs. Among the RAMP subisoforms, only RAMP2 knockout mice (−/−) reproduce the phenotype of embryonic lethality of AM−/−, illustrating the importance of the AM-RAMP2-signaling system. Although AM and RAMP2 are abundantly expressed in kidney, their function there remains largely unknown. We used genetically modified mice to assess the pathophysiological functions of the AM-RAMP2 system. RAMP2+/− mice and their wild-type littermates were used in a streptozotocin (STZ)-induced renal injury model. The effect of STZ on glomeruli did not differ between the 2 types of mice. On the other hand, damage to the proximal urinary tubules was greater in RAMP2+/−. Tubular injury in RAMP2+/− was resistant to correction of blood glucose by insulin administration. We examined the effect of STZ on human renal proximal tubule epithelial cells (RPTECs), which express glucose transporter 2 (GLUT2), the glucose transporter that specifically takes up STZ. STZ activated the endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK). AM suppressed PERK activation, its downstream signaling, and CCAAT/enhancer-binding homologous protein (CHOP)-induced cell death. We confirmed that the tubular damage was caused by ER stress-induced cell death using tunicamycin (TUN), which directly evokes ER stress. In RAMP2+/− kidneys, TUN caused severe injury with enhanced ER stress. In wild-type mice, TUN-induced tubular damage was reversed by AM administration. On the other hand, in RAMP2+/−, the rescue effect of exogenous AM was lost. These results indicate that the AM-RAMP2 system suppresses ER stress-induced tubule cell death, thereby exerting a protective effect on kidney. The AM-RAMP2 system thus has the potential to serve as a therapeutic target in kidney disease.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after percutaneous coronary intervention. Calcitonin gene-related peptide (CGRP) is produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. Originally identified as a strongly vasodilatory neuropeptide, CGRP is now known to be a pleiotropic peptide widely distributed in various organs and tissues. Our aim was to investigate the possibility that CGRP acts as an endogenous vasoprotective molecule. We compared the effect of CGRP deficiency on neointimal formation after wire-induced vascular injury in wild-type and CGRP knockout (CGRP-/-) mice. We found that neointimal formation after vascular injury was markedly enhanced in CGRP-/- mice, which also showed a higher degree of oxidative stress, as indicated by reduced expression of nitric oxide synthase, increased expression of p47phox, and elevated levels of 4HNE, as well as greater infiltration of macrophages. In addition, CGRP-deficiency led to increased vascular smooth muscle cell (VSMC) proliferation within the neointima. By contrast, bone marrow-derived cells had little or no effect on neointimal formation in CGRP-/-mice. In vitro analysis showed that CGRP-treatment suppressed VSMC proliferation, migration, and ERK1/2 activity. These results clearly demonstrate that endogenous CGRP suppresses the oxidative stress and VSMC proliferation induced by vascular injury. As a vasoprotective molecule, CGRP could be an important therapeutic target in cardiovascular disease.

Many reports advocate two-step drainage as the treatment for globe ruptures associated with suprachoroidal hemorrhage (SCH) involving the macula [1–3]. The first surgery is to close the ocular perforation. The second surgery is a combination of SCH drainage through sclerotomy and vitrectomy for secondary retinal detachment, which is unavoidable in perforating ocular trauma. Clot lysis, which is indispensable for drainage, can be determined by dynamic B-mode ultrasonography, which shows a freely moving liquefied blood clot in the suprachoroidal space [3]. However, the visual acuity outcomes of this two-step strategy remain generally poor, especially when the SCH is massive, showing a kissing configuration [1–3]. Previous reports suggest that early drainage of massive SCH is essential for good visual prognosis [2–4], but natural blood clot lysis, which is indispensable before drainage, takes 1–2 weeks [3]. To our knowledge, this is the first report on the clinical use of tissue-type plasminogen activator (tPA) for the drainage of a massive SCH demonstrating a kissing configuration. This treatment provided an excellent visual outcome. Case report

Background Fat embolism in the deep retinal capillary plexus is one of the reported mechanisms underlying central/paracentral scotoma in patients with Purtscher’s retinopathy. Here we report the clear delineation of capillary dropout in the deep capillary plexus using optical coherence tomography angiography (OCTA) in a chronic case of unexplained scotoma that developed after femoral fracture. The patient exhibited normal fluorescein angiography (FA) findings and a normal retinal appearance. Case presentation A 42-year-old Japanese man with a history of bilateral, unexplained paracentral scotoma that developed after femoral fracture and pulmonary fat embolism due to a car accident 20 years ago was referred to our outpatient clinic. Initial ophthalmological examination revealed unremarkable retinal findings. Goldmann perimetry, FA, and full field electroretinography showed no pathological changes. Although fat embolism in the retinal vasculature was suspected, psychosomatic visual field defects could not be ruled out. We performed OCTA, which clearly delineated capillary dropout in the deep retinal capillary plexus. A final diagnosis of paracentral acute middle maculopathy secondary to Purtscher’s retinopathy was made on the basis of this finding. Conclusions Our findings suggest that OCTA clearly and noninvasively delineates the deep retinal capillary plexus and the superficial capillary plexus. Because conventional FA provides limited depth resolution, capillary dropout restricted within the deep capillary plexus cannot be detected, particularly when the superficial capillary plexus is well preserved. Thus, OCTA can be a useful tool for the detection of capillary dropout in the deep retinal capillary plexus.