Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Keijiro Ishikawa Last modified date:2022.05.17

Assistant Professor / Department of Clinical Medicine / Faculty of Medical Sciences


Papers
1. Yosuke Fukuda, Shintaro Nakao, Ri-Ichiro Kohno, Keijiro Ishikawa, Sakurako Shimokawa, Satomi Shiose, Atsunobu Takeda, Yuki Morizane, Koh-Hei Sonoda, Postoperative follow-up of submacular hemorrhage displacement treated with vitrectomy and subretinal injection of tissue plasminogen activator: ultrawide-field fundus autofluorescence imaging in gas-filled eyes., Japanese journal of ophthalmology, 10.1007/s10384-022-00910-7, 66, 3, 264-270, 2022.05, PURPOSE: To investigate the utility of Optos ultrawide-field fundus autofluorescence (UWF-FAF) imaging for postoperative follow-up of gas-filled eyes after vitrectomy with subretinal tissue-plasminogen activator (t-PA) injection for subretinal hemorrhage (SRH) displacement. STUDY DESIGN: Retrospective consecutive case series. METHODS: This study included 24 eyes with SRH. Vitrectomy with subretinal t-PA injection was performed, followed by postoperative prone positioning. FAF images acquired using Optos California were examined and the SRH occupancy in the macula was calculated. The main outcome measures were displacement rate and direction of SRH for 3 days postoperatively, and postoperative best-corrected visual acuity (BCVA). RESULTS: The postoperative BCVA ranged from improvement (23 eyes; 95.8%) to no change (one eye; 4.2%). Analysis was done using postoperative Optos FAF images for 20 eyes (83.3%). Postoperative SRH occupancy was significantly reduced, by 27.4%, compared with the preoperative occupancy (P = 0.03). A statistically significant reduction was found between the preoperative and postoperative day (POD)1 (P = 0.04), but not between POD1 and POD2 (P = 0.7), or between POD2 and POD3 (P = 1.0). CONCLUSION: UWF-FAF imaging is useful for postoperative follow-up of gas-filled eyes after vitrectomy with subretinal t-PA injection for SRH displacement..
2. Atsunobu Takeda, Eiichi Hasegawa, Nobuyo Yawata, Shoji Notomi, Keijiro Ishikawa, Yusuke Murakami, Toshio Hisatomi, Kazuhiro Kimura, Koh-Hei Sonoda, Increased vitreous levels of B cell activation factor (BAFF) and soluble interleukin-6 receptor in patients with macular edema due to uveitis related to Behçet's disease and sarcoidosis., Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 10.1007/s00417-022-05600-1, 2022.03, PURPOSE: Uveitis accounts for 10-15% of all cases of blindness in the developed world. Uveitic macular edema (UME) is a primary cause of permanent visual impairment in patients with uveitis. Because proinflammatory mediators elicit inflammation and lead to UME, we determined the profiles of proinflammatory mediators associated with complications, such as ME, in the vitreous humor of patients with panuveitis related to Behçet's disease (BD) and sarcoidosis. METHODS: In this retrospective study, we enrolled 21 patients with uveitis, including 6 with BD and 15 with sarcoidosis, and 15 patients with idiopathic epiretinal membrane (iERM) at the Department of Ophthalmology, Kyushu University Hospital, between January 2008 and April 2016. Vitreous concentrations of 32 proinflammatory mediators, including cytokines and soluble receptors of tumor necrosis factor (TNF) and interleukin (IL)-6 families, were assessed using a bead-based multiplex assay and their association with clinical data was examined. RESULTS: The levels of proinflammatory mediators, including a proliferation-inducing ligand (APRIL), B cell activating factor belonging to the TNF family (BAFF), soluble cluster of differentiation 30 (sCD30), soluble TNF receptor-1 (sTNFR1), sTNFR2, TNF-α, IL-6, and soluble IL-6 receptor-α (sIL-6Rα), were significantly higher in patients with uveitis. With regard to clinical parameters in patients with uveitis, vitreous levels of BAFF and sIL-6Rα were prominently elevated in patients with UME compared to in those without UME (P < 0.01, respectively). CONCLUSIONS: Our results suggest that elevated vitreous levels of BAFF and sIL-6Rα are associated with the pathogenesis of UME in patients with panuveitis related to BD and sarcoidosis..
3. Sumihiro Kawano, Takumi Imai, Taiji Sakamoto, Differences in primary retinal detachment surgery conducted on holidays and workdays analyzed using the Japan Retinal Detachment Registry., Japanese journal of ophthalmology, 10.1007/s10384-022-00911-6, 66, 3, 271-277, 2022.05, PURPOSE: To investigate the characteristics of retinal detachment (RD) and compare the outcomes of surgical interventions, such as scleral buckling (SB), pars plana vitrectomy (PPV), or PPV combined with SB, conducted on holidays and on workdays to determine the optimal surgical timing for primary RD treatment in clinical practice. STUDY DESIGN: Retrospective cohort study. METHODS: The cohort included 3178 patients with primary RD registered in the Japan Retinal Detachment Registry between February 2016 and March 2017. Surgery data were divided into holiday and workday groups. A descriptive analysis of primary RD characteristics was performed, and the outcomes for each surgical intervention were assessed. The primary outcome was anatomical failure at 6 months post-surgery classified as follows: level 1, inoperable state; level 2, anatomical recovery with silicone-oil use; and level 3, additional surgery required for RD repair. RESULTS: The holiday group comprised 108 and the workday, 3070 cases of primary RD. Compared with those in the workday group, surgery in the holiday group took longer (PPV, P < 0.0001; SB, P = 0.047) and was performed by less experienced surgeons (P = 0.014). However, there were no statistically significant differences in surgical failure 6 months post-surgery between the workday and holiday groups. CONCLUSION: Although surgery conducted on holidays and workdays was not significantly different in terms of outcome, some surgery should be postponed with proper preoperative interim measures to limit RD progress until it can be conducted on workdays by a well-prepared team..
4. Keijiro Ishikawa, Masato Akiyama, Kenichiro Mori, Takahito Nakama, Shoji Notomi, Shintaro Nakao, Ri-Ichiro Kohno, Atsunobu Takeda, Koh-Hei Sonoda, Drainage Retinotomy Confers Risk of Epiretinal Membrane Formation After Vitrectomy for Rhegmatogenous Retinal Detachment Repair., American journal of ophthalmology, 10.1016/j.ajo.2021.07.028, 234, 20-27, 2022.02, PURPOSE: To describe the factors associated with epiretinal membrane (ERM) formation in eyes treated with pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RRD). DESIGN: Nation-wide, multi-center, clinical cohort study based on registry data. METHODS: Subjects: The 2239 cases treated with PPV for RRD repair registered in the Japan-Retinal Detachment Registry between February 2016 and March 2017. PROCEDURES: Associations of 13 baseline characteristics and eight surgical procedures with ERM formation were evaluated using univariate analysis. We conducted a propensity score-matched analysis for the significantly associated clinical factor(s). MAIN OUTCOME MEASURES: ERM formation after six months of vitrectomy. RESULTS: ERM had developed in 104 cases (4.6%) by six months. We found that a drainage retinotomy was significantly associated with ERM after multiple testing correction (odds ratio [OR] = 2.22, 95% confidence interval [CI] = 1.50-3.31, P < 0.001). In the propensity score-matched analysis (N = 492 in each group), we confirmed a significant difference in the incidence of ERM after six months of vitrectomy (8.3% and 2.6% in cases with and without drainage retinotomy, respectively; OR = 3.35, 95% CI 1.77-6.33; P < 0.001). CONCLUSIONS: Eyes treated with PPV combined with drainage retinotomy are more likely to develop ERM postoperatively..
5. Kenichiro Mori, Keijiro Ishikawa, Yosuke Fukuda, Rui Ji, Iori Wada, Yuki Kubo, Masato Akiyama, Shoji Notomi, Yusuke Murakami, Shintaro Nakao, Satoshi Arakawa, Satomi Shiose, Toshio Hisatomi, Shigeo Yoshida, Ram Kannan, Koh-Hei Sonoda, TNFRSF10A downregulation induces retinal pigment epithelium degeneration during the pathogenesis of age-related macular degeneration and central serous chorioretinopathy., Human molecular genetics, 10.1093/hmg/ddac020, 2022.02, Age-related macular degeneration (AMD) and central serous chorioretinopathy (CSC) are common diseases that can cause vision loss in older and younger populations. These diseases share pathophysiological conditions derived from retinal pigment epithelium (RPE) dysfunction. Tumor necrosis factor receptor superfamily 10A (TNFRSF10A)-LOC389641 with the same lead single-nucleotide polymorphism (SNP) (rs13278062) is the only overlapped susceptibility locus found in both AMD and CSC through genome-wide association studies. This lead SNP has been reported to alter the transcriptional activity of TNFRSF10A. This study aimed to elucidate the function of TNFRSF10A in RPE degeneration using human primary RPE cells and Tnfrsf10 knockout (Tnfrsf10-/-) mice. TNFRSF10A was found to be localized in human RPE. In vitro assays revealed that a T allele of rs13278062, the risk allele for AMD and CSC, downregulated TNFRSF10A transcription in RPE, leading to decreased cell viability and increased apoptosis through protein kinase C-α (PKCA) downregulation. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, rescued the cell viability. Morphological RPE abnormality were found in the retina of Tnfrsf10-/- mice. Our data suggest that downregulation of TNFRSF10A expression inactivates PKCA signaling and causes cellular vulnerability of the RPE, which may contribute to the pathogenesis of AMD and CSC..
6. Iori Wada, Satomi Shiose, Keijiro Ishikawa, Kumiko Kano, Shoji Notomi, Kenichiro Mori, Masato Akiyama, Shintaro Nakao, Koh-Hei Sonoda, One-year efficacy of "rescue photodynamic therapy" for patients with typical age-related macular degeneration, polypoidal choroidal vasculopathy, and pachychoroid neovasculopathy refractory to anti-vascular endothelial growth factor therapy., Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 10.1007/s00417-022-05553-5, 260, 6, 2029-2036, 2022.01, PURPOSE: This study aimed to evaluate the one-year outcomes of photodynamic therapy (PDT) as a rescue treatment for age-related macular degeneration (AMD) refractory to anti-vascular endothelial growth factor (VEGF) therapy. METHODS: Patients with AMD refractory to anti-VEGF therapy, treated with "rescue-PDT" were retrospectively investigated. The time of PDT was defined as the baseline value. Baseline characteristics including sex, age, best-corrected visual acuity (BCVA), central macular thickness (CMT), and foveal choroidal thickness (FCT) were examined. The changes in BCVA, CMT, and recurrence were also assessed at the 1-year follow-up. The logMAR VA change of 0.3 or more was defined as "improved" or "declined." RESULTS: Twenty-three consecutive eyes (typical AMD: 10 eyes, polypoidal choroidal vasculopathy: 10 eyes, and pachychoroid neovasculopathy: 3 eyes), which underwent "rescue-PDT," were analyzed in this study. The BCVA was improved in three patients and maintained in 20 patients at 12 months after PDT (mean BCVA change: 0.11 ± 0.19). The CMT improved in 19 patients (82.6%), and the mean CMT changed from 318.5 ± 93.7 μm to 225.9 ± 51.6 μm (p < 0.01) 12 months after PDT. "Retreatment" of anti-VEGF drug injections was considered if the retinal fluid or retinal hemorrhage recurred after PDT. The baseline FCT of the "retreatment group (15 eyes)" was significantly lower than that of the "no retreatment group (8 eyes)" (206.3 ± 50.7 μm vs 293.9 ± 85.7 μm: p = 0.033). CONCLUSIONS: PDT could be an effective treatment option for anti-VEGF refractory AMD to maintain visual acuity and control retinal fluid for up to 12 months..
7. Shintaro Nakao, Yoshihiro Kaizu, Juun Horie, Iori Wada, Mitsuru Arima, Yosuke Fukuda, Keijiro Ishikawa, Koh-Hei Sonoda, Volumetric Three-Dimensional Optical Coherence Tomography Angiography of Retinal Neovascularization in Proliferative Diabetic Retinopathy., Retinal cases & brief reports, 10.1097/ICB.0000000000001183, 2021.07, PURPOSE: To evaluate structural and angiographic neovascularization in patients with proliferative diabetic retinopathy (PDR) using volumetric three-dimensional optical coherence tomography angiography (OCTA). METHODS: This prospective, observational cross-sectional study included 29 eyes of 27 patients with PDR. The angiogenic structure, feeding vessel (epicenter), flow volume, and flow volume density (FVD) of the neovasculatures were evaluated using three-dimensional OCTA imaging. The flow area and the flow area density (FAD) were also measured using en face OCTA imaging. RESULTS: Sites of neovascularization were imaged successfully in 17 of the 29 eyes (58.6%). Three proposed types of neovascularization were identified on the basis of structural features seen on the three-dimensional OCTA images. Neovascularization of the adhesion type (9 of 17, 52.9%) adhered to the retinal vasculature. Those of the traction type (5 of 17, 29.4%) were partially separated from the retinal vascular plexus. Those of the mushroom type (3 of 17, 17.6%) were connected to the retinal vasculature via several epicenters. There was a significant difference between highly leaky (active) and faintly leaky (inactive) neovascularization for FVD, but not for flow area, flow volume, or FAD (P=0.01, 0.9, 0.6, and 0.1, respectively). CONCLUSIONS: Volumetric three-dimensional OCTA revealed three types of neovascularization in PDR, and may be useful for assessing neovascular activity and planning vitrectomies..
8. Shoji Notomi, Satomi Shiose, Keijiro Ishikawa, Yosuke Fukuda, Kumiko Kano, Kenichiro Mori, Iori Wada, Yoshihiro Kaizu, Hidetaka Matsumoto, Masato Akiyama, Koh-Hei Sonoda, Drusen and pigment abnormality predict the development of neovascular age-related macular degeneration in Japanese patients, PLOS ONE, 10.1371/journal.pone.0255213, 16, 7, e0255213-e0255213, 2021.07, Drusen are known to be the important hallmark to predict the development of age-related macular degeneration (AMD). The prevalence of drusen is lower in Asians compared with Caucasians so that the role of signs constituting early AMD is not well established in Asian populations as in Western countries. In this study, we retrospectively investigated clinical characteristics and 5-year incidence of neovascular AMD (nAMD) in the fellow eye of unilateral nAMD patients. Of 296 consecutive unilateral nAMD patients who had been followed up more than 5 years, 170 typical AMD, 119 polypoidal choroidal vasculopathy, and 7 retinal angiomatous proliferation were included. To examine factors associated with nAMD occurrence in the fellow eye, drusen and pigmentary abnormality in the fellow eye were classified into 4 categories; Category 1: no or small drusen < 63 μm (37.2%), Category 2: 63–125 μm medium drusen or pigmentary abnormality (22.2%), Category 3: large drusen > 125 μm (25.0%), Category P: pachydrusen (15.5%). The mean sub-foveal choroidal thickness (SFCT) was Category 1: 276 μm, Category 2: 308 μm, Category 3: 246 μm, and Category P: 302 μm, respectively. Of note, SFCT in Category 2 and Category P was significantly larger than those of Category 3. Finally, the 5-year incidence of nAMD in the fellow eye was 32/296 (10.8%); Category 1: 0/110 (0%), Category 2: 12/66 (18.2%), Category 3: 20/74 (27.0%), and Category P: 0/46 (0%). Thus, signs of intermediate AMD (large drusen) as well as those of early AMD, especially the pigmentary abnormality, may contribute to development of bilateral nAMD in Japanese patients..
9. Atsunobu Takeda, Eiichi Hasegawa, Shoji Notomi, Keijiro Ishikawa, Mitsuru Arima, Yusuke Murakami, Shintaro Nakao, Toshio Hisatomi, Koh-Hei Sonoda, Surgical Outcomes of Contrast Sensitivity and Visual Acuity in Uveitis-Associated Cataract, Clinical Ophthalmology, 10.2147/opth.s314173, Volume 15, 2665-2673, 2021.06, PURPOSE: To evaluate the pre- and post-operative outcomes of phacoemulsification in patients with uveitis-associated cataract in remission, such as conventional visual acuity (VA), photopic and mesopic contrast visual acuity (CVA), and flares in the anterior chamber objectively assessed as intraocular inflammation. PATIENTS AND METHODS: This prospective study included 26 eyes of 19 patients with uveitis and 45 eyes of 26 controls who underwent cataract surgery at the Kyushu University Hospital and Kyushu Medical Center in Fukuoka, Japan, from October 2016 to December 2018. Conventional VA and flare values in the anterior chamber were evaluated preoperatively and 1 and 3 months postoperatively. Photopic and mesopic CVAs were assessed preoperatively and 3 months postoperatively. RESULTS: The best-corrected VA (BCVA) was improved significantly from baseline to 1 and 3 months postoperatively in both groups (P < 0.01 in both groups). The mean preoperative 100% and 10% CVAs under the photopic condition were significantly lower in the uveitis group than in the control group (P < 0.05 for both CVA), whereas the mean preoperative 100% CVA under the mesopic condition was comparable between the two groups. Although the mean preoperative 100% and 10% CVAs improved significantly from baseline under both photopic and mesopic conditions in both groups (P < 0.01 in both groups), the postoperative contrast sensitivities under both photopic and mesopic conditions remained lower in the uveitis group than in the control group (P < 0.01 for both conditions). The postoperative complications included recurrence of active inflammation in five eyes and cystoid macular edema in one eye and were managed by topical steroid therapy alone. CONCLUSION: Cataract surgery for uveitis-associated cataracts during remission is well tolerated. However, the present results suggest that amelioration of hemeralopia and/or nyctalopia is not as good as expected after cataract surgery in patients with uveitis..
10. Kenichiro Mori, Keijiro Ishikawa, Iori Wada, Yuki Kubo, Yoshiyuki Kobayashi, Takahito Nakama, Masatoshi Haruta, Masato Akiyama, Shintaro Nakao, Shigeo Yoshida, Koh-Hei Sonoda, Changes in metamorphopsia after the treat-and-extend regimen of anti-VEGF therapy for macular edema associated with branch retinal vein occlusion, PLOS ONE, 10.1371/journal.pone.0241343, 15, 10, e0241343-e0241343, 2020.10, This study aims to investigate the changes in metamorphopsia after administering the treat-and-extend regimen of anti-vascular endothelial growth factor therapy for branch retinal vein occlusion-associated macular edema. We retrospectively examined 27 patients (27 eyes) with macula edema due to branch retinal vein occlusion who received intravitreal injections of anti-vascular endothelial growth factor agents using the treat-and-extend regimen for ≥18 months. We evaluated best-corrected visual acuity, central macular thickness, macular edema recurrence, and amount of metamorphopsia quantified by M-CHARTS. The best-corrected visual acuity (logarithm of minimum angle of resolution) and central macular thickness significantly improved at 18 months compared to baseline, the median value (interquartile range [IQR]), 0.30 (0.15-0.52) and 459 (373-542) μm at baseline, and 0 (-0.08-0.16) and 267 (232-306) μm at 18 months. The M-CHARTS score (the mean of vertical and horizontal scores) significantly decreased at 1, 6, and 12 months compared to baseline, but worsened at 18 month, the median value (IQR), 0.45 (0.250-0.925), 0.4 (0.15-0.70), 0.4 (0.150-0.625), 0.4 (0.225-0.550) and 0.45 (0.225-0.750) at baseline, 1 month, 6 months, 12 months and 18 months, respectively. The median cumulative number of macular edema recurrences was 2 (IQR, 0.5-3.0) at 18 months. Simple linear regression and multivariate analyses revealed that the change in the mean M-CHARTS score at 18 months was significantly correlated with the baseline score and the cumulative number of macular edema recurrences. Anti-vascular endothelial growth factor therapy using the treat-and-extend regimen improved metamorphopsia in branch retinal vein occlusion-related macular edema in the short to mid-term follow-up period, but not in the long term. Macular edema recurrence may be associated with persistent metamorphopsia..
11. Atsunobu Takeda, Eiichi Hasegawa, Shintaro Nakao, Keijiro Ishikawa, Yusuke Murakami, Toshio Hisatomi, Mitsuru Arima, Nobuyo Yawata, Yoshinao Oda, Kazuhiro Kimura, Hiroshi Yoshikawa, Koh-Hei Sonoda, Vitreous levels of interleukin-35 as a prognostic factor in B-cell vitreoretinal lymphoma., Scientific reports, 10.1038/s41598-020-72962-z, 10, 1, 15715-15715, 2020.09, Vitreoretinal lymphoma (VRL) is a rare disease of B-cell origin with poor prognosis. Regulatory cytokines promote tumor development by suppressing antitumor immunity in several cancer types, including B-cell malignancies. To identify the regulatory cytokines associated with poor prognosis in patients with B-cell VRL, we determined the regulatory cytokines profiles in the vitreous humor of patients with VRL. This retrospective study included 22 patients with VRL, 24 with non-infectious uveitis (NIU), and 20 with idiopathic epiretinal membrane (control). Vitreous concentrations of regulatory cytokines were assessed using a cytometric beads assay and association with clinical data was examined. IL-35 and soluble IL-2 receptor α levels were significantly higher in patients with VRL and NIU than those in the control group. The 5-year overall survival (OS) rates for the group with high intravitreal IL-35 was significantly poorer than those for the group with low intravitreal IL-35, who were diagnosed with VRL at the onset (P = 0.024, log-rank test). The 5-year OS rates with intravitreal IL-35 levels above and below the median were 40.0% and 83.3%, respectively. Our results suggest that high intravitreal IL-35 levels indicate poor prognosis for patients diagnosed with B-cell VRL at the onset..
12. Yuki Kubo, Keijiro Ishikawa, Kenichiro Mori, Yoshiyuki Kobayashi, Takahito Nakama, Mitsuru Arima, Shintaro Nakao, Toshio Hisatomi, Masatoshi Haruta, Koh-Hei Sonoda, Shigeo Yoshida, Periostin and tenascin-C interaction promotes angiogenesis in ischemic proliferative retinopathy., Scientific reports, 10.1038/s41598-020-66278-1, 10, 1, 9299-9299, 2020.06, Ischemic proliferative retinopathy (IPR), such as proliferative diabetic retinopathy (PDR), retinal vein occlusion and retinopathy of prematurity is a major cause of vision loss. Our previous studies demonstrated that periostin (PN) and tenascin-C (TNC) are involved in the pathogenesis of IPR. However, the interactive role of PN and TNC in angiogenesis associated with IPR remain unknown. We found significant correlation between concentrations of PN and TNC in PDR vitreous humor. mRNA and protein expression of PN and TNC were found in pre-retinal fibrovascular membranes excised from PDR patients. Interleukin-13 (IL-13) promoted mRNA and protein expression of PN and TNC, and co-immunoprecipitation assay revealed binding between PN and TNC in human microvascular endothelial cells (HRECs). IL-13 promoted angiogenic functions of HRECs. Single inhibition of PN or TNC and their dual inhibition by siRNA suppressed the up-regulated angiogenic functions. Pathological pre-retinal neovessels of oxygen-induced retinopathy (OIR) mice were attenuated in PN knock-out, TNC knock-out and dual knock-out mice compared to wild-type mice. Both in vitro and in vivo, PN inhibition had a stronger inhibitory effect on angiogenesis compared to TNC inhibition, and had a similar effect to dual inhibition of PN and TNC. Furthermore, PN knock-out mice showed scant TNC expression in pre-retinal neovessels of OIR retinas. Our findings suggest that interaction of PN and TNC facilitates pre-retinal angiogenesis, and PN is an effective therapeutic target for IPR such as PDR..
13. Keijiro Ishikawa, Takuma Fukui, Shintaro Nakao, Satomi Shiose, Koh-Hei Sonoda, Vitrectomy with peripapillary internal limiting membrane peeling for macular retinoschisis associated with normal-tension glaucoma., American journal of ophthalmology case reports, 10.1016/j.ajoc.2020.100663, 18, 100663-100663, 2020.06, Purpose: Although vitrectomy has been reported to be effective for the treatment of macular retinoschisis associated with glaucoma in a few case series, the surgical techniques have yet to be established. This article aimed to describe the cases of two patients with macular retinoschisis who underwent vitrectomy with peripapillary internal limiting membrane peeling around the defective area of the retinal nerve fiber layer. Observations: Both patients had been diagnosed with normal tension glaucoma and treated with eye drops to stabilize intraocular pressure. Progression of macular retinoschisis and accompanied vision loss were observed in both cases. Twelve months after the surgery, both patients had resolution of the retinoschisis and improvement in best corrected visual acuity. Conclusions and importance: Our surgical technique may be effective for the resolution of macular retinoschisis in eyes with normal tension glaucoma..
14. Kenichiro Mori, Shigeo Yoshida, Yoshiyuki Kobayashi, Keijiro Ishikawa, Shintaro Nakao, Toshio Hisatomi, Masatoshi Haruta, Tatsuro Isihibashi, Koh-Hei Sonoda, Decrease in the number of microaneurysms in diabetic macular edema after anti-vascular endothelial growth factor therapy: implications for indocyanine green angiography-guided detection of refractory microaneurysms., Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 10.1007/s00417-020-04608-9, 258, 4, 735-741, 2020.04, PURPOSE: We evaluated changes in the numbers of microaneurysms (MAs) on fluorescein angiography (FA) and indocyanine green angiography (IA) in eyes with diabetic macular edema (DME) following intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents. METHODS: Twenty-one eyes of 16 patients with DME were included in this retrospective study. All patients received an initial loading dose of three monthly injections of anti-VEGF agents; thereafter, they received a pro re nata regimen for at least 12 months of follow-up. FA and IA images were obtained before and at 6 months after the initial injection. RESULTS: The median numbers of MAs significantly decreased from six (interquartile range [IQR] 3-7) MAs in early-phase FA, three (IQR 3-5) leaky MAs in late-phase FA, and two (IQR 1-4) MAs in late-phase IA at baseline to two (IQR 1-3) MAs in early-phase FA, one (IQR 0-2) leaky MA in late-phase FA, and one (IQR 0-2) MA in late-phase IA at 6 months (P < 0.0001 for all). Only the median numbers of MAs in late-phase IA at baseline and at 6 months were significantly higher in the recurrent DME group (13 eyes) than in the non-recurrent DME group (five eyes) (three [IQR 2-4] vs one [IQR 1-2], one [IQR 0.5-2] vs zero [P = 0.0185 and P = 0.009]). CONCLUSION: Intravitreal injection of anti-VEGF agents reduced the numbers of MAs in patients with DME. The numbers of MAs detected by late-phase IA might be useful predictors of DME recurrence..
15. Keijiro Ishikawa, Ri-Ichiro Kohno, Kenichiro Mori, Yusuke Murakami, Shintaro Nakao, Masato Akiyama, Shigeo Yoshida, Koh-Hei Sonoda, Increased expression of periostin and tenascin-C in eyes with neovascular glaucoma secondary to PDR., Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 10.1007/s00417-019-04574-x, 258, 3, 621-628, 2020.03, PURPOSE: To investigate periostin (PN) and tenascin-C (TNC) expression in the aqueous humor and trabeculectomy specimens of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR). METHODS: This study enrolled 37 eyes of 37 patients who were grouped into (1) NVG secondary to PDR (NVG; n = 8); (2) PDR without NVG (PDR; n = 9); (3) primary open-angle glaucoma (POAG; n = 11); and (4) cataract surgery patients as a control group (CG; n = 9). Aqueous humor samples were collected from the anterior chamber at the start of surgery or intravitreal injection of anti-VEGF drug. The concentrations of PN, TNC, VEGF, and TGF-β2 (transforming growth factor-beta 2) were measured by ELISA. Sclerostomy tissues containing trabecular meshwork were obtained from two NVG patients and a POAG patient who underwent trabeculectomy surgery. Immunohistochemical analyses were performed to determine the localization of PN and TNC expression in the sclerostomy tissues. RESULTS: PN and TNC-C levels were below detection threshold in the POAG and CG groups. The NVG group had significantly higher levels of PN and TNC compared with the PDR group (84.7 ng/ml vs 2.2 ng/ml and 18.5 ng/ml vs 4.6 ng/ml, respectively; p < 0.05). There was a significant correlation between the levels of PN and TNC-C in the NVG group (r = 0.86, p < 0.05). We found significant expression of PN in the trabecular meshwork and Schlemm's canal of sclerostomy tissues excised from patients with NVG. CONCLUSIONS: Increased PN and TNC expression suggests their possible involvement in the pathogenesis of NVG secondary to PDR..
16. Yoshihiro Kaizu, Shintaro Nakao, Iori Wada, Mitsuru Arima, Muneo Yamaguchi, Keijiro Ishikawa, Masato Akiyama, Junji Kishimoto, Toshio Hisatomi, Koh-Hei Sonoda, Microaneurysm Imaging Using Multiple En Face OCT Angiography Image Averaging: Morphology and Visualization., Ophthalmology. Retina, 10.1016/j.oret.2019.09.010, 4, 2, 175-186, 2020.02, PURPOSE: In diabetic retinopathy (DR), OCT angiography (OCTA) could not image all fluorescein angiography (FA)-detected microaneurysms. We investigated whether multiple image averaging could enhance the microaneurysm detection capability of OCTA in patients with DR. DESIGN: Prospective and cross-sectional observational study. PARTICIPANTS: Consecutive 31 patients (n = 62 eyes) with DR. METHODS: All eyes underwent FA and 3 × 3 mm fovea-centered OCTA images were obtained using 2 devices: RTVue XR Avanti (Optovue Inc, Fremont, CA) and OCT HS-100 (Canon Inc, Toyko, Japan). OCTA imaging (HS-100) was performed 10 consecutive times. Microaneurysm detection capability was compared among 5 OCTA images (single image, ×3, ×5, and ×10 averaged images and single scan image with the RTVue XR Avanti device). MAIN OUTCOME MEASURES: Microaneurysm detection capability and the correlation between microaneurysm clinical characteristics or morphology and the extent of image averaging required for OCTA detection. RESULTS: A total of 415 microaneurysms could be analyzed in 31 eyes from 25 patients. Microaneurysms detected on single image, ×3, ×5, and ×10 averaged OCTA images were 144 (34.7%), 227 (54.7%), 285 (68.7%), and 306 (73.7%), respectively. Microaneurysm detection capability was significantly increased with increased image averaging. Microaneurysm detection with OCTA was not correlated with retinal thickness, FA leakiness, and indocyanine green angiogram detection or the number of averaged images, whereas there was significant correlation between microaneurysm morphology and microaneurysm visibility by the image-averaging process for 4 morphologies, particular the focal bulge types (P < 0.01). CONCLUSIONS: In DR, multiple image averaging is useful for increasing the microaneurysm detection capability of OCTA, especially for focal bulge-type microaneurysms..
17. Yusuke Murakami, Keijiro Ishikawa, Shintaro Nakao, Koh-Hei Sonoda, Innate immune response in retinal homeostasis and inflammatory disorders., Progress in retinal and eye research, 10.1016/j.preteyeres.2019.100778, 74, 100778-100778, 2020.01, Innate immune cells such as neutrophils, monocyte-macrophages and microglial cells are pivotal for the health and disease of the retina. For the maintenance of retinal homeostasis, these cells and immunosuppressive molecules in the eye actively regulate the induction and the expression of inflammation in order to prevent excessive activation and subsequent tissue damage. In the disease context, these regulatory mechanisms are modulated genetically and/or by environmental stimuli such as damage-associated molecular patterns (DAMPs), and a chronic innate immune response regulates or contributes to the formation of diverse retinal disorders such as uveitis, retinitis pigmentosa, retinal vascular diseases and retinal fibrosis. Here we summarize the recent knowledge regarding the innate immune response in both ocular immune regulation and inflammatory retinal diseases, and we describe the potential of the innate immune response as a biomarker and therapeutic target..
18. Yoshihiro Kaizu, Shintaro Nakao, Mitsuru Arima, Iori Wada, Muneo Yamaguchi, Haruka Sekiryu, Takehito Hayami, Keijiro Ishikawa, Yasuhiro Ikeda, Koh-Hei Sonoda, Capillary dropout is dominant in deep capillary plexus in early diabetic retinopathy in optical coherence tomography angiography., Acta ophthalmologica, 10.1111/aos.14041, 97, 5, e811-e812, 2019.08.
19. Yoshihiro Kaizu, Shintaro Nakao, Mitsuru Arima, Takehito Hayami, Iori Wada, Muneo Yamaguchi, Haruka Sekiryu, Keijiro Ishikawa, Yasuhiro Ikeda, Koh-Hei Sonoda, Flow Density in Optical Coherence Tomography Angiography is Useful for Retinopathy Diagnosis in Diabetic Patients., Scientific reports, 10.1038/s41598-019-45194-z, 9, 1, 8668-8668, 2019.06, Our study evaluated the diagnostic capability of flow density (FD) in OCT angiography (OCTA) for diabetic retinopathy (DR) detection in diabetic patients. We studied 93 eyes of 68 diabetic patients who underwent OCTA (36 and 57 eyes without and with DR, respectively). Retinal capillary FD of a 2.6 × 2.6 mm2 area and four divided areas at the superficial (SCP) and deep capillary plexus (DCP) were measured. Predictions were evaluated using the area under the receiver operating characteristic curve (AUC). The diagnostic capabilities of the FDs in discriminating between eyes without DR and eyes with total or early DR were compared. Furthermore, predictions with foveal avascular zone (FAZ) area, hemoglobin A1c (HbA1c), and DM duration were also compared with FD. Prediction using FD AUC in the temporal side in the DCP (0.83) was the highest and significantly better than all other AUCs examined (P < 0.05), including discriminating between eyes without DR and with early DR (P < 0.01). Prediction using this particular AUC was also significantly better than that by FAZ area and HbA1c (P < 0.001 and <0.001, respectively). Area-divided FD in OCTA may be valuable for diagnosing retinopathy in diabetic patients..
20. Keijiro Ishikawa, Ri-ichiro Kohno, Eiichi Hasegawa, Shintaro Nakao, Shigeo Yoshida, Koh-Hei Sonoda, Preoperative estimation of distance between retinal break and limbus with wide-field fundus imaging: Potential clinical utility for conventional scleral buckling, PLOS ONE, 10.1371/journal.pone.0212284, 14, 2, e0212284-e0212284, 2019.02, OBJECTIVE: Accurate scleral marking of retinal breaks is essential for successful scleral buckling. This study aimed to investigate the use of wide-field fundus images obtained with an Optos for preoperative estimation of the distance from the limbus to the retinal breaks. METHODS AND ANALYSIS: This is a retrospective review of 29 eyes from 26 patients with rhegmatogenous retinal detachment who received scleral buckling with anatomically successful repair. They underwent wide-field fundus photography with Optos California. In the pre- and postoperative fundus images, we measured distances from the macula to the retinal tears (TM), to the center of the vortex veins (VM), to the optic disc (DM), and to the posterior edge of the scleral buckle (BM). RESULTS: (BM-VM) / DM was significantly correlated with the distance from the limbus to the posterior edge of the scleral buckle that had been determined intraoperatively. (r = 0.705; p<0.001) We applied a regression line derived from this correlation with the value of (TM -VM) / DM in order to calculate estimated distances between retinal breaks and the limbus. The calculated distances were all within the range of distances from the limbus to the anterior and posterior edges of the scleral buckles. CONCLUSION: Preoperative analysis of Optos images may be useful for estimating the distance from the limbus to retinal breaks, which might aid scleral marking during scleral buckling surgery..
21. Fumiyuki Sasaki, Tomoaki Koga, Mai Ohba, Kazuko Saeki, Toshiaki Okuno, Keijiro Ishikawa, Takahito Nakama, Shintaro Nakao, Shigeo Yoshida, Tatsuro Ishibashi, Hamid Ahmadieh, Mozhgan Rezaei Kanavi, Ali Hafezi-Moghadam, Josef M Penninger, Koh-Hei Sonoda, Takehiko Yokomizo, Leukotriene B4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models., JCI insight, 10.1172/jci.insight.96902, 3, 18, 2018.09, Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD..
22. Shigeo Yoshida, Takahito Nakama, Keijiro Ishikawa, Shintaro Nakao, Koh-Hei Sonoda, Tatsuro Ishibashi, Periostin in vitreoretinal diseases., Cellular and molecular life sciences : CMLS, 10.1007/s00018-017-2651-5, 74, 23, 4329-4337, 2017.12, Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-β2 (TGF-β2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases..
23. María Berdasco, Antonio Gómez, Marcos J Rubio, Jaume Català-Mora, Vicente Zanón-Moreno, Miguel Lopez, Cristina Hernández, Shigeo Yoshida, Takahito Nakama, Keijiro Ishikawa, Tatsuro Ishibashi, Amina M Boubekeur, Lotfi Louhibi, Miguel A Pujana, Sergi Sayols, Fernando Setien, Dolores Corella, Carmen de Torres, Andreu Parareda, Jaume Mora, Ling Zhao, Kang Zhang, Matilde E Lleonart, Javier Alonso, Rafael Simó, Josep M Caminal, Manel Esteller, DNA Methylomes Reveal Biological Networks Involved in Human Eye Development, Functions and Associated Disorders., Scientific reports, 10.1038/s41598-017-12084-1, 7, 1, 11762-11762, 2017.09, This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients..
24. Yedi Zhou, Shigeo Yoshida, Yuki Kubo, Takeru Yoshimura, Yoshiyuki Kobayashi, Takahito Nakama, Muneo Yamaguchi, Keijiro Ishikawa, Yuji Oshima, Tatsuro Ishibashi, Different distributions of M1 and M2 macrophages in a mouse model of laser-induced choroidal neovascularization., Molecular medicine reports, 10.3892/mmr.2017.6491, 15, 6, 3949-3956, 2017.06, Choroidal neovascularization (CNV) is a serious complication of age‑related macular degeneration. The aim of the present study was to investigate the expression and distribution of M1 and M2 macrophages in a laser‑induced CNV adult mouse model. The mRNA expression levels of M1, M2 and pan macrophage markers, and macrophage‑associated angiogenic cytokines, were determined by reverse transcription‑quantitative polymerase chain reaction. Immunofluorescence studies were performed to determine the location of the macrophages. The expression levels of M1 macrophage markers increased to a greater extent compared with M2 markers in the retinal pigment epithelium (RPE)‑choroid complexes following laser photocoagulation. By contrast, the expression levels of M2 macrophage markers increased primarily in the retinas. Immunofluorescence studies revealed that the increased number of cluster of differentiation (CD)206‑positive cells were located primarily in the retina, whereas the CD80‑positive cells were located around the site of CNVs in the RPE‑choroid. In addition, the M1‑associated cytokines increased to a greater extent in the RPE‑choroid complexes, whereas the M2‑associated cytokines were highly expressed in the retinas. These findings indicate that M1 and M2 macrophage numbers increased following CNV; however, the locations were different in this mouse model of laser‑induced CNV. The results of the present study suggest that M1 macrophages have a more direct role in inhibiting the development of CNV..
25. Takahito Nakama, Shigeo Yoshida, Keijiro Ishikawa, Yuki Kubo, Yoshiyuki Kobayashi, Yedi Zhou, Shintaro Nakao, Toshio Hisatomi, Yasuhiro Ikeda, Kazumasa Takao, Kazunori Yoshikawa, Akira Matsuda, Junya Ono, Shoichiro Ohta, Kenji Izuhara, Akira Kudo, Koh-Hei Sonoda, Tatsuro Ishibashi, Therapeutic Effect of Novel Single-Stranded RNAi Agent Targeting Periostin in Eyes with Retinal Neovascularization., Molecular therapy. Nucleic acids, 10.1016/j.omtn.2017.01.004, 6, 279-289, 2017.03, Retinal neovascularization (NV) due to retinal ischemia remains one of the principal causes of vision impairment in patients with ischemic retinal diseases. We recently reported that periostin (POSTN) may play a role in the development of preretinal fibrovascular membranes, but its role in retinal NV has not been determined. The purpose of this study was to examine the expression of POSTN in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of POSTN on retinal NV using Postn KO mice and human retinal endothelial cells (HRECs) in culture. In addition, we used a novel RNAi agent, NK0144, which targets POSTN to determine its effect on the development of retinal NV. Our results showed that the expression of POSTN was increased in the vascular endothelial cells, pericytes, and M2 macrophages in ischemic retinas. POSTN promoted the ischemia-induced retinal NV by Akt phosphorylation through integrin αvβ3. NK0144 had a greater inhibitory effect than canonical double-stranded siRNA on preretinal pathological NV in vivo and in vitro. These findings suggest a causal relationship between POSTN and retinal NV, and indicate a potential therapeutic role of intravitreal injection of NK0144 for retinal neovascular diseases..
26. Takahito Nakama, Shigeo Yoshida, Keijiro Ishikawa, Yoshiyuki Kobayashi, Takaya Abe, Hiroshi Kiyonari, Go Shioi, Naruto Katsuragi, Tatsuro Ishibashi, Ryuichi Morishita, Yoshiaki Taniyama, Different roles played by periostin splice variants in retinal neovascularization., Experimental eye research, 10.1016/j.exer.2016.10.012, 153, 133-140, 2016.12, Retinal neovascularization (NV) due to retinal ischemia is one of the major causes of vision reduction in patients with different types of retinal diseases although anti-vascular endothelial growth factor (anti-VEGF) therapy can partially reduce the size of the retinal NV. We recently reported that periostin plays an important role in the development of NV and the formation of preretinal fibrovascular membranes, but the role of the splice variants of periostin on retinal NV has not been determined. We examined the expressions of periostin splice variants in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of periostin splice variants on retinal NV using periostin knock out mice, and the effects of anti-periostin antibodies on retinal NV. Our results showed that the expressions of the periostin splice variants were increased in ischemic retinas. The degree of increase of periostin lacking exon 17 was the highest among the periostin splice variants examined. Both genetic ablation of periostin exons 17 and 21 and antibodies for periostin exons 17 and 21 affected preretinal pathological NV. Inhibition of exon 17 of periostin had the greatest effect in reducing preretinal pathological NV. These findings suggest a causal link between periostin splice variants and retinal NV, and an intravitreal injection of antibody for exon 17 and exon 21 of periostin should be considered to inhibit preretinal pathological NV..
27. Yoshiyuki Kobayashi, Shigeo Yoshida, Yedi Zhou, Takahito Nakama, Keijiro Ishikawa, Yuki Kubo, Mitsuru Arima, Shintaro Nakao, Toshio Hisatomi, Yasuhiro Ikeda, Akira Matsuda, Koh-Hei Sonoda, Tatsuro Ishibashi, Tenascin-C secreted by transdifferentiated retinal pigment epithelial cells promotes choroidal neovascularization via integrin αV., Laboratory investigation; a journal of technical methods and pathology, 10.1038/labinvest.2016.99, 96, 11, 1178-1188, 2016.11, Tenascin-C is expressed in choroidal neovascular (CNV) membranes in eyes with age-related macular degeneration (AMD). However, its role in the pathogenesis of CNV remains to be elucidated. Here we investigated the role of tenascin-C in CNV formation. In immunofluorescence analyses, tenascin-C co-stained with α-SMA, pan-cytokeratin, CD31, CD34, and integrin αV in the CNV membranes of patients with AMD and a mouse model of laser-induced CNV. A marked increase in the expression of tenascin-C mRNA and protein was observed 3 days after laser photocoagulation in the mouse CNV model. Tenascin-C was also shown to promote proliferation and inhibit adhesion of human retinal pigment epithelial (hRPE) cells in vitro. Moreover, tenascin-C promoted proliferation, adhesion, migration, and tube formation in human microvascular endothelial cells (HMVECs); these functions were, however, blocked by cilengitide, an integrin αV inhibitor. Exposure to TGF-β2 increased tenascin-C expression in hRPE cells. Conditioned media harvested from TGF-β2-treated hRPE cell cultures enhanced HMVEC proliferation and tube formation, which were inhibited by pretreatment with tenascin-C siRNA. The CNV volume was significantly reduced in tenascin-C knockout mice and tenascin-C siRNA-injected mice. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of CNV via integrin αV in a paracrine manner. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of CNV development associated with AMD..
28. Yedi Zhou, Shigeo Yoshida, Yuki Kubo, Yoshiyuki Kobayashi, Takahito Nakama, Muneo Yamaguchi, Keijiro Ishikawa, Shintaro Nakao, Yasuhiro Ikeda, Tatsuro Ishibashi, Koh-Hei Sonoda, Interleukin-12 inhibits pathological neovascularization in mouse model of oxygen-induced retinopathy., Scientific reports, 10.1038/srep28140, 6, 28140-28140, 2016.06, Hypoxia-induced retinal neovascularization is a major pathological condition in many vision-threatening diseases. In the present study, we determined whether interleukin (IL)-12, a cytokine that regulates angiogenesis, plays a role in the neovascularization in a mouse model of oxygen-induced retinopathy (OIR). We found that the expressions of the mRNAs of both IL-12p35 and IL-12p40 were significantly reduced in the OIR retinas compared to that of the room air-raised control. The sizes of the avascular areas and neovascular tufts were larger in IL-12p40 knock-out (KO) mice than that in wild type (WT) mice. In addition, an intravitreal injection of recombinant IL-12 reduced both avascular areas and neovascular tufts. IL-12 injection enhanced the expressions of interferon-gamma (IFN-γ) and other downstream chemokines. In an in vitro system, IL-12 had no significant effect on tube formation of human retinal microvascular endothelial cells (HRECs). Moreover, a blockade of IFN-γ suppressed the inhibitory effect of IL-12 on pathological neovascularization. These results suggest that IL-12 plays important roles in inhibiting pathological retinal neovascularization..
29. Keijiro Ishikawa, Parameswaran G Sreekumar, Christine Spee, Hossein Nazari, Danhong Zhu, Ram Kannan, David R Hinton, αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition., The American journal of pathology, 10.1016/j.ajpath.2015.11.014, 186, 4, 859-73, 2016.04, Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro, overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-β2-induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration..
30. Yoshiyuki Kobayashi, Shigeo Yoshida, Yedi Zhou, Takahito Nakama, Keijiro Ishikawa, Mitsuru Arima, Shintaro Nakao, Yukio Sassa, Atsunobu Takeda, Toshio Hisatomi, Yasuhiro Ikeda, Akira Matsuda, Koh-Hei Sonoda, Tatsuro Ishibashi, Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy., Molecular vision, 22, 436-45, 2016.04, PURPOSE: We previously demonstrated that tenascin-C was highly expressed in the fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy (PDR). However, its role in the pathogenesis of FVMs has not been determined. The purpose of this study was to investigate what role tenascin-C plays in the formation and angiogenesis of FVMs. METHODS: The level of tenascin-C was determined by sandwich enzyme-linked immunosorbent assay in the vitreous samples collected from patients with PDR and with a macular hole as control. The locations of tenascin-C, α- smooth muscle actin (SMA), CD34, glial fibrillary acidic protein (GFAP), and integrin αV in the FVMs from PDR patients were determined by immunohistochemistry. We also measured the in vitro expression of the mRNA and protein of tenascin-C in vascular smooth muscle cells (VSMCs) stimulated by interleukin (IL)-13. The effects of tenascin-C on cell proliferation, migration, and tube formation were determined in human retinal endothelial cells (HRECs) in culture. RESULTS: The mean vitreous levels of tenascin-C were significantly higher in patients with PDR than in patients with a macular hole (p<0.001). Double immunofluorescence analyses of FVMs from PDR patients showed that tenascin-C co-stained FVMs with α-SMA, CD34, and integrin αV but not with GFAP. In addition, IL-13 treatment increased both the expression and secretion of tenascin-C by VSMCs in a dose-dependent manner. Tenascin-C exposure promoted proliferation, migration, and tube formation in HRECs. Tenascin-C neutralizing antibody significantly blocked the tube formation by HRECs exposed to VSMC-IL-13-conditioned medium. CONCLUSIONS: Our findings suggest that tenascin-C is secreted from VSMCs and promotes angiogenesis in the FVMs associated with PDR..
31. Parameswaran G Sreekumar, Keijiro Ishikawa, Chris Spee, Hemal H Mehta, Junxiang Wan, Kelvin Yen, Pinchas Cohen, Ram Kannan, David R Hinton, The Mitochondrial-Derived Peptide Humanin Protects RPE Cells From Oxidative Stress, Senescence, and Mitochondrial Dysfunction., Investigative ophthalmology & visual science, 10.1167/iovs.15-17053, 57, 3, 1238-53, 2016.03, PURPOSE: To investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress-induced cell death, mitochondrial bioenergetics, and senescence. METHODS: Humanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5-10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress-induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment. RESULTS: A prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress-induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death. CONCLUSIONS: Humanin protected RPE cells against oxidative stress-induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD..
32. Takashi Tachibana, Shigeo Yoshida, Yuki Kubo, Yoshiyuki Koayashi, Takahito Nakama, Keijiro Ishikawa, Shintaro Nakao, Kenji Izuhara, Toshihiro Kono, Tatsuro Ishibashi, Reduced vitreal concentration of periostin after vitrectomy in patients with proliferative diabetic retinopathy., Acta ophthalmologica, 10.1111/aos.12752, 94, 1, e81-2, 2016.02.
33. Keijiro Ishikawa, Ram Kannan, David R Hinton, Molecular mechanisms of subretinal fibrosis in age-related macular degeneration., Experimental eye research, 10.1016/j.exer.2015.03.009, 142, 19-25, 2016.01, Subretinal fibrosis is a result of a wound healing response that follows choroidal neovascularization in neovascular age-related macular degeneration (nAMD). Although anti-vascular endothelial growth factor therapy has become a standard treatment that improves visual acuity in many nAMD patients, unsuccessful treatment outcomes have often been attributed to the progression of subretinal fibrosis. In this review, we summarize the cellular and extracellular components of subretinal fibrous membranes and also discuss the possible molecular mechanisms including the functional involvement of growth factors and the inflammatory response in the process. Moreover, we present an murine animal model of subretinal fibrosis that might facilitate greater understanding of the pathophysiology and the development of novel therapeutic strategies for the inhibition of subretinal fibrosis in nAMD..
34. Takashi Tachibana, Shigeo Yoshida, Yoshiyuki Kobayashi, Takahito Nakama, Keijiro Ishikawa, Akihito Sengoku, Shintaro Nakao, Yuji Oshima, Tatsuro Ishibashi, Differential improvement of vertical and horizontal metamorphopsia scores after epiretinal membrane vitrectomy with ILM peeling., Acta ophthalmologica, 10.1111/aos.12734, 93, 8, e681-2, 2015.12.
35. Huiming Zhang, Shikun He, Christine Spee, Keijiro Ishikawa, David R Hinton, SIRT1 mediated inhibition of VEGF/VEGFR2 signaling by Resveratrol and its relevance to choroidal neovascularization., Cytokine, 10.1016/j.cyto.2015.06.019, 76, 2, 549-552, 2015.12, SIRT1, a NAD(+) -dependent histone deacetylase, has been shown to act as a key regulator of angiogenesis. The purpose of this study was to determine the effects of resveratrol (RSV, a SIRT1 activator) on the vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway and to establish its relevance to choroidal neovascularization (CNV), a blinding complication of age-related macular degeneration. Western blot and ELISA assay showed that RSV inhibited hypoxia-inducible factor (HIF)-1α accumulation and VEGF secretion induced by cobalt chloride (CoCl2) through SIRT1 in human retinal pigment epithelial (hRPE) cells. Furthermore, RSV down-regulated VEGFR2 phosphorylation and activation induced by VEGF in endothelial cells via SIRT1. Thus, the inhibitory effect of RSV on the HIF-1α/VEGF/VEGFR2 signaling axis is mediated, at least in part, through SIRT1. The results suggest that targeting SIRT1 could have therapeutic potential for the treatment of CNV..
36. Keijiro Ishikawa, Shikun He, Hiroto Terasaki, Hossein Nazari, Huiming Zhang, Christine Spee, Ram Kannan, David R Hinton, Resveratrol inhibits epithelial-mesenchymal transition of retinal pigment epithelium and development of proliferative vitreoretinopathy., Scientific reports, 10.1038/srep16386, 5, 16386-16386, 2015.11, Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and ocular trauma, and its recurrence may lead to irreversible vision loss. Epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of PVR, which is characterized by fibrotic membrane formation and traction retinal detachment. In this study, we investigated the potential impact of resveratrol (RESV) on EMT and the fibrotic process in cultured RPE cells and further examined the preventive effect of RESV on PVR development using a rabbit model of PVR. We found that RESV induces mesenchymal to epithelial transition (MET) and inhibits transforming growth factor-β2(TGF-β2)-induced EMT of RPE cells by deacetylating SMAD4. The effect of RESV on MET was dependent on sirtuin1 activation. RESV suppressed proliferation, migration and fibronectin synthesis induced by platelet-derived growth factor-BB or TGF-β2. In vivo, RESV inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that RESV reduced fibrotic membrane formation and decreased α-SMA expression in the epiretinal membranes. These results suggest the potential use of RESV as a therapeutic agent to prevent the development of PVR by targeting EMT of RPE..
37. Yedi Zhou, Shigeo Yoshida, Shintaro Nakao, Takeru Yoshimura, Yoshiyuki Kobayashi, Takahito Nakama, Yuki Kubo, Kohta Miyawaki, Muneo Yamaguchi, Keijiro Ishikawa, Yuji Oshima, Koichi Akashi, Tatsuro Ishibashi, Erratum. M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.14-16012a, 56, 11, 6990-6990, 2015.10.
38. Shikun He, Ernesto Barron, Keijiro Ishikawa, Hossein Nazari Khanamiri, Chris Spee, Peng Zhou, Satoru Kase, Zhuoshi Wang, Laurie Diane Dustin, David R Hinton, Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.14-16258, 56, 9, 5579-89, 2015.08, PURPOSE: The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-β-induced retinal pigment epithelial (RPE) cell transdifferentiation. METHODS: Expression of MeCP2 and its colocalization with cytokeratin and α-smooth muscle actin (α-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2'-deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylation-specific PCR. Effects of 5-AZA-dC on expression of α-SMA, fibronectin (FN), and TGF-β receptor 2 (TGF-β R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of α-SMA and FN induced by TGFβ was determined. RESULTS: MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and α-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of α-SMA, FN, TGF-β R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-β induced expression of α-SMA, and FN was suppressed by knock-down of MeCP2. CONCLUSIONS: MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR..
39. Shigeo Yoshida, Yuki Kubo, Yoshiyuki Kobayashi, Yedi Zhou, Takahito Nakama, Muneo Yamaguchi, Takashi Tachibana, Keijiro Ishikawa, Ryoichi Arita, Shintaro Nakao, Yukio Sassa, Yuji Oshima, Toshihiro Kono, Tatsuro Ishibashi, Increased vitreous concentrations of MCP-1 and IL-6 after vitrectomy in patients with proliferative diabetic retinopathy: possible association with postoperative macular oedema., The British journal of ophthalmology, 10.1136/bjophthalmol-2014-306366, 99, 7, 960-6, 2015.07, PURPOSE: To determine whether vitreal concentrations of MCP-1, IL-6 and IL-8 are altered after vitrectomy in patients with proliferative diabetic retinopathy (PDR) and to investigate whether the altered levels of these cytokines are associated with postoperative macular oedema. METHODS: Vitreous samples were collected from 36 eyes of 33 patients with PDR before pars plana vitrectomy without intraocular lens (IOL) implantation, and also from the same 36 eyes during IOL implantation surgery approximately 7 months after the initial vitrectomy. Levels of MCP-1, IL-6, IL-8 and vascular endothelial growth factor were measured by flow cytometry using cytometric bead array (CBA) technology. RESULTS: The mean vitreous levels of MCP-1, IL-6 and IL-8 in the samples collected before vitrectomy were significantly higher in patients with PDR than in control patients (p<0.0001). The levels of MCP-1 and IL-6 in the samples collected at the time of IOL implantation were significantly higher than those collected before vitrectomy (p<0.05). In contrast, the level of IL-8 was significantly lower after vitrectomy (p<0.05). The levels of IL-6 and IL-8, but not MCP-1, in the vitreous from eyes with PDR were inversely correlated with the interval between the initial vitrectomy and the time of implantation surgery. Among the vitrectomised patients, the mean vitreous level of MCP-1 in eyes with diabetic macular oedema (DME) was significantly higher than in those without DME (p=0.028). CONCLUSIONS: The elevated levels of MCP-1 and IL-6 may indicate prolonged inflammation even after successful vitrectomy, which can cause postoperative DME..
40. Yedi Zhou, Shigeo Yoshida, Shintaro Nakao, Takeru Yoshimura, Yoshiyuki Kobayashi, Takahito Nakama, Yuki Kubo, Kohta Miyawaki, Muneo Yamaguchi, Keijiro Ishikawa, Yuji Oshima, Koichi Akashi, Tatsuro Ishibashi, M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.14-16012, 56, 8, 4767-77, 2015.07, PURPOSE: To investigate the roles played by M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR). METHODS: Oxygen-induced retinopathy was induced in C57BL/6J mice by exposing postnatal day seven (P7) pups to 75% oxygen and then returning them to room air at P12. Real-time RT-PCR and immunofluorescence staining were used to assess the levels and distributions of different macrophage markers. Bone marrow-derived M1 and M2 macrophages and mannosylated clodronate liposomes (MCLs) were injected into the vitreous on P12 to examine the effects at P17. M2 macrophages were cocultured with human retinal endothelial cells (HRECs) to examine their effects on proliferation and tube formation. RESULTS: The results showed that the M2 macrophages, rather than M1 phenotype, were highly expressed in OIR mice. The number of M2 macrophages had increased significantly at P17, and the increase was closely associated with the presence of neovascular tufts in the OIR retinas. Selective depletion of M2 macrophages suppressed the pathological neovascularization and promoted physiological revascularization. In contrast, intravitreal injection of bone marrow-derived M2 macrophages or the culture supernatants promoted pathological neovascularization and inhibited physiological revascularization. In an in vitro coculture system, M2-polarized macrophages significantly promoted proliferation and tube formation of HRECs. CONCLUSIONS: These results indicated that M2 macrophages, rather than M1, play an important role in promoting retinal pathological neovascularization probably by producing secreted factors. Thus, targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization..
41. Shigeo Yoshida, Yoshiyuki Kobayashi, Takahito Nakama, Yedi Zhou, Keijiro Ishikawa, Ryoichi Arita, Shintaro Nakao, Masanori Miyazaki, Yukio Sassa, Yuji Oshima, Kenji Izuhara, Toshihiro Kono, Tatsuro Ishibashi, Increased expression of M-CSF and IL-13 in vitreous of patients with proliferative diabetic retinopathy: implications for M2 macrophage-involving fibrovascular membrane formation., The British journal of ophthalmology, 10.1136/bjophthalmol-2014-305860, 99, 5, 629-34, 2015.05, PURPOSE: We recently demonstrated that M2 macrophages were involved in the development of fibrovascular membranes (FVM) associated with proliferative diabetic retinopathy (PDR) possibly through the induction of periostin. The purpose of this study was to determine whether macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-13, inducers of the M2 polarisation of macrophages from monocytes, are elevated in the vitreous of patients with PDR, and whether M2-polarised macrophages induce periostin production. METHODS: We measured the levels of M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, IL-13, soluble (s)CD163, periostin and vascular endothelial growth factor by sandwich ELISA in vitreous samples collected from 61 eyes of 47 patients with PDR, and 39 eyes of 36 patients with non-diabetic ocular diseases (control group). Human monocytes were polarised in vitro with GM-CSF, interferon-γ, and lipopolysaccharide for M1 macrophages, and M-CSF, IL-4, and IL-13 for M2 macrophages. Quantitative real-time PCR was used to determine the mRNA level of periostin. RESULTS: The concentrations of M-CSF and IL-13 in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p<0.0001). There was a strong positive correlation between the vitreous concentrations of M-CSF and sCD163 and periostin. The mean vitreous level of IL-13 was significantly higher in eyes with FVMs than in those without FVMs (epicentre only). In vitro studies showed that M2-polarlised macrophages significantly increased the expression of the mRNA of periostin. CONCLUSIONS: These findings indicate that the M2 polarisation of macrophages is induced by M-CSF and IL-13 in diabetic retinas. The presence of M-CSF and IL-13 would then promote FVM formation by periostin production..
42. Yoshiyuki Kobayashi, Shigeo Yoshida, Takahito Nakama, Yedi Zhou, Keijiro Ishikawa, Ryoichi Arita, Shintaro Nakao, Masanori Miyazaki, Yukio Sassa, Yuji Oshima, Kenji Izuhara, Toshihiro Kono, Tatsuro Ishibashi, Overexpression of CD163 in vitreous and fibrovascular membranes of patients with proliferative diabetic retinopathy: possible involvement of periostin., The British journal of ophthalmology, 10.1136/bjophthalmol-2014-305321, 99, 4, 451-6, 2015.04, AIM: To determine whether CD163, a specific marker for M2 macrophages, is involved in the formation of preretinal fibrovascular membranes (FVMs) present in eyes with proliferative diabetic retinopathy (PDR). METHODS: We measured the levels of soluble (s)CD163, periostin and vascular endothelial growth factor by sandwich ELISA in vitreous samples from 74 eyes of 62 patients with PDR, 20 eyes of 18 patients with proliferative vitreoretinopathy, and 56 eyes of 54 patients with non-diabetic ocular diseases (control group). Immunohistochemical analyses were performed to determine the expressions of CD68, CD163 and periostin in the surgically resected FVMs and idiopathic epiretinal membranes (ERMs). RESULTS: The concentrations of sCD163 and periostin in the vitreous were significantly higher in patients with PDR than in non-diabetic controls (p<0.0001). There was a strong correlation between the vitreous concentrations of sCD163 and periostin. The mean vitreous level of sCD163 was significantly higher in eyes with FVMs than in those without FVMs (epicentre only). The number and percentage of CD163+ macrophages were significantly higher in the FVMs than in the idiopathic ERMs. Immunohistochemical analysis showed co-localisation of CD163 and periostin in FVM cells. CONCLUSIONS: These findings indicate that the overexpression of CD163 by macrophages may be involved in the development of FVMs partly through periostin production..
43. Souska Zandi, Shintaro Nakao, Kwang-Hoon Chun, Paolo Fiorina, Dawei Sun, Ryoichi Arita, Ming Zhao, Enoch Kim, Olivier Schueller, Stewart Campbell, Mahdi Taher, Mark Ivan Melhorn, Alexander Schering, Francesca Gatti, Sara Tezza, Fang Xie, Andrea Vergani, Shigeo Yoshida, Keijiro Ishikawa, Muneo Yamaguchi, Fumiyuki Sasaki, Ruth Schmidt-Ullrich, Yasuaki Hata, Hiroshi Enaida, Mitsuko Yuzawa, Takehiko Yokomizo, Young-Bum Kim, Paul Sweetnam, Tatsuro Ishibashi, Ali Hafezi-Moghadam, ROCK-isoform-specific polarization of macrophages associated with age-related macular degeneration., Cell reports, 10.1016/j.celrep.2015.01.050, 10, 7, 1173-86, 2015.02, Age 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..
44. Keijiro Ishikawa, Shigeo Yoshida, Yoshiyuki Kobayashi, Yedi Zhou, Takahito Nakama, Shintaro Nakao, Yukio Sassa, Yuji Oshima, Hiroaki Niiro, Koichi Akashi, Toshihiro Kono, Tatsuro Ishibashi, Microarray analysis of gene expression in fibrovascular membranes excised from patients with proliferative diabetic retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.14-15589, 56, 2, 932-46, 2015.01, PURPOSE: We determined the profile of genes expressed in fibrovascular membranes (FVMs). METHODS: Six FVMs were surgically removed from patients with proliferative diabetic retinopathy (PDR) during pars plana vitrectomy with membrane peeling. The FVMs were classified into three active FVMs or three inactive FVMs according to the presence or absence of neovascularization (NV) in the membranes. Total RNA was isolated from the six FVMs and also from three normal human retinas. The DNA microarray analysis was performed to compare the genes expressed in the FVMs to those in normal human retinas, and also between active and inactive FVMs. Ingenuity pathway analysis (IPA) was used to determine the key biological networks related to the genes that were significantly altered. Quantitative RT-PCR and immunohistochemistry were performed to validate the microarray analyses. RESULTS: There were 87 genes expressed at significantly higher levels in FVMs than in normal human retinas. Functional classification of these genes showed that the most clustered genes were those related to extracellular matrix formation. The top biological network generated by the IPA was cellular assembly and organization involving nodes of genes related to extracellular matrix formation. These networks included the collagen family and matricellular proteins, THBS2, POSTN, and TNC. There were 91 genes significantly upregulated in active FVMs, and the most clustered functional category was angiogenesis. In contrast, 89 genes were significantly upregulated in inactive FVMs, and the most clustered functional category was metabolism. The IPA revealed that the top biological network related to the genes that were significantly altered in this comparison was cell-to-cell signaling, and interactions involving the PDGF and TGFβ families. The results of quantitative RT-PCR analyses and immunohistochemistry for several selected molecules were in good agreement with the microarray data. CONCLUSIONS: Our data indicate that extracellular matrix-related molecules such as POSTN, TNC, TGFβ, and angiogenic factors have important roles in promoting the development of FVMs associated with PDR..
45. Keijiro Ishikawa, Shigeo Yoshida, Shintaro Nakao, Takahito Nakama, Takeshi Kita, Ryo Asato, Yukio Sassa, Ryoichi Arita, Masanori Miyazaki, Hiroshi Enaida, Yuji Oshima, Noboru Murakami, Hiroaki Niiro, Junya Ono, Akira Matsuda, Yoshinobu Goto, Koichi Akashi, Kenji Izuhara, Akira Kudo, Toshihiro Kono, Ali Hafezi-Moghadam, Tatsuro Ishibashi, Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy., FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 10.1096/fj.13-229740, 28, 1, 131-42, 2014.01, Proliferative vitreoretinopathy (PVR) is a severe, vision-threatening disorder characterized by the fibrous membrane formation that leads to tractional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up-regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αV-mediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFβ2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR..
46. Shintaro Nakao, Souska Zandi, Ri-ichiro Kohno, Dawei Sun, Takahito Nakama, Keijiro Ishikawa, Shigeo Yoshida, Hiroshi Enaida, Tatsuro Ishibashi, Ali Hafezi-Moghadam, Lack of lymphatics and lymph node-mediated immunity in choroidal neovascularization., Investigative ophthalmology & visual science, 10.1167/iovs.12-10341, 54, 6, 3830-6, 2013.06, 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..
47. Shintaro Nakao, Keijiro Ishikawa, Shigeo Yoshida, Ri-Ichiro Kohno, Masanori Miyazaki, Hiroshi Enaida, Toshihiro Kono, Tatsuro Ishibashi, Altered vascular microenvironment by bevacizumab in diabetic fibrovascular membrane., Retina (Philadelphia, Pa.), 10.1097/IAE.0b013e3182753b41, 33, 5, 957-63, 2013.05, PURPOSE: The purpose of this study was to evaluate the impact of intravitreal bevacizumab (IVB) on three cellular components (vascular endothelial cells, pericytes, and myofibroblasts) of the vascular microenvironment in fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy. METHODS: Immunohistological studies with antibodies of CD34, αSMA, and transforming growth factor-β were performed on 20 surgical specimens obtained during a pars plana vitrectomy from 8 IVB-treated eyes, whereas 12 remained untreated. Four different indexes of vascular phenotype (vascular area, vascular major axis, CD34 endothelial area, and blood vessel density) and αSMA expression in vascular and stromal components were quantitatively analyzed. RESULTS: The intraluminal area of blood vessels, CD34 endothelial area, and the blood vessel density in IVB-treated FVMs were significantly less than in untreated FVMs. The number of CD34 blood vessels in IVB-treated FVMs was similar to that in untreated FVMs. Intravitreal bevacizumab could not affect vascular and stromal αSMA area significantly. However, the ratio of vascular αSMA area/CD34 area was significantly higher in IVB-treated FVMs than in untreated FVMs. Transforming growth factor-β expression could be observed in the IVB-treated FVM. CONCLUSION: Intravitreal bevacizumab might primarily affect blood vessels, and the effects on pericytes and myofibroblasts might be secondary. Intravitreal bevacizumab treatment regulates vascular microenvironment by the contraction of blood vessels, the increasing pericyte ratio, and transforming growth factor-β expression in FVMs of patients with proliferative diabetic retinopathy..
48. Noriko Yoshida, Yasuhiro Ikeda, Shoji Notomi, Keijiro Ishikawa, Yusuke Murakami, Toshio Hisatomi, Hiroshi Enaida, Tatsuro Ishibashi, Clinical evidence of sustained chronic inflammatory reaction in retinitis pigmentosa., Ophthalmology, 10.1016/j.ophtha.2012.07.006, 120, 1, 100-5, 2013.01, PURPOSE: To study the nature of inflammatory reaction in eyes of patients with retinitis pigmentosa (RP) and its possible role in the pathogenesis of RP. DESIGN: Retrospective, observational study. PARTICIPANTS AND CONTROLS: Three hundred seventy-one consecutive patients diagnosed with typical RP were included in this study. We included 165 patients without active inflammatory diseases, including 20 patients diagnosed with cataract, and 36 patients diagnosed with idiopathic epiretinal membrane as controls. METHODS: Density of the inflammatory cells in the anterior vitreous cavity was measured and graded by slit-lamp biomicroscopy. A multiplex enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the concentration of cytokines and chemokines in aqueous humor and vitreous fluid of patients with RP and controls. In addition, we investigated the relationship between visual function and anterior vitreous cells in these patients. MAIN OUTCOME MEASURES: Slit-lamp biomicroscopic analysis, best-corrected visual acuity, visual field analysis, and multiplex ELISA. RESULTS: In 190 of 509 eyes with RP (37.3%), "1+" (5-9 cells per field) or more cells were observed in the anterior vitreous cavity. Strong inflammatory reaction with "2+" cells (10-30 cells per field) was associated with younger age. In the elderly patients with RP, significantly decreased visual function was seen in a group with "1+" or more cells (P<0.05). Moreover, the levels of a variety of proinflammatory cytokines and chemokines, including monocyte chemotactic protein-1, were increased both in the aqueous humor and vitreous fluid of RP patients compared with the levels in control patients. CONCLUSIONS: Sustained chronic inflammatory reaction may underlie the pathogenesis of RP, suggesting interventions for ocular inflammatory reaction as a potential treatment for patients with RP. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article..
49. Noriko Yoshida, Yasuhiro Ikeda, Shoji Notomi, Keijiro Ishikawa, Yusuke Murakami, Toshio Hisatomi, Hiroshi Enaida, Tatsuro Ishibashi, Laboratory evidence of sustained chronic inflammatory reaction in retinitis pigmentosa., Ophthalmology, 10.1016/j.ophtha.2012.07.008, 120, 1, e5-12, 2013.01, PURPOSE: To study the nature of retinal inflammatory response in rd10 mice, an animal model of retinitis pigmentosa (RP), and to investigate the effect of an antioxidant on retinal inflammation and photoreceptor apoptosis. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: This study included 42 untreated rd10 mice, 30 N-acetylcysteine (NAC)-treated rd10 mice, and 20 C57BL/6 mice as controls. METHODS: Real-time polymerase chain reaction (PCR) was performed to evaluate the expression levels of inflammatory factors (proinflammatory cytokines and chemokines) in rd10 mouse retinas. Rd10 mice were treated with an antioxidant NAC, and its effect on retinal inflammation and photoreceptor apoptosis were examined by immunohistochemistry. MAIN OUTCOME MEASURES: Real-time PCR and immunohistochemistry. RESULTS: We demonstrated sequential events involving increased expression of proinflammatory cytokines and chemokines, activation of microglia, and photoreceptor apoptosis during retinal degeneration of rd10 mice. Furthermore, antioxidant treatment with NAC prevented the photoreceptor cell death along with suppression of inflammatory factors and microglial activation. CONCLUSIONS: Sustained chronic inflammatory reaction may contribute to the pathogenesis of retinal degeneration in rd10 mice, suggesting interventions for ocular inflammatory reaction using antioxidants as a potential treatment for patients with RP. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article..
50. Shigeo Yoshida, Takahito Nakama, Keijiro Ishikawa, Mitsuru Arima, Takashi Tachibana, Shintaro Nakao, Yukio Sassa, Miho Yasuda, Hiroshi Enaida, Yuji Oshima, Toshihiro Kono, Tatsuro Ishibashi, Antiangiogenic shift in vitreous after vitrectomy in patients with proliferative diabetic retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.12-9671, 53, 11, 6997-7003, 2012.10, PURPOSE: We determined whether the concentrations of VEGF, erythropoietin, and endostatin in the vitreous are altered after vitrectomy in patient with proliferative diabetic retinopathy (PDR). METHODS: We measured the levels of VEGF, erythropoietin, and endostatin by sandwich ELISA in vitreous samples collected from 38 eyes of 33 patients with PDR before pars plana vitrectomy (without IOL implantation) and the same 38 eyes during IOL implantation 3.1 to 25.7 (mean 6.7) months after the initial vitrectomy. RESULTS: The mean vitreous levels of VEGF (964.5 pg/mL) and erythropoietin (1359.5 pg/mL) in the samples collected before vitrectomy were significantly higher in patients with PDR than in the control patients (0.68 and 70.7 pg/mL, respectively; P < 0.01). The levels of VEGF (292.5 pg/mL) and erythropoietin (557.9 pg/mL) in the samples from eyes with PDR collected at the time of IOL implantation were significantly lower than those collected before vitrectomy (P < 0.01). In contrast, the changes in the level of endostatin were not significant after vitrectomy. The VEGF and erythropoietin levels in the vitreous fluid from patients with PDR were correlated inversely with the interval between the initial vitrectomy and the time of the IOL implantation. CONCLUSIONS: The significant decrease in the intravitreal concentration of VEGF and erythropoietin, and an absence of a significant change in the endostatin indicated a shift in the antiangiogenic balance in the vitreous of patients with PDR after successful vitrectomy..
51. Mitsuru Arima, Shigeo Yoshida, Takahito Nakama, Keijiro Ishikawa, Shintaro Nakao, Takeru Yoshimura, Ryo Asato, Yukio Sassa, Takeshi Kita, Hiroshi Enaida, Yuji Oshima, Akira Matsuda, Akira Kudo, Tatsuro Ishibashi, Involvement of periostin in regression of hyaloidvascular system during ocular development., Investigative ophthalmology & visual science, 53, 10, 6495-503, 2012.09, PURPOSE: A timely regression of the hyaloid vascular system (HVS) is required for the normal ocular development. Although macrophages have a critical role in this process, the exact mechanism remains undetermined. Periostin is a matricellular protein involved in tissue and vascular remodeling. The purpose of our study was to determine whether periostin is involved in the HVS regression. METHODS: We used wild type (WT) and periostin knockout (KO) mice. Indocyanine green angiography and immunohistochemistry with isolectin B4 were used to evaluate the HVS regression. TUNEL-labeling was used to quantify the number of apoptotic hyaloid vascular endothelial cells. F4/80 and Iba-1 staining was performed to determine the number and location of macrophages in the vitreous. The location of periostin also was investigated by immunohistochemistry. To determine the functional role of periostin, the degree of adhesion of human monocytes to fibronectin was measured by an adhesion assay. RESULTS: The HVS regression and peak in the number of TUNEL-positive apoptotic endothelial cells were delayed in periostin KO mice. The number of F4/80 positive cells in the vitreous was higher in periostin KO mice. Only a small number of Iba-1-positive cells near the hyaloid vessels was co-stained with periostin, and peripheral blood monocytes were not stained with periostin. Adhesion assay showed that periostin increased the degree of attachment of monocytes to fibronectin. CONCLUSIONS: These results suggest that periostin, which is secreted by the intraocular macrophages, enhances the HVS regression by intensifying the adhesion of macrophages to hyaloid vessels..
52. Shintaro Nakao, Mitsuru Arima, Keijiro Ishikawa, Riichiro Kohno, Shuhei Kawahara, Masanori Miyazaki, Shigeo Yoshida, Hiroshi Enaida, Ali Hafezi-Moghadam, Toshihiro Kono, Tatsuro Ishibashi, Intravitreal anti-VEGF therapy blocks inflammatory cell infiltration and re-entry into the circulation in retinal angiogenesis., Investigative ophthalmology & visual science, 10.1167/iovs.11-9119, 53, 7, 4323-8, 2012.06, PURPOSE: Anti-VEGF-A antibody (Ab) (e.g., bevacizumab, ranibizumab) is widely used as a treatment against retinal angiogenesis and edema. The purpose of this study was to evaluate whether intravitreal anti-VEGF Ab injection modulates inflammatory cells in retinal angiogenesis. METHODS: To investigate whether intravitreal bevacizumab injections affect the number of inflammatory cells in proliferative diabetic retinopathy (PDR) membranes in patients, immunohistochemical staining with CD45 Ab (pan-leukocyte marker) was performed using the surgically obtained membranes in pars plana vitrectomy with or without pretreatment with bevacizumab. To check whether anti-VEGF-A Ab affects leukocytes going in and out of blood vessels during retinal angiogenesis, the authors performed their novel leukocyte transmigration assay and CD45 immunostaining in a mouse model of oxygen-induced retinopathy (OIR). RESULTS: The authors' new imaging approach revealed that intravitreal injection of anti-VEGF-A Ab blocks leukocyte infiltration as well as angiogenesis. The Ab injection inhibited leukocyte transmigration before affecting the angiogenenic area. CD45 staining showed no significant difference in the leukocyte number in the angiogenic retina or the human PDR membranes between the anti-VEGF-A Ab injected group and the control group. Furthermore, VEGF-A inhibition also affected leukocytes going out from the retina. CONCLUSIONS: Intravitreal injection of anti-VEGF-A Ab could inhibit leukocyte trafficking in the retina, suggesting that anti-VEGF-A therapy could serve as a treatment in retinal inflammation..
53. Keijiro Ishikawa, Shigeo Yoshida, Shintaro Nakao, Yukio Sassa, Ryo Asato, Riichiro Kohno, Mitsuru Arima, Takeshi Kita, Ayako Yoshida, Kenoki Ohuchida, Tatsuro Ishibashi, Bone marrow-derived monocyte lineage cells recruited by MIP-1β promote physiological revascularization in mouse model of oxygen-induced retinopathy., Laboratory investigation; a journal of technical methods and pathology, 10.1038/labinvest.2011.141, 92, 1, 91-101, 2012.01, Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1β (MIP-1β) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1β in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1β was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1β reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1β-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1β is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1β induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases..
54. Shigeo Yoshida, Keijiro Ishikawa, Ryo Asato, Mitsuru Arima, Yukio Sassa, Ayako Yoshida, Hiroshi Yoshikawa, Keisuke Narukawa, Satoshi Obika, Junya Ono, Shoichiro Ohta, Kenji Izuhara, Toshihiro Kono, Tatsuro Ishibashi, Increased expression of periostin in vitreous and fibrovascular membranes obtained from patients with proliferative diabetic retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.10-6625, 52, 8, 5670-8, 2011.07, PURPOSE: Preretinal fibrovascular membranes (FVMs) form as a sequela to proliferative diabetic retinopathy (PDR), and their presence can lead to a severe decrease of vision. The purpose of this study was to determine whether periostin, a matricellular protein that plays a role in cell adhesion and migration, is associated with the formation of FVMs. METHODS: One hundred six vitreous samples and 15 FVMs were obtained during vitrectomy on patients with PDR. Semiquantitative RT-PCR was performed to determine the periostin level of the mRNA. Immunohistochemical analyses were performed to determine the sites of periostin expression in the FVMs. ELISA was used to measure the concentrations of periostin, bFGF, and VEGF in the vitreous. RESULTS: The periostin level of the mRNA was high in 10 of 10 FVMs tested but was barely detectable in the control retinas. Sequencing of the periostin PCR products revealed three splice variants of the FVMs. Immunohistochemical analysis showed colocalization of periostin and α-SMA in FVM cells. The concentration of periostin in the vitreous was significantly higher in patients with PDR than in the 31 eyes of patients with a macular hole or an epiretinal membrane (P < 0.001). Among the PDR patients, the mean vitreous level of periostin in eyes with FVMs was significantly higher than in those without FVMs (epicenter only; P < 0.001). The correlation between the vitreous concentrations of periostin and of bFGF and VEGF was not significant. CONCLUSIONS: These findings indicate that periostin may be involved in the development of FVMs..
55. Keijiro Ishikawa, Shigeo Yoshida, Koji Kadota, Takanori Nakamura, Hiroaki Niiro, Satoshi Arakawa, Ayako Yoshida, Koichi Akashi, Tatsuro Ishibashi, Gene expression profile of hyperoxic and hypoxic retinas in a mouse model of oxygen-induced retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.09-4605, 51, 8, 4307-19, 2010.08, PURPOSE: To determine a profile of gene expression in retinas of a murine model of oxygen-induced retinopathy (OIR). METHODS: OIR was induced in C57BL/6N mice by exposing postnatal day (P)7 pups to 75% oxygen for 5 days and then returning them to room air at P12. Gene microarrays containing more than 47,000 transcripts were used to study the changes in gene expression in retinas isolated immediately (P12) and at 12 hours (P12.5) after exposure to hyperoxia. The retinas of P12 mice raised under normoxic conditions served as control subjects. Quantitative RT-PCR and multiplex ELISA were performed to validate the microarray analyses. RESULTS: The expression of 83 gene transcripts was significantly altered in the hyperoxic P12 retinas. These genes were classified as cellular components or were associated with development, metabolism, transport, stress response, cell adhesion, inflammation, or vision. The genes related to retinal growth, such as Pdgfb and Robo4, which are associated with vascular development, were downregulated. In contrast, the expression levels of 95 genes were significantly altered in the hypoxic P12.5 retinas, which contained several known hypoxia-regulated genes including Vegfa and Hif1a. The differentially expressed genes were broadly clustered into the development, inflammation, metabolism, signaling, antiapoptosis, cellular component, transport, glycolysis, and vision groups. Those associated with organogenesis (e.g., Vegfa, Igfbp3, Tnfrsf12a, and Nestin) and to inflammation (e.g., Ccl3, Ccl4, and MHCs) were upregulated. The results of quantitative RT-PCR and multiplex ELISA were in agreement with the microarray data. CONCLUSIONS: These alterations in gene expression may determine the hyperoxic growth retardation, postischemic inflammation, neovascularization, and remodeling in retinas of murine OIR..
56. Shigeo Yoshida, Atsushi Ogura, Keijiro Ishikawa, Ayako Yoshida, Richiro Kohno, Yoko Yamaji, Kazuho Ikeo, Takashi Gojobori, Toshihiro Kono, Tatsuro Ishibashi, Gene expression profile of fibrovascular membranes from patients with proliferative diabetic retinopathy., The British journal of ophthalmology, 10.1136/bjo.2009.167072, 94, 6, 795-801, 2010.06, BACKGROUND/AIMS: The purpose of this study was to generate a profile of genes expressed in preretinal fibrovascular membranes (FVMs) from patients with proliferative diabetic retinopathy. METHODS: A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from FVMs obtained during vitrectomy. The sequence from the 5' end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). Functional annotation was retrieved from Ensemble database and analysed by FatiGO. The web-based VisANT software was used to identify the molecular networks within the FVMs. RESULTS: A total of 2816 ESTs were assembled in 625 non-redundant clusters. Among these, 515 matched the human cDNA database. The 515 clusters were subdivided by functional subsets of genes related to ribosomal activity, oxidative phosphorylation, focal adhesion, cell adhesion and other functions. Querying against the VisANT database yielded 3175 possible physical relationships to other genes/proteins, which included an additional 2480 genes that were not detected in the FVM library. CONCLUSIONS: The cDNA library constructed from human FVMs will be a valuable source of information. It should facilitate a wide range of studies that can establish the molecular mechanisms underlying the development of FVMs..
57. Shigeo Yoshida, Keijiro Ishikawa, Tokiko Matsumoto, Ayako Yoshida, Tatsuro Ishibashi, Toshihiro Kono, Reduced concentrations of angiogenesis-related factors in vitreous after vitrectomy in patients with proliferative diabetic retinopathy., Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 10.1007/s00417-010-1301-5, 248, 6, 799-804, 2010.06, BACKGROUND: The purpose of this study was to determine whether vitrectomy alters the angiogenic profile in the vitreous of eyes with proliferative diabetic retinopathy (PDR). METHODS: We measured the levels of angiopoietin-2, HGF, bFGF, PDGF, TIMP-1, and TIMP-2 by sandwich enzyme linked immunosorbent assay (ELISA) in vitreous samples from 27 eyes of 26 patients with PDR before pars plana vitrectomy (without IOL implantation) and in 12 fluid samples from 12 patients with PDR obtained during an IOL implantation 3.5 to 9 (mean 4.9) months after an earlier vitrectomy. The levels of these factors were also measured in 12 vitreous samples obtained from 12 eyes that had undergone epiretinal membrane (ERM) or macular hole (MH) surgeries. RESULTS: The mean vitreous levels of both angiopietin-2 (103 pg/ml) and HGF (1091 pg/ml) in the sample from eyes with PDR collected at the time of the IOL implantation were significantly lower than in those collected before the vitrectomy (P < 0.01). On the other hand, the changes in the levels of TIMP-1 and TIMP-2 were both not significant after vitrectomy. CONCLUSION: The significant decrease of angiopietin-2 and HGF in the vitreous fluid after vitrectomy suggests that vitrectomy shifts the eye towards an anti-angiogenic environment..
58. Shigeo Yoshida, Aki Miyazaki, Keijiro Ishikawa, Yasuhiro Ikeda, Kimihiko Fujisawa, Tatsuro Ishibashi, Clinical course during 40-year follow-up of Axenfeld-Rieger syndrome in a Japanese family., Oman journal of ophthalmology, 10.4103/0974-620X.60021, 3, 1, 34-5, 2010.01.
59. Satomi Shiose, Shigeo Yoshida, Keijiro Ishikawa, Tatsuro Ishibashi, VMD2 mutational analysis in a Japanese family with Best macular dystrophy., Oman journal of ophthalmology, 10.4103/0974-620X.57317, 2, 3, 143-4, 2009.09.
60. Yoko Yamaji, Shigeo Yoshida, Keijiro Ishikawa, Akihito Sengoku, Kota Sato, Ayako Yoshida, Rumi Kuwahara, Kenoki Ohuchida, Eiji Oki, Hiroshi Enaida, Kimihiko Fujisawa, Toshihiro Kono, Tatsuro Ishibashi, TEM7 (PLXDC1) in neovascular endothelial cells of fibrovascular membranes from patients with proliferative diabetic retinopathy., Investigative ophthalmology & visual science, 10.1167/iovs.07-1249, 49, 7, 3151-7, 2008.07, PURPOSE: Proliferative diabetic retinopathy (PDR) results from the formation of fibrovascular membranes (FVMs) in the posterior fundus that can lead to a severe decrease of vision. Tumor endothelial marker 7 (TEM7) is a protein that is highly expressed in the endothelial cells of tumors, but whether it plays a role in FVMs is unknown. The purpose of this study was to determine whether TEM7 is associated with the formation of FVMs. METHODS: FVMs were obtained during vitrectomy from patients with PDR. RT-PCR was performed to determine the level of expression of the mRNA of TEM7. The splice variants of TEM7 were identified by direct sequencing. Immunohistochemical analyses and in situ hybridization was performed to determine the sites of TEM7 in the FVMs. RESULTS: The level of the mRNA of TEM7 was high in 10 of 10 FVMs but was barely detectable in the five idiopathic epiretinal membranes. Direct sequencing of subcloned TEM7 PCR products revealed several splice variants (intracellular, secreted, and membrane-bound forms of TEM7) in the FVMs. Immunohistochemical analysis showed a colocalization of TEM7 and CD34, an endothelial cell marker, in most of the neovascular endothelial cells in the FVMs. Immunoelectron microscopy revealed that membrane-bound TEM7 was expressed on the luminal surfaces of the vascular endothelial cells of FVMs. CONCLUSIONS: This study indicates that TEM7 may play a significant role in the proliferation and maintenance of neovascular endothelial cells in the FVMs. If correct, TEM7 may be a molecular target for new diagnostic and therapeutic strategies for PDR..