Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein.

As the acute lymphoblastic leukaemia (ALL) develops, expression of L-asparaginase (ASNase) protein is known to decrease. Therefore, deficiency of the ASNase protein would be regarded as one of the significant indications of the ALL. For the treatment of ALL, recombinant ASNase protein derived from bacterial origin is used which causes cytotoxicity by deprivation of Asn. However, short half-life of the protein is an obstacle for medical use. In order to overcome this limit, recombinant ASNase was fused to 30Kc19 with protein-stabilizing and cell-penetrating properties. As the 30Kc19 protein may induce steric hindrance, we further added a PLGLAG linker sequence (LK) between the ASNase and 30Kc19. The treatment of ASNase-LK-30Kc19 fusion protein demonstrated enhanced stability, cell-penetrating property, and anti-cancer activity. Intracellular delivery of both the non-cleaved and cleaved forms of the protein were observed, suggesting that ASNase acted both internally and externally, performing high anti-cancer activity by effective depletion of intracellular Asn. Additionally, ASNase-LK-30Kc19 showed high selectivity towards cancer cells. In terms of the dosage, releasable ASNase from ASNase-LK-30Kc19 reached the same half-maximal inhibitory concentration at a concentration five times lower than non-releasable ASNase-30Kc19. Altogether, the findings suggest that this fusion approach has potential applications in the treatment of ALL.

A standardized extract of Rhynchosia volubilis Lour. exerts a protective effect on benzalkonium chloride-induced mouse dry eye model.

ETHNOPHARMACOLOGICAL RELEVANCE: In contrast to other leguminous plants generally used as food, Rhynchosia volubilis Loureiro, a small soybean with a black seed coat, has been used as a traditional oriental remedy for various human diseases in Eastern Asia. In this study, we demonstrated the protective effect of R. volubilis against dry eye disease. AIM OF THE STUDY: We aimed to investigate whether a standardized ethanol extract of R. volubilis (EERV) can protect the cornea in a benzalkonium chloride (BAC)-induced mouse dry eye model. MATERIALS AND METHODS: Experimental dry eye was induced by the instillation of 0.2% BAC on mouse cornea. A standardized ethanol extract of R. volubilis (EERV) was orally administered following BAC treatment. The positive control group was treated with commercial eye drops. Fluorescein staining, tear break-up time (BUT), and hematoxylin and eosin staining were evaluated on the ocular surface. Squamous metaplasia and apoptosis in the corneal epithelial layer were detected by immunostaining. Furthermore, the protein expression of cytochrome c, Bcl-2, and Bax was determined. RESULTS: EERV treatment significantly improved fluorescein scoring, BUT, and smoothness in the cornea compared to the vehicle group. In addition, EERV inhibited squamous metaplasia and apoptosis in the cornea. The expression of cytochrome c and Bax was upregulated, while that of Bcl-2 was downregulated in the vehicle group compared with that in the control group. However, EERV treatment inhibited the expression of cytochrome c and Bax, while that of Bcl-2 was improved. CONCLUSION: Standardized EERV could be a beneficial candidate for the treatment of dry eye disease.

Subconjunctival Injection of Viscoelastic Material for Leaking Sclerotomy in Transconjunctival Sutureless Vitrectomy.

Aim. To evaluate the effectiveness of subconjunctivally injected viscoelastic material (VEM) for the self-sealing of leaking sclerotomy in transconjunctival sutureless vitrectomy (TSV). Methods. This was a prospective interventional series. Subconjunctival injection of VEM was performed in eyes showing leaking sclerotomy at the end of TSV in selected cases. This procedure was performed in 24 consecutive eyes from 24 patients scheduled for 23- or 25-gauge TSV with phacoemulsification for various vitreoretinal diseases combined with cataracts. Results. Among the 24 eyes, 13 cases were scheduled for 23-gauge TSV, while 11 cases were scheduled for 25-gauge TSV. The average number of injection sites per eye was 1.7 ± 0.9 in the 23-gauge cases and 1.5 ± 0.7 in the 25-gauge cases. Leakage was most commonly observed at the vitrector site of the sclerotomy, while little leakage was observed at the illuminator site. There were no cases of postoperative hypotony. Conclusion. Subconjunctival injection of VEM was simple and effective for the self-sealing of leaking sclerotomy after TSV in selected cases.

The leaves of Diospyros kaki exert beneficial effects on a benzalkonium chloride-induced murine dry eye model.

PURPOSE: In this study, the beneficial effects of the oral administration of ethanol extract of Diospyros kaki (EEDK) were tested on a mouse dry eye model induced by benzalkonium chloride (BAC). METHODS: A solution of 0.2% BAC was administered topically to mouse eyes for 14 days, twice daily, to induce dry eye. Various concentrations of EEDK were administrated daily by oral gavage for 14 days after BAC treatment. Preservative-free eye drops were instilled in the positive-control group. The tear secretion volume (Schirmer's test), tear break-up time (BUT), and fluorescein score were measured on the ocular surface. BAC-induced corneal damage was tested with hematoxylin-eosin staining. Moreover, apoptotic cell death in the corneal epithelial layer was investigated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The protein expression level of interleukin-1alpha (IL-1α), IL-1ß, IL-6, tumor necrosis factor- alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) was determined with western blot analysis. Furthermore, squamous metaplasia in the corneal epithelial layer was detected with immunofluorescent staining for cytokeratine-10. The cellular proliferation in the cornea was examined with immunohistochemical staining for Ki-67. RESULTS: EEDK treatment resulted in prolonged BUT, decreased fluorescein score, increased tear volume, and smoother epithelial cells compared with BAC treatment alone in the cornea. Moreover, EEDK treatment inhibited the inflammatory response and corneal epithelial cell death in a BAC-induced murine dry eye model, and changes in squamous cells were inhibited. Proliferative activity in the corneal epithelium cells was improved with EEDK. CONCLUSIONS: EEDK could be a potential therapeutic agent in the clinical treatment of dry eye.

Effect of Posterior Subtenon Triamcinolone Acetonide Injection on Diabetic Macular Edema Refractory to Intravitreal Bevacizumab Injection.

PURPOSE: To evaluate the effects of posterior subtenon triamcinolone acetonide injection on refractory diabetic macular edema (DME) after intravitreal bevacizumab (IVB) injection failure. METHODS: Patients with DME and central subfield thickness (CST) >300 µm who did not respond to IVB injections were retrospectively included. Specifically, we enrolled patients who were diagnosed with refractory DME and who experienced an increase in CST after 1 to 2 IVB injections or no decrease after ≥3 consecutive IVB injections. One clinician injected 20 mg of triamcinolone acetonide into the posterior subtenon space. All patients received ophthalmic examinations at baseline and at 2, 4, and 6 months post-baseline. Examinations included Snellen visual acuity, intraocular pressure, and spectral-domain optical coherence tomography. RESULTS: Forty eyes of 34 patients were included. The average baseline CST was 476 µm. The average CST decreased to 368 µm at 2 months, 374 µm at 4 months, and 427 µm at 6 months (p < 0.001 for all results, Wilcoxon signed-rank test). The average intraocular pressure increased from 15.50 to 16.92 mmHg at 2 months but decreased to 16.30 mmHg at 4 months and 15.65 mmHg at 6 months. Logarithm of the minimum angle of resolution visual acuity improved from 0.56 to 0.50 at 2 months (p = 0.023), 0.50 at 4 months (p = 0.083), and 0.48 at 6 months (p = 0.133, Wilcoxon signed-rank test). No complications were detected. CONCLUSIONS: Posterior subtenon triamcinolone acetonide is an effective and safe treatment for reducing CST in DME refractory to IVB.

Diospyros kaki Extract Inhibits Alkali Burn-Induced Corneal Neovascularization.

The purpose of this study was to evaluate the effect of ethanol extract of Diospyros kaki (EEDK) leaves on corneal neovascularization (CoNV) in rats. One week after the alkali burns in the corneas, the CoNV area coverage in the CoNV-positive control group, 100 mg/kg EEDK group, and 200 mg/kg EEDK group was 43.3% ± 5.5%, 337.7% ± 2.5%, and 27.2% ± 4.3%, respectively. The areas of CoNV in the EEDK-treated groups were significantly different from those of the CoNV group. EEDK significantly attenuated the upregulation of vascular endothelial growth factor, fibroblast growth factor, interleukin-6, and matrix metalloproteinase-2 (MMP-2) protein levels. Orally administrated D. kaki inhibited CoNV development in rats.

Choroidal Thickness in Eyes with Unilateral Ocular Ischemic Syndrome.

Aim. To analyze the subfoveal choroid thickness and choroidal volume in unilateral ocular ischemic syndrome (OIS). Methods. A retrospective review was conducted for all patients with unilateral OIS from October 2010 through June 2014. The subfoveal choroidal thickness (SFChT) and choroidal volume of both eyes were compared. Results. 19 unilateral OIS patients were included in this study. The mean SFChT of OIS eyes was significantly lower than that of fellow eyes (OIS eyes: 208.89 ± 82.62 µm and fellow eyes: 265.31 ± 82.77 µm, P < 0.001). The choroidal volume of OIS eyes was significantly smaller than that of fellow eyes (OIS eyes: 0.16 ± 0.05 mm(3) and fellow eyes: 0.21 ± 0.05 mm(3), P < 0.001). Conclusion. The choroidal thickness and volume of OIS eyes were smaller than those of unaffected fellow eyes. Decreased choroidal circulation caused by carotid artery stenosis might affect the discordance of choroidal thickness and choroidal volume.

Leaves of Persimmon (Diospyros kaki Thunb.) Ameliorate N-Methyl-N-nitrosourea (MNU)-Induced Retinal Degeneration in Mice.

The purpose of the study was to investigate the protective effects of the ethanol extract of Diospyros kaki (EEDK) persimmon leaves to study N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in mice. EEDK was orally administered after MNU injection. Retinal layer thicknesses were significantly increased in the EEDK-treated group compared with the MNU-treated group. The outer nuclear layer was preserved in the retinas of EEDK-treated mice. Moreover, EEDK treatment reduced the MNU-dependent up-regulation of glial fibrillary acidic protein (GFAP) and nestin expression in Müller and astrocyte cells. EEDK treatment also inhibited MNU-dependent down-regulation of rhodopsin expression. Quercetin exposure significantly attenuated the negative effects of H2O2 in R28 cells, suggesting that quercetin can act in an antioxidative capacity. Thus, EEDK may be considered as an agent for treating or preventing degenerative retinal diseases, such as retinitis pigmentosa and age-related macular degeneration.

The association between choroidal thickness variations and response to intravitreal bevacizumab in central serous chorioretinopathy.

PURPOSE: To analyze differences in the subfoveal choroidal thickness (SFChT) between bevacizumab responders (BevRs) and nonresponders (BevNRs) in patients with idiopathic central serous chorioretinopathy (CSC). METHODS: The medical records of 30 unilateral chronic CSC patients who were treated with intravitreal bevacizumab (IVB) as a first line treatment were reviewed. Patients were categorized as BevNRs when CSC did not completely resolve after a minimum of 3 IVB treatments. Enhanced depth imaging-optical coherence tomography was used and SFChT was measured before and after treatment. Choroidal hyperpermeability was also evaluated using indocyanine angiography. RESULTS: Twenty and 10 eyes were classified as BevRs or BevNRs, respectively. The mean number of IVB treatments was 2.22 ± 0.89 in BevRs, and 4.80 ± 1.03 in BevNRs. Compared with BevNRs, BevRs demonstrated significantly greater pretreatment SFChT (441.25 ± 88.09 vs. 364.10 ± 61.97 µm); SFChT reduction following IVB was significantly greater in BevRs than BevNRs. SFChT in the unaffected eyes was also greater in BevRs than BevNRs. Choroidal hyperpermeability was detected less frequently in BevNRs (hypofluorescence on late-phase, 0.0% and 33.3% in BevNRs and BevRs, respectively; p= 0.049). CONCLUSIONS: Compared with CSC eyes that did not respond well to IVB, BevRs demonstrated significantly thicker SFChT at baseline, greater reduction in SFChT after IVB treatment, and hyperfluorescence on late-phase indocyanine green angiography. We recommend IVB injection as the first-line therapy for CSC eyes with relatively high SFChT and hyperfluorescence on late-phase indocyanine green angiography.

Response to Comment on "Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA".

Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand.

Baicalin attenuates laser-induced choroidal neovascularization.

PURPOSE: To determine whether intravitreally-injected baicalin inhibits the growth of choroidal neovascularization (CNV) experimentally induced via laser photocoagulation through analysis of angiogenic factors. MATERIALS AND METHODS: Six CNVs were induced in the left eyes of 8-week-old male Brown Norway rats. Immediately after the induction of CNV, 4 µl of baicalin solution (0.1, 1 or 5 nmol) and 4 µl of a solution containing 100 µg of bevacizumab were slowly injected into the vitreous cavity under direct observation with an operating microscope. At 14 days after CNV induction, fluorescein angiography (FA) was performed, and choroidal flat mounts were produced for quantitative assessment of CNV. The levels of the anti-angiogenic proteins vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and matrix metalloproteinase-2 (MMP-2) were determined via Western blot analysis. RESULTS: FA of bevacizumab- and baicalin-treated rats showed significantly reduced CNV and leakage from the CNV lesions compared to control rats at day 14. Choroidal flat mounts revealed that baicalin inhibited the growth of CNV lesions in a dose-dependent manner. Western blot analysis demonstrated that baicalin significantly attenuated the up-regulation of VEGF, PDGF and MMP-2. CONCLUSION: Baicalin suppressed laser-induced CNV formation in rats. These results suggest that baicalin should be considered as a candidate drug for treating exudative age-related macular degeneration.

Cytokine levels of the aqueous humour in central serous chorioretinopathy.

BACKGROUND: This study aimed to investigate the levels of aqueous humour cytokines in the eyes of patients with central serous chorioretinopathy (CSC), who received an intravitreal injection of bevacizumab, and to evaluate the differences in cytokine levels between bevacizumab responders and non-responders. METHODS: Aqueous humour samples were collected from 39 eyes of 39 patients with CSC and 25 eyes of 25 control patients undergoing cataract surgery and were analysed using Multiplex bead assays. We compared the cytokine levels in patients with CSC (responders and non-responders) and control subjects. After intravitreal injections of bevacizumab in patients with CSC, we evaluated the changes in visual acuity and optical coherence tomographic parameters. RESULTS: Significantly increased levels of interferon gamma-induced protein (IP-10) were detected in the aqueous humour of eyes from patients with CSC compared to controls (p = 0.036). Vascular endothelial growth factor (VEGF) levels were significantly higher in bevacizumab responders than in non-responders (p = 0.021). The concentrations of the other cytokines investigated did not differ significantly between patients with CSC and control subjects or between responders and non-responders. CONCLUSION: The results of the present study suggest that IP-10 and VEGF may contribute to the pathogenesis of CSC. It is clear that intravitreal bevacizumab was helpful in some of the patients with CSC, who showed higher levels of VEGF in the aqueous humour. However, the true effect and the rationale of anti-VEGF injection treatment in patients with CSC remain to be elucidated. Our results suggest that patients with CSC may be more susceptible to VEGF and/or other cytokines than the normal population.

Association of aqueous humor cytokines with the development of retinal ischemia and recurrent macular edema in retinal vein occlusion.

PURPOSE: We evaluated the association of angiogenic and inflammatory cytokine levels in the aqueous humor with development of retinal ischemia and recurrent macular edema in retinal vein occlusion (RVO) patients. METHODS: This was a retrospective cross-sectional study, and patients with RVO (n = 41) and age-matched control subjects (n = 25) were included. The concentrations of angiogenic and inflammatory cytokines, including VEGF, PDGF-AA, IL-1a, IL-6, IL-8, MCP-1, TNF-α, and IP-10, in the aqueous humor were measured before intravitreal injection of bevacizumab using suspension array technology. After retinal hemorrhage disappeared, fluorescein angiography (FA) images were obtained. Based on FA data, RVO patients were divided into a nonischemic group and an ischemic group. We investigated the presence of recurrent macular edema using optical coherent tomography (OCT) during the follow-up period. We compared the levels of cytokines between RVO patients and control subjects, between nonischemic and ischemic groups, and between patients with and without recurrent macular edema. RESULTS: The levels of VEGF, PDGF-AA, IL-1a, IL-6, IL-8, MCP-1, TNF-α, and IP-10 in the aqueous humor were higher in the RVO group than in the control group. The levels of IL-8, PDFGF-AA, TNF-α, and VEGF in the aqueous humor were significantly higher in the ischemic RVO group than in the nonischemic RVO group. We did not observe any association between cytokine levels and recurrent macular edema. CONCLUSIONS: Angiogenic and inflammatory cytokines were overexpressed in RVO patients. Additionally, increased levels of IL-8, PDFGF-AA, TNF-α, and VEGF in the aqueous humor at the onset of RVO were associated with the development of future retinal ischemia in RVO patients.

Effect of combined cataract surgery and ranibizumab injection in postoperative macular edema in nonproliferative diabetic retinopathy.

PURPOSE: To evaluate whether intravitreal ranibizumab injection at cataract surgery prevents postoperative diabetic macular edema (PME) in patients with stable diabetic retinopathy without significant macular edema. METHODS: Eighty patients with cataract, stable diabetic retinopathy, and no significant macular edema were randomized to a sham group (cataract surgery only) or a group undergoing cataract surgery plus intraoperative ranibizumab injection. Best-corrected visual acuities, central subfield thickness, and total macular volume were assessed at baseline and 1 week, 1, 3, and 6 months postoperatively by spectral domain optical coherence tomography. Clinically meaningful PME (central subfield thickness increase >60 µm relative to baseline) was computed. RESULTS: The groups did not differ in baseline best-corrected visual acuity, central subfield thickness, and total macular volume. Compared with the ranibizumab injection group, the sham group had significantly larger central subfield thickness increases relative to baseline at 1 week and 1 month; larger total macular volume increases at all time points (P = 0.012, P = 0.005, P < 0.001, P < 0.001, P = 0.005, P = 0.017, respectively); higher PME frequency at 1 month (P = 0.019); and poorer best-corrected visual acuity improvement from baseline to 6 months after surgery (P = 0.046). CONCLUSION: In patients with stable diabetic retinopathy without significant macular edema, intravitreal ranibizumab injection at cataract surgery may prevent the postoperative worsening of macular edema and may improve the final visual outcome without affecting safety.

Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA.

Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo- and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature's repertoire of cellulose digestion paradigms remain only partially discovered and understood.

Structural recovery of the detached macula after retinal detachment repair as assessed by optical coherence tomography.

PURPOSE: To investigate correlations between preoperative and postoperative foveal microstructures in patients with macula-off rhegmatogenous retinal detachment (RRD). METHODS: We reviewed the records of 31 eyes from 31 patients with macula-off RRD who had undergone successful re-attachment surgery. We analyzed data obtained from complete ophthalmologic examinations and optical coherence tomography (OCT) before and 9 to 12 months after surgery. All postoperative OCT measurements were taken with spectral-domain OCT, but a subset of preoperative OCT measurements were taken with time-domain OCT. RESULTS: The mean duration of macular detachment was 15.5 ± 15.2 days, and mean preoperative best-corrected visual acuity (BCVA, logarithm of the minimum angle of resolution) was 1.03 ± 0.68. Preoperative visual acuity was correlated with retinal detachment height (p < 0.001) and the existence of intraretinal separation (IRS) along with outer layer undulation (OLU) (p = 0.022), but not with macula-off duration. The final BCVA was significantly correlated with integrity of the junction between the photoreceptor inner and outer segments (IS/OS) combined with the continuity of external limiting membrane (ELM) (p = 0.025). The presence of IRS and OLU on a detached macula were highly correlated with the final postoperative integrity of the IS/OS junction and the ELM (p = 0.017). CONCLUSIONS: Eyes preoperatively exhibiting IRS and OLU showed a higher incidence of disruption to the photoreceptor IS/OS junction and the ELM at final follow-up. Such a close correlation between preoperative and postoperative structural changes may explain why ultimate visual recovery in such eyes is poor.

Changes of choroidal thickness, intraocular pressure and other optical coherence tomographic parameters after haemodialysis.

BACKGROUND: The aim was to evaluate changes in choroidal thickness, intraocular pressure (IOP) and other parameters of optical coherence tomography, such as central foveal thickness (CFT), macular volume, peripapillary retinal nerve fibre layer (RNFL) thickness after haemodialysis (HD). METHODS: Thirty-four eyes of 34 chronic renal failure patients (9 eyes with diabetic patients) who underwent haemodialysis in the Dialysis Unit of Gangneung Asan Hospital were included. CFT, macular volume, peripapillary RNFL and choroidal thickness were measured before and after haemodialysis by optical coherence tomography (OCT, Spectralis; Heidelberg Engineering). Changes in the IOP were evaluated with Goldmann applanation tonometry before and after haemodialysis. RESULTS: The average choroidal thickness decreased significantly, from 233.1 ± 77.5 µm to 219.1 ± 76.8 µm (paired t-test, p < 0.001). This change correlated with the amount of body weight lost (Pearson's correlation coefficient = 0.348, p = 0.044). The mean IOP decreased from 15.1 ± 2.6 mmHg to 13.9 ± 2.2 mmHg after haemodialysis (paired t-test, p = 0.03). The change in macular volume and the small decrease in CFT (214.0 ± 21.0 µm to 213.8 ± 21.8 µm) were not significant. The measured overall change in RNFL thickness from 93.6 ± 15.5 µm to 94.7 ± 14.6 µm was not significant. CONCLUSIONS: Haemodialysis can affect various ocular parameters, particularly choroidal thickness and IOP.

Enzymatic synthesis of piceid glucosides using maltosyltransferase from Caldicellulosiruptor bescii DSM 6725.

Piceid is widely used in food, cosmetics, and pharmaceuticals because of its therapeutic benefits. However, the use of piceid as a drug is limited because of its low solubility. To increase solubility, we synthesized piceid glucosides using maltosyltransferase from Caldicellulosiruptor bescii. The MTase gene was cloned and expressed in Escherichia coli. The enzyme had a unique transfer specificity to the transfer of maltosyl units. Four piceid transglycosylation products were present and identified by thin-layer chromatography and recycling preparative high-performance liquid chromatography. The major product was purified by C(18) and gel filtration chromatography, and its molecular structure was determined using nuclear magnetic resonance spectroscopy to be α-D-maltosyl-(1→4)-piceid. The solubility of maltosyl piceid was 8.54 × 10(3) and 1.86 × 10(3) times those of natural resveratrol and piceid, respectively, suggesting that the transglycosylation greatly increased the water solubility. This suggests that dietary intake of this compound can enhance the bioavailability of resveratrol in the human body.

Recurrent submacular hemorrhage in patients with neovascular age-related macular degeneration.

PURPOSE: To describe the incidence, risk factors for, and long-term visual outcomes of recurrent submacular hemorrhage in the context of age-related macular degeneration. METHODS: Medical records of patients with neovascular age-related macular degeneration with or without polypoidal choroidal vasculopathy showing submacular hemorrhage at their first visit to our institution were reviewed. The required minimum follow-up period was 24 months, and any newly developed submacular hemorrhage larger than 1 disk area after near-complete resolution of initial hemorrhage was defined as recurrence. RESULTS: A total of 47 eyes of 47 patients were eligible for inclusion. Twenty-four patients showed recurrent submacular hemorrhage during the follow-up period (Group I). Patients without recurrent submacular hemorrhage were included in Group II. The time to recurrent submacular hemorrhage in Group I patients was 21.4 ± 9.2 months. Polypoidal choroidal vasculopathy was present in 50% of Group I patients (n = 12) and 13% of Group II patients (n = 3) (P = 0.025). Intravitreal anti-vascular endothelial growth factor injection was performed during the follow-up period in 70.8% of Group I patients (n = 17) and 95.7% of Group II patients (n = 22) (P = 0.048). Visual acuity change during the follow-up period did not significantly differ between the two groups. CONCLUSION: In patients with neovascular age-related macular degeneration presenting with submacular hemorrhage at their first visit, the incidence of recurrent submacular hemorrhage was 51.1% in our retrospective long-term follow-up study. The presence of polypoidal choroidal vasculopathy was associated with an increased risk of recurrent submacular hemorrhage. Use of anti-vascular endothelial growth factor agents was correlated with a reduced risk of such hemorrhage. Visual acuity was stably maintained over 2 years regardless of hemorrhage recurrence.