Plan complexity metrics for head and neck VMAT competition plans.

Previous plan competitions have largely focused on dose metric assessments. However, whether the submitted plans were realistic and reasonable from a quality assurance (QA) perspective remains unclear. This study aimed to investigate the relationship between aperture-based plan complexity metrics (PCM) in volumetric modulated arc therapy (VMAT) competition plans and clinical treatment plans verified through patient-specific QA (PSQA). In addition, the association of PCMs with plan quality was examined. A head and neck (HN) plan competition was held for Japanese institutions from June 2019 to July 2019, in which 210 competition plans were submitted. Dose distribution quality was quantified based on dose-volume histogram (DVH) metrics by calculating the dose distribution plan score (DDPS). Differences in PCMs between the two VMAT treatment plan groups (HN plan competitions held in Japan and clinically accepted HN VMAT plans through PSQA) were investigated. The mean (± standard deviation) DDPS for the 98 HN competition plans was 158.5 ± 20.6 (maximum DDPS: 200). DDPS showed a weak correlation with PCMs with a maximum r of 0.45 for monitor unit (MU); its correlation with some PCMs was "very weak." Significant differences were found in some PCMs between plans with the highest 20% DDPSs and the remaining plans. The clinical VMAT and competition plans revealed similar distributions for some PCMs. Deviations in PCMs for the two groups were comparable, indicating considerable variability among planners regarding planning skills. The plan complexity for HN VMAT competition plans increased for high-quality plans, as shown by the dose distribution. Direct comparison of PCMs between competition plans and clinically accepted plans showed that the submitted HN VMAT competition plans were realistic and reasonable from the QA perspective. This evaluation may provide a set of criteria for evaluating plan quality in plan competitions.

Clinicopathological findings in refractory diabetic macular edema: A case report.

The present study describes the case of a patient with refractory diabetic cystoid macular edema who underwent vitrectomy with en bloc removal of the cystoid lesion component. The current study also performed histopathological and immunohistochemical analyses of the cystoid lesion component to assess fibrin/fibrinogen and advanced glycation end-products (AGEs) immunoreactivity. A 69-year-old Japanese man presented with visual loss in the left eye due to residual cystoid macular edema (CME) refractory to anti-vascular endothelial growth factor therapy. Best-corrected visual acuity was 1.2 in the right eye (OD) and 0.5 in the left eye (OS). Fundus examination showed dot hemorrhages and hard exudates in the peri-macular region with pan-retinal photocoagulation scars in both eye. Swept-source optical coherence tomography revealed CME with slight hyperreflectivity in the cyst OS. A total of 3 months after the initial visit, pars plana vitrectomy was performed, and the translucent solidified component within the cystoid lesion was isolated. Histopathologically, the excised component was elliptical in shape, measuring 0.7x0.4 mm and exhibited homogeneous eosinophilic material without cellular components. No membranous structure was observed surrounding the component. Immunohistochemistry demonstrated that the tissue was positive for fibrin/fibrinogen and weakly positive for AGEs, but was negative for glial fibrillary acidic protein, type 1 collagen and receptor for AGEs. To the best of our knowledge, the present case report is the first to histopathologically examine the contents of refractory CME, and to immunohistochemically demonstrate that fibrin in diabetic CME may be post-translationally modified by AGEs. These results suggested that fibrin in CME may escape degradation by plasmin due to post-translational modifications.

Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.

PURPOSE: Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. METHODS: We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. RESULTS: The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. CONCLUSIONS: Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

Phosphorylation of αB-Crystallin Involves Interleukin-1ß-Mediated Intracellular Retention in Retinal Müller Cells: A New Mechanism Underlying Fibrovascular Membrane Formation.

Purpose: Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of the key elements. The phosphorylation of αB-crystallin/CRYAB modulates its molecular dynamics and chaperone activity, and attenuates αB-crystallin secretion via exosomes. In this study, we investigated the effect of phosphorylated αB-crystallin in retinal Müller cells on diabetic mimicking conditions, including interleukin (IL)-1ß stimuli. Methods: Human retinal Müller cells (MIO-M1) were used to examine gene and protein expressions with real-time quantitative PCR, enzyme linked immunosorbent assay (ELISA), and immunoblot analyses. Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling staining. Retinal tissues isolated from the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic animal model with obesity, and fibrovascular membranes from patients with PDR were examined by double-staining immunofluorescence. Results: CRYAB mRNA was downregulated in MIO-M1 cells with the addition of 10 ng/mL IL-1ß; however, intracellular αB-crystallin protein levels were maintained. The αB-crystallin serine 59 (Ser59) residue was phosphorylated with IL-1ß application in MIO-M1 cells. Cell apoptosis in MIO-M1 cells was induced by CRYAB knockdown. Immunoreactivity for Ser59-phosphorylated αB-crystallin and glial fibrillary acidic protein was colocalized in glial cells of SDT fatty rats and fibrovascular membranes. Conclusions: The Ser59 phosphorylation of αB-crystallin was modulated by IL-1ß in Müller cells under diabetic mimicking inflammatory conditions, suggesting that αB-crystallin contributes to the pathogenesis of PDR through an anti-apoptotic effect.

Effects of Mirogabalin on Hyperalgesia and Chronic Ocular Pain in Tear-Deficient Dry-Eye Rats.

Purpose: Patients with dry eye disease (DED) sometimes complain of ocular pain. DED-related ocular pain has many similarities with neuropathic pain. Mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, is approved for treating neuropathic pain in Japan. This study aimed to investigate the effect of mirogabalin on hyperalgesia and chronic ocular pain in a rat DED model. Methods: DED was induced in female Sprague Dawley rats by unilaterally excising the external lacrimal gland (ELG) and Harderian gland (HG). After 4 weeks of ELG and HG removal, tear production (pH threads) and corneal epithelial damage (fluorescein staining) were evaluated. Corneal hyperalgesia and chronic pain were analyzed, respectively, by measuring capsaicin-induced eye-wiping behavior and c-Fos expression in the trigeminal nucleus. Mirogabalin (10 or 3 mg/kg) was evaluated for effects on DED-induced hyperalgesia and chronic ocular pain. Results: Tear production was significantly lower in DED-induced eyes than in control eyes. Corneal damage was significantly higher in DED eyes than in control eyes. Hyperalgesia and chronic ocular pain were detected 4 weeks after ELG and HG removal. Five days of mirogabalin administration significantly suppressed capsaicin-induced eye-wiping behavior, which indicated the suppression of ocular hyperalgesia. Administration of 10 mg/kg mirogabalin significantly reduced c-Fos expression in the trigeminal nucleus, which indicated the amelioration of chronic ocular pain. Conclusions: Mirogabalin suppressed DED-induced hyperalgesia and chronic ocular pain in a rat DED model. Our findings suggested that mirogabalin might effectively alleviate chronic ocular pain in patients with DED.

Relationship between Brachial-Ankle Pulse Wave Velocity and Fundus Arteriolar Area Calculated Using a Deep-Learning Algorithm.

PURPOSE: Retinal vessels reflect alterations related to hypertension and arteriosclerosis in the physical status. Previously, we had reported a deep-learning algorithm for automatically detecting retinal vessels and measuring the total retinal vascular area in fundus photographs (VAFP). Herein, we investigated the relationship between VAFP and brachial-ankle pulse wave velocity (baPWV), which is the gold standard for arterial stiffness assessment in clinical practice. METHODS: Retinal photographs (n = 696) obtained from 372 individuals who visited the Keijinkai Maruyama Clinic for regular health checkups were used to analyze VAFP. Additionally, the baPWV was measured for each patient. Automatic retinal-vessel segmentation was performed using our deep-learning algorithm, and the total arteriolar area (AA) and total venular area (VA) were measured. Correlations between baPWV and several parameters, including AA and VA, were assessed. RESULTS: The baPWV was negatively correlated with AA (R = -0.40, n = 696, P < 2.2e-16) and VA (R = -0.36, n = 696, P < 2.2e-16). Independent variables (AA, sex, age, and systolic blood pressure) selected using the stepwise method showed a significant correlation with baPWV. The estimated baPWV, calculated using a regression equation with variables including AA, showed a better correlation with the measured baPWV (R = 0.70, n = 696, P < 2.2e-16) than the estimated value without AA (R = 0.68, n = 696, P < 2.2e-16). CONCLUSIONS: AA and VA were significantly correlated with baPWV. Moreover, baPWV estimated using AA correlated well with the actual baPWV. VAFP may serve as an alternative biomarker for evaluating systemic arterial stiffness.

Placental growth factor stabilizes VEGF receptor-2 protein in retinal pigment epithelial cells by downregulating glycogen synthase kinase 3 activity.

Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family of proteins that participate in angiogenesis and vasculogenesis. Anti-VEGF therapy has become the standard treatment for ocular angiogenic disorders in ophthalmological practice. However, there is emerging evidence that anti-VEGF treatment may increase the risk of atrophy of the retinal pigment epithelium (RPE), which is important for the homeostasis of retinal tissue. Whereas the cytoprotective role of VEGF family molecules, particularly that of VEGF A (VEGFA) through its receptor VEGF receptor-2 (VEGFR-2), has been recognized, the physiological role of PlGF in the retina is still unknown. In this study, we explored the role of PlGF in the RPE using PlGF-knockdown RPE cells generated by retrovirus-based PlGF-shRNA transduction. We show that VEGFA reduced apoptosis induced by serum starvation in RPE cells, whereas the antiapoptotic effect of VEGFA was abrogated by VEGFR-2 knockdown. Furthermore, PlGF knockdown increased serum starvation-induced cell apoptosis and unexpectedly reduced the protein level of VEGFR-2 in the RPE. The antiapoptotic effect of VEGFA was also diminished in PlGF-knockdown RPE cells. In addition, we found that glycogen synthase kinase 3 activity was involved in proteasomal degradation of VEGFR-2 in RPE cells and inactivated by PlGF via AKT phosphorylation. Overall, the present data demonstrate that PlGF is crucial for RPE cell viability and that PlGF supports VEGFA/VEGFR-2 signaling by stabilizing the VEGFR-2 protein levels through glycogen synthase kinase 3 inactivation.

Serum advanced glycation end-products and αB-crystallin in diabetic retinopathy patients.

αB-crystallin, one of the small heat shock proteins, which is also known as HSPB5, has cytoprotective effects under inflammatory conditions. Advanced glycation end-products (AGE) are produced through non-enzymatic glycation under conditions of hyperglycemia and they contribute to angiogenesis and inflammation. The aim of this study was to examine the levels of serum αB-crystallin and AGE concentrations in blood samples collected from proliferative diabetic retinopathy (PDR) patients. Blood samples were collected from seven diabetic patients with PDR and eight patients without diabetes mellitus who underwent vitrectomy due to PDR and idiopathic macular diseases, respectively, in a single center. The levels of serum αB-crystallin and AGE were measured by ELISA and correlations were assessed statistically. The serum levels (mean ± SEM) of AGE were significantly higher in PDR patients (28.41±0.46 µg/ml) than in patients with non-diabetic macular diseases (25.76±0.60 µg/ml; P=0.015), whereas there was no significant difference in serum αB-crystallin levels. There was one patient with an extremely high level of αB-crystallin, who was treated with systemic corticosteroid due to chronic autoimmune inflammatory diseases. The current prospective study showed that serum AGE levels were significantly higher in PDR patients; however, no correlations between serum AGE and αB-crystallin levels were identified.

Downregulation of AlphaB-crystallin in Retinal Pigment Epithelial Cells Exposed to Diabetes-related Stimuli In Vivo and In Vitro.

BACKGROUND/AIM: AlphaB-crystallin plays a pivotal role in many diseases. However, the involvement of alphaB-crystallin in retinal pigment epithelial (RPE) cells with diabetes stimuli remains unknown. The aim of this study is to examine the alterations of RPE cells and alphaB-crystallin expression in diabetic models in vivo and in vitro. MATERIALS AND METHODS: Diabetic conditions in mice were induced by streptozotocin (STZ). The thickness of the RPE/choroid complex was measured by optical coherence tomography (OCT). Periodic acid-Schiff (PAS) staining was used to investigate the choriocapillaris in histological sections of murine eyeballs and oxidative stress was evaluated using immunofluorescence with anti-4-hydroxynonenal (HNE) antibody. AlphaB-crystallin expression was examined in the RPE/choroid complex using ELISA. Real-Time PCR was performed to evaluate the alphaB-crystallin expression in cultured human RPE cells with high glucose or following advanced glycation end-products (AGE) stimulation. RESULTS: In diabetic mice, OCT-based RPE/choroidal layers were thickened 2 months after STZ stimulation, where PAS-positive dilated choriocapillaris was noted. Immunoreactivity of 4-HNE was strongly observed in the RPE layer, from which a significant number of RPE cells was lost. Meanwhile, alphaB-crystallin expression in 2-month STZ mice was significantly lower compared to controls. In accordance with these results, in vitro data showed that the alphaB-crystallin expression was also significantly lower in RPE cells with high glucose or following AGE stimulation compared to untreated cells. CONCLUSION: In both types of diabetic models the expression of alphaB-crystallin was found to be downregulated in RPE cells and was associated with increased levels of oxidative stress.

ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

Objective: Acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently modulates cellular function. Our previous study demonstrated that acrolein induces glial cell migration, a pathological hallmark of diabetic retinopathy; however, the detailed cellular mechanism remains unclear. The purpose of this study was to investigate the role of acrolein in retinal glial cell migration by focusing on rho-associated coiled-coil-containing protein kinases (ROCKs). Methods: Immunofluorescence staining for ROCK isoforms was performed using sections of fibrovascular tissue obtained from the eyes of patients with proliferative diabetic retinopathy (PDR). Rat retinal Müller glial cell line, TR-MUL5, was stimulated with acrolein and the levels of ROCK1 were evaluated using real-time PCR and western blotting. Phosphorylation of the myosin-binding subunit of myosin light chain phosphatase [myosin phosphatase target subunit 1, (MYPT1)] and myosin light chain 2 (MLC2) was assessed. The cell migration rate of TR-MUL5 cells exposed to acrolein and/or ripasudil, a non-selective ROCK inhibitor, was measured using the Oris cell migration assay. Results: ROCK isoforms, ROCK1 and ROCK2, were positively stained in the cytosol of glial cells in fibrovascular tissues. In TR-MUL5 cells, the mRNA expression level of Rock1, but not Rock2, was increased following acrolein stimulation. In line with the PCR data, western blotting showed increase in ROCK1 and cleaved ROCK1 protein in TR-MUL5 cells stimulated with acrolein. N-acetylcysteine (NAC) suppressed acrolein-associated Rock1 upregulation in TR-MUL5 cells. Acrolein augmented the phosphorylation of MYPT1 and MLC2 and increased the cell migration rate of TR-MUL5 cells, both of which were abrogated by ripasudil. Conclusions: Our study demonstrated that ROCK1 mediates the migration of retinal glial cells promoted by the unsaturated aldehyde acrolein.

A Deep Learning Architecture for Vascular Area Measurement in Fundus Images.

Purpose: To develop a novel evaluation system for retinal vessel alterations caused by hypertension using a deep learning algorithm. Design: Retrospective study. Participants: Fundus photographs (n = 10 571) of health-check participants (n = 5598). Methods: The participants were analyzed using a fully automatic architecture assisted by a deep learning system, and the total area of retinal arterioles and venules was assessed separately. The retinal vessels were extracted automatically from each photograph and categorized as arterioles or venules. Subsequently, the total arteriolar area (AA) and total venular area (VA) were measured. The correlations among AA, VA, age, systolic blood pressure (SBP), and diastolic blood pressure were analyzed. Six ophthalmologists manually evaluated the arteriovenous ratio (AVR) in fundus images (n = 102), and the correlation between the SBP and AVR was evaluated manually. Main Outcome Measures: Total arteriolar area and VA. Results: The deep learning algorithm demonstrated favorable properties of vessel segmentation and arteriovenous classification, comparable with pre-existing techniques. Using the algorithm, a significant positive correlation was found between AA and VA. Both AA and VA demonstrated negative correlations with age and blood pressure. Furthermore, the SBP showed a higher negative correlation with AA measured by the algorithm than with AVR. Conclusions: The current data demonstrated that the retinal vascular area measured with the deep learning system could be a novel index of hypertension-related vascular changes.

Involvement of Müller Glial Autoinduction of TGF-ß in Diabetic Fibrovascular Proliferation Via Glial-Mesenchymal Transition.

Purpose: Müller glial-mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-ß-SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-ß in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production. Methods: Surgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-ß downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-ß signal activation. Results: Müller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-ß receptors. In vitro administration of TGF-ß1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-ß1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-ß1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues. Conclusions: This study underscored the significance of Müller glial autoinduction of TGF-ß as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-ß-driven GMT and VEGF-A-driven angiogenesis.

Pathological Role of Unsaturated Aldehyde Acrolein in Diabetic Retinopathy.

With increasing prevalence of diabetes and a progressively aging society, diabetic retinopathy is emerging as one of the global leading causes of blindness. Recent studies have shown that vascular endothelial growth factor (VEGF) plays a central role in the pathogenesis of diabetic retinopathy and anti-VEGF agents have become the first-line therapy for the vision-threatening disease. However, recent studies have also demonstrated that diabetic retinopathy is a multifactorial disease and that VEGF-independent mechanism(s) also underlie much of the pathological changes in diabetic retinopathy. Acrolein is a highly reactive unsaturated aldehyde and is implicated in protein dysfunction. As acrolein is common in air pollutants, previous studies have focused on it as an exogenous causative factor, for instance, in the development of respiratory diseases. However, it has been discovered that acrolein is also endogenously produced and induces cell toxicity and oxidative stress in the body. In addition, accumulating evidence suggests that acrolein and/or acrolein-conjugated proteins are associated with the molecular mechanisms in diabetic retinopathy. This review summarizes the pathological roles and mechanisms of endogenous acrolein production in the pathogenesis of diabetic retinopathy.

Cytoprotective Effect of Astaxanthin in a Model of Normal Intraocular Pressure Glaucoma.

Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies. Lowering intraocular pressure is currently the only way to treat glaucoma, but it is often insufficient to inhibit the progression of the disease. Glaucoma is a multifactorial disease, and the involvement of oxidative stress has recently received much attention. In the present study, we investigated the cytoprotective effect of astaxanthin (AST) on RGC degeneration using a normal-tension glaucoma (NTG) mouse model, which lacks the glutamate/aspartate transporter (Glast) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure. Three-week-old Glast± mice were given intraperitoneal injections of AST at 10, 30, or 60 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. Five weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. We also used a retrograde labeling technique to quantify the number of RGCs. Additionally, the phosphorylated (p) IκB/total IκB ratio and the 4-hydroxynonenal (HNE) were measured in retinal tissues. The number of RGCs in Glast± mice was significantly decreased compared with that in control mice. RGC loss was suppressed by the administration of AST at 60 mg/kg/day, compared with vehicle alone. Following AST administration, the concentration of 4-HNE in the retina was also suppressed, but the pIκB/IκB ratio did not change. Our study revealed that the antioxidative stress effects of AST inhibit RGC degeneration in the retina and may be useful in the treatment of NTG.

Diabetic Cataract in Spontaneously Diabetic Torii Fatty Rats.

Spontaneously Diabetic Torii (SDT) fatty rat is a novel animal model of type 2 diabetes with obesity. SDT fatty rats develop hyperglycemia, dyslipidemia, and other diabetic complications including ocular disorders; however, diabetic cataract formation in SDT fatty rats has not been fully investigated. The aim of the current study was to investigate the characteristics of cataract in the SDT fatty rats. The mean body weight of SDT fatty rats is larger than that of age-matched Sprague-Dawley (SD) rats and control animals until 8 weeks of age, and thereafter the growing speed decreased until the end of observation at 16 weeks of age. Blood glucose levels in SDT fatty rats were significantly higher than those in SD rats throughout the observational period. Slit-lamp examination revealed that no rats showed cataract formation at 5 weeks of age; however, SDT fatty rats gradually developed cortical cataract and posterior subcapsular cataract, both of which are the common types of cataract in patients with type 2 diabetes. The levels of glucose, sorbitol, and fructose were higher in the lens tissues of SDT fatty rats in comparison with that of SD rats. Furthermore, the level of 4-hydroxynonenal (4-HNE) was higher in the lens of SDT fatty rats than in that of SD rats. By contrast, total glutathione (GSH) concentration was lower in the lens of SDT fatty rats than in that of SD rats. The present study demonstrated that the cataractogenesis in SDT fatty rats resembled human diabetic cataract formation, indicating that SDT fatty rats serve as a potential animal model in researches on human cataract associated with type 2 diabetes and obesity.

Blockade of Platelet-Derived Growth Factor Signaling Inhibits Choroidal Neovascularization and Subretinal Fibrosis in Mice.

Neovascular age related macular degeneration (nAMD) leads to severe vision loss worldwide and is characterized by the formation of choroidal neovascularization (CNV) and fibrosis. In the current study, we aimed to investigate the effect of blockade for platelet derived growth factor receptor-ß (PDGFR-ß) on the formation of choroidal neovascularization and fibrosis in the laser-induced CNV model in mice. Firstly, the presence of PDGFR-ß in CNV lesions were confirmed. Intravitreal injection of PDGFR-ß neutralizing antibody significantly reduced the size of CNV and subretinal fibrosis. Additionally, subretinal hyperreflective material (SHRM), a landmark feature on OCT as a risk factor for subretinal fibrosis formation in nAMD patients was also suppressed by PDGFR-ß blockade. Furthermore, pericytes were abundantly recruited to the CNV lesions during CNV formation, however, blockade of PDGFR-ß significantly reduced pericyte recruitment. In addition, PDGF-BB stimulation increased the migration of the rat retinal pericyte cell line, R-rPCT1, which was abrogated by the neutralization of PDGFR-ß. These results indicate that blockade of PDGFR-ß attenuates laser-induced CNV and fibrosis through the inhibition of pericyte migration.

Regulation of Spermine Oxidase through Hypoxia-Inducible Factor-1α Signaling in Retinal Glial Cells under Hypoxic Conditions.

Purpose: Acrolein, a highly reactive unsaturated aldehyde, is known to facilitate glial cell migration, one of the pathological hallmarks in diabetic retinopathy. However, cellular mechanisms of acrolein generation in retinal glial cells remains elusive. In the present study, we investigated the role and regulation of spermine oxidase (SMOX), one of the enzymes related to acrolein generation, in retinal glial cells under hypoxic condition. Methods: Immunofluorescence staining for SMOX was performed using sections of fibrovascular tissues obtained from patients with proliferative diabetic retinopathy. Expression levels of polyamine oxidation enzymes including SMOX were analyzed in rat retinal Müller cell line 5 (TR-MUL5) cells under either normoxic or hypoxic conditions. The transcriptional activity of Smox in TR-MUL5 cells was evaluated using the luciferase assay. Levels of acrolein-conjugated protein, Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), and hydrogen peroxide were measured. Results: SMOX was localized in glial cells in fibrovascular tissues. Hypoxia induced SMOX production in TR-MUL5 cells, which was suppressed by silencing of hypoxia-inducible factor-1α (Hif1a), but not Hif2a. Transcriptional activity of Smox was regulated through HIF-1 binding to hypoxia response elements 2, 3, and 4 sites in the promoter region of Smox. Generation of FDP-Lys and hydrogen peroxide increased in TR-MUL5 cells under hypoxic condition, which was abrogated by SMOX inhibitor MDL72527. Conclusions: The current data demonstrated that hypoxia regulates production of SMOX, which plays a role in the generation of oxidative stress inducers, through HIF-1α signaling in Müller glial cells under hypoxic condition.

Glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced Müller glial galectin-1 expression via HIF-1α destabilization.

Galectin-1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin-1ß-driven inflammatory pathway for galectin-1 expression in vitro and in vivo. Here, we show glucocorticoid-mediated inhibitory mechanism against hypoxia-inducible factor (HIF)-1α-involved galectin-1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia-induced increases in galectin-1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia-stimulated cells decreased HIF-1α protein, but not mRNA, together with its DNA-binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co-immunoprecipitation revealed that glucocorticoid-transactivated TSC22D3 interacted with HIF-1α, leading to degradation of hypoxia-stabilized HIF-1α via the ubiquitin-proteasome pathway. Silencing TSC22D3 reversed glucocorticoid-mediated ubiquitination of HIF-1α and subsequent down-regulation of HIF-1α and galectin-1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes-induced retinal HIF-1α and galectin-1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co-localization of galectin-1 and HIF-1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced retinal glial galectin-1/LGALS1 expression via HIF-1α destabilization, highlighting therapeutic implications for DR in the era of anti-vascular endothelial growth factor treatment.

Unsaturated Aldehyde Acrolein Promotes Retinal Glial Cell Migration.

Purpose: To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Methods: Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. Results: At a high concentration, acrolein (100 µM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 µM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Conclusions: Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.

A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death.

Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.