PAI-1 is a vascular cell-specific HIF-2-dependent angiogenic factor that promotes retinal neovascularization in diabetic patients.

For patients with proliferative diabetic retinopathy (PDR) who do not respond adequately to pan-retinal laser photocoagulation (PRP) or anti-vascular endothelial growth factor (VEGF) therapies, we hypothesized that vascular cells within neovascular tissue secrete autocrine/paracrine angiogenic factors that promote disease progression. To identify these factors, we performed multiplex ELISA angiogenesis arrays on aqueous fluid from PDR patients who responded inadequately to anti-VEGF therapy and/or PRP and identified plasminogen activator inhibitor-1 (PAI-1). PAI-1 expression was increased in vitreous biopsies and neovascular tissue from PDR eyes, limited to retinal vascular cells, regulated by the transcription factor hypoxia-inducible factor (HIF)-2α, and necessary and sufficient to stimulate angiogenesis. Using a pharmacologic inhibitor of HIF-2α (PT-2385) or nanoparticle-mediated RNA interference targeting Pai1, we demonstrate that the HIF-2α/PAI-1 axis is necessary for the development of retinal neovascularization in mice. These results suggest that targeting HIF-2α/PAI-1 will be an effective adjunct therapy for the treatment of PDR patients.

Progression Patterns of Myopic Traction Maculopathy in the Fellow Eye After Pars Plana Vitrectomy of the Primary Eye.

Purpose: This retrospective study investigated the patterns and risk factors of progression of myopic traction maculopathy (MTM) of fellow eyes after pars plana vitrectomy (PPV) of primary eyes. Methods: The study population comprised 153 patients with MTM in both myopic eyes who sequentially underwent PPV (2006-2021). Observation periods were from PPV of the primary eye (baseline) to PPV of the fellow (end). MTM was graded based on optical coherence tomography (OCT) images and the ATN (atrophy [A], traction [T], and neovascularization [N]) system. An increase in T grade was considered MTM progression. Results: MTM progressed in 43.8% of fellow eyes during 34.57 ± 34.08 months. The progression of fellow eyes correlated with T grade of primary eyes (P < 0.001). Risk factors for the progression of MTM in fellow eyes were primary eyes in T4-T5, age at baseline <60 years, and fellow eyes with partial posterior vitreous detachment (PVD; P < 0.001, P = 0.042, and P = 0.002, respectively). Fellow eyes in T1/T2 at baseline progressed faster compared with those in T0 (P < 0.001); the annual rate of progression to T3-T5 of the T0 (T1-T2) groups was 9.98% (24.59%). Conclusions: Risk factors for the progression of MTM in fellow eyes included PPV when relatively young, primary eye at high T grade, and partial PVD of the fellow eye. Personalized follow-up for fellow eyes should be based on the severity of MTM of both eyes.

HIF-1α and HIF-2α redundantly promote retinal neovascularization in patients with ischemic retinal disease.

Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2-specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.

Effects of intravitreal conbercept before panretinal photocoagulation on lipid exudates in diabetic macular documented by optical coherence tomography.

AIM: To evaluate the effects of intravitreal conbercept (IVC) as adjunctive treatments before panretinal photocoagulation (PRP) to decrease hyperreflective dots (HRDs) in Chinese proliferative diabetic retinopathy (PDR) patients. METHODS: Fifty-nine enrolled patients were categorized into 2 groups: single dose IVC (0.5 mg/0.05 mL) 1wk before PRP (Plus group) or PRP only (PRP group). Six months later, we measured the best corrected visual acuity (BCVA), central macula thickness (CMT) by optical coherence tomography and counted the number of HRDs in different retina layers. RESULTS: The average CMT significantly decreased in Plus group but increased in PRP group. The average BCVA in the Plus group was also significantly better than that in the PRP group. Total HRDs decreased in the Plus group but increased in PRP group significantly. IVC pre-treatment has beneficial effects on reducing HRDs forming in the inner retina layer while the PRP alone increased the HRDs in the outer retina layer. CONCLUSION: IVC is a promising adjunctive treatment to PRP in the treatment of PDR. Single dose IVC one week before PRP is suggested to improve retina blood-retina barrier, decrease lipid exudate and inhibit HRDs development in PDR.

Downregulation of circRNA DMNT3B contributes to diabetic retinal vascular dysfunction through targeting miR-20b-5p and BAMBI.

BACKGROUND: Diabetic retinopathy, a vascular complication of diabetes mellitus, is the leading cause of visual impairment and blindness. circRNAs act as competing endogenous RNA, sponging target miRNA and thus influencing mRNA expression in vascular diseases. We investigated whether and how circDNMT3B is involved in retinal vascular dysfunction under diabetic conditions. METHODS: qRT-PCR was performed to detect expression of circDNMT3B, miR-20b-5p, and BAMBI in retinal microvascular endothelial cells under diabetic conditions. Western blot, Cell Counting Kit-8, Transwell, Matrigel tube formation, and retinal trypsin digestion assays were conducted to explore the roles of circDNMT3B/miR-20b-5p/BAMBI in retinal vascular dysfunction. Bioinformatics analysis and luciferase reporter, siRNA, and overexpression assays were used to reveal the mechanisms of the circDNMT3B/miR-20b-5p/BAMBI interaction. Electroretinograms were used to evaluate visual function. FINDINGS: Upregulation of miR-20b-5p under diabetic conditions promoted proliferation, migration, and tube formation of human retinal microvascular endothelial cells (HRMECs), which was mediated by downregulated BAMBI. Under diabetic conditions, circDNMT3B, which acts as a sponge of miR-20b-5p, is downregulated. circDNMT3B overexpression reduced retinal acellular capillary number and alleviated visual damage in diabetic rats. Changes in expression of circDNMT3B and miR-20b-5p were confirmed in the proliferative fibrovascular membranes of patients with diabetic retinopathy. INTERPRETATION: Downregulation of circDNMT3B contributes to vascular dysfunction in diabetic retinas through regulating miR-20b-5p and BAMBI, providing a potential treatment strategy for diabetic retinopathy. FUNDING: National Natural Science Foundation of China, National Key Basic Research Program of China, Shanghai Municipal Science and Technology Major Project, and ZJLab.

Single-Dose Intravitreal Conbercept before Panretinal Photocoagulation as an Effective Adjunctive Treatment in Chinese Proliferative Diabetic Retinopathy.

PURPOSE: This study evaluates the efficacy and patient satisfaction of intravitreal conbercept (IVC) as adjunctive treatment before panretinal photocoagulation (PRP) for Chinese proliferative diabetic retinopathy (PDR) with or without clinically significant macular edema. METHODS: We enrolled 94 patients and categorized them into 2 groups: eyes that received PRP with single-dose IVC (0.5 mg/0.05 mL) 1 week before PRP (Plus group) or PRP only (PRP group). We measured the central macular thickness (CMT) by optical coherence tomography and best-corrected visual acuity. Satisfaction of PRP after 3 months was evaluated by a satisfaction questionnaire. RESULTS: Single-dose IVC 1 week before PRP significantly increased the PRP completion rate and satisfaction of treatment after 3 months in PDR patients. The average CMT significantly decreased in the Plus group but increased in the PRP group. No average visual changes were detected in the Plus group, but significant average visual loss was detected in the PPR group. The most important factors that determined satisfaction were the PRP completion rate in the short term and better vision gain after PRP. CONCLUSIONS: IVC is a promising adjunctive treatment to PRP in the treatment of PDR. Single-dose IVC 1 week before PRP was suggested to improve PRP completion rate and patient satisfaction in the short term.

Hypoxia-Inducible Factor-Dependent Expression of Angiopoietin-Like 4 by Conjunctival Epithelial Cells Promotes the Angiogenic Phenotype of Pterygia.

Purpose: Disappointing results from clinical studies assessing the efficacy of therapies targeting vascular endothelial growth factor (VEGF) for the treatment of pterygia suggest that other angiogenic mediators may also play a role in its development. We therefore explore the relative contribution of VEGF, hypoxia-inducible factor (HIF)-1α (the transcription factor that regulates VEGF expression in ocular neovascular disease), and a second HIF-regulated mediator, angiopoietin-like 4 (ANGPTL4), to the angiogenic phenotype of pterygia. Methods: Expression of HIF-1α, VEGF, and ANGPTL4 were examined in surgically excised pterygia, and in immortalized human (ih) and primary rabbit (pr) conjunctival epithelial cells (CjECs). Endothelial cell (EC) tubule formation assays using media conditioned by ihCjECs in the presence or absence of inducers/inhibitors of HIF-1 or RNA interference (RNAi) targeting VEGF, ANGPTL4, or both were used to assess their relative contribution to the angiogenic potential of these cells. Results: HIF-1α and VEGF expression were detected in 6/6 surgically excised pterygia and localized to CjECs. Accumulation of HIF-1α in was confirmed in ihCjECs and prCjECs, including stratified prCjECs grown on collagen vitrigel, and resulted in expression of VEGF and the promotion of EC tubule formation; the latter effect was partially blocked using RNAi targeting VEGF mRNA expression. We demonstrate expression of a second HIF-regulated angiogenic mediator, ANGPTL4, in CjECs in culture and in surgically excised pterygia. RNAi targeting ANGPTL4 inhibited EC tubule formation and was additive to RNAi targeting VEGF. Conclusions: Our results support the development of therapies targeting both ANGPTL4 and VEGF for the treatment of patients with pterygia.

Characteristics of retinal vein occlusion with final vision better than 78 letters after sequential therapy with ranibizumab and triamcinolone acetate.

AIM: To analyze the reasons that may lead to the different vision result by combining the ranibizumab and triamcinolone acetate (TA) in sequence to treat macular edema in retinal vein occlusion (RVO). METHODS: Ranibizumab and TA were combined in sequence to treat 43 patients with macular edema secondary to RVO. Six months after the treatment, patients with central fovea thickness (CFT) less than 300 µm in optical coherence tomography (OCT) were collected into Groups I and II, based on vision acuity (VA) better than 78 letters or less than 60 letters. The age, baseline VA, duration from onset to treatment, CFT at the baseline, sub-retinal fluid (SRF), sub-foveal exudates and injection times of TA and ranibizumab were taken into comparison. RESULTS: The mean age of the subjects was 46.4y in Group I but 57.5y in Group II. The difference of age was significant between groups (P<0.01). The mean baseline VA was 51.4 letters in Group I and 43.9 letters in Group II (P<0.05). The baseline CFT were 670.9 µm in Group I with SRF in 54.3% patients and 678.1 µm in Group II with SRF in 52.9% (P>0.05). The mean number of injections of TA was 0.9 and the mean number of injections of ranibizumab was 2.3 in Group I but 1.7 and 2.9 respectively in Group II. The treatment times of ranibizumab had no difference between the 2 groups (P>0.05) but the difference of TA injection times was significant, P<0.05. Subfoveal exudates at final stage happened in no subjects in Group I but in 45.83% subjects in Group II. CONCLUSION: This combined treatment is safer than TA injection and cheaper than ranibizumab injection alone. Younger patients and earlier treatment will help to get better vision outcome. Subfoveal exudates at the final stage have significant relationship with vision outcome. No relationship existed between the baseline CFT, SRF and the vision outcome.

Pharmacologic induction of heme oxygenase-1 plays a protective role in diabetic retinopathy in rats.

PURPOSE: We investigated the protective effects of HO-1, induced by hemin, in the retinas of streptozotocin (STZ)-induced diabetic rats, and document the possible anti-inflammatory, anti-apoptotic, and anti-proliferative mechanisms underlying the protection. METHODS: Sprague-Dawley (SD) rats were induced to diabetes by intraperitoneal injection of STZ (60 mg/kg). Later, some of the rats were given intraperitoneal injections of hemin (20 mg/kg) to induce expression of HO-1. The protective effects of hemin were evaluated by examining the hemoglobin concentration (Hb) and the glycosylated hemoglobin (HbA1c) level of blood from the rats, further calculating the numbers of TUNEL positive cells in retinal ganglion cell (RGC) layer and diI-labeled RGCs. We also documented the expressions of HO-1, HIF-1α, SOD-1, VEGF, p53, and bcl-2 by Western blot analysis and real-time quantitative PCR. Expressions of Nrf2, tERK 1/2, and pERK 1/2 were detected only by Western blot analysis. HO-1, Nrf2, pERK, and GFAP proteins were detected by immunofluorescence. RESULTS: The Hb level was higher in hemin-treated rat blood than nontreated diabetic group, while the HbA1c level was lower. Hemin significantly activated HO-1 expression in the retinas of diabetic rats, combined with accordant changes of Nrf2/pERK protein expression, and upregulated the expression of GFAP in retina. Retinal ganglion cells displayed greater sensitivity to apoptosis when the HO-1 level was lower. Overexpression of HO-1 was associated with an increase in the activation of SOD-1 and bcl-2, and a decrease of the expression of HIF-1α, VEGF, and p53. CONCLUSIONS: HO-1 is an important positive modulator of the Nrf2/ERK-related signaling. Overexpression of HO-1 by hemin induction protected retinal ganglion cells in diabetic retinopathy through anti-inflammatory, anti-apoptotic, and anti-proliferative effects.

Aquaporin changes during diabetic retinopathy in rats are accelerated by systemic hypertension and are linked to the renin-angiotensin system.

PURPOSE: We explored the relationship between the renin-angiotensin system (RAS) and aquaporins (AQP1 and AQP4 in Müller glia and astrocytes) in diabetic retinopathy (DR) with and without systemic hypertension. METHODS: Diabetes was induced in spontaneously hypertensive rats (SHR) and normotensive control Wistar Kyoto (WKY) rats by intraperitoneal injections of streptozotocin. The diabetic and control non-diabetic rats were assigned randomly to receive no anti-hypertension treatment, or to be treated with the angiotensin II receptor blocker (ARB), valsartan (40 mg/kg/d) or the beta-blocker, metoprolol (50 mg/kg/day). Eight weeks later, retinas were evaluated by immunohistochemistry and Western blot to detect changes in the expression of AQP1, AQP4, and glial fibrillary acidic protein (GFAP). RESULTS: Hypertension increased expression of glial GFAP and AQP4 (P < 0.01), but not AQP1 (P > 0.05) in diabetic rats. Valsartan and metoprolol decreased GFAP, AQP1, and AQP4 expression in diabetic SHR rats (P < 0.01). Valsartan decreased GFAP and AQP1 expression in diabetic WKY rats (P < 0.01), while metoprolol did not. CONCLUSIONS: Activation of Müller glia and astrocytes was involved in the mechanism by which systemic hypertension affects DR. AQPs and macroglia were linked to changes in the RAS in DR. Changes in aquaporin expression in DR were increased by hypertension. This provides additional support for the early use of an ARB in the treatment of DR, especially in cases with retinal edema.

ERK1/2 signaling pathways involved in VEGF release in diabetic rat retina.

PURPOSE: Vascular endothelial growth factor (VEGF) is one of the major factors promoting diabetic retinopathy (DR). A better understanding of the signaling pathway in VEGF regulation is of clinical importance to identify more precise therapeutic targets for diabetic retinopathy. The ERK1/2 signaling pathway has been shown to play a key role in some oncoma and hematologic diseases by mediating VEGF release. This research was conducted to determine whether the ERK1/2 signaling pathway also plays a major role in VEGF release in DR development. METHODS: One hundred Sprague-Dawley (SD) rats were induced to diabetes by streptozotocin (STZ) injection and monitored at several time points (1, 2, 3, 4, 8, and 12 weeks) for ERK1/2 phosphorylation, Activator protein (AP)-1 activity and concentration, and VEGF protein and mRNA expression, using immunohistochemical and biochemical methods. RESULTS. The ERK1/2 signaling pathway was rapidly activated 1 week after diabetes was induced. AP-1, the downstream transcription factor of ERK1/2, was also activated, and VEGF became highly regulated in a similar trend. U0126, an inhibitor of ERK1/2, also downregulated VEGF expression, in addition to ERK1/2 and AP-1 activity. CONCLUSIONS: ERK1/2 signaling pathway is involved in VEGF release in diabetic rat retina; therefore, ERK1/2 may be a potential therapeutic target of DR.

Regional macular light sensitivity changes in myopic Chinese adults: an MP1 study.

PURPOSE: To investigate the variation of macular light sensitivity (MLS) in myopic Chinese adults by using microperimetry. METHODS: MLS was recorded with the MP1 microperimeter (Nidek Technologies, Padova, Italy) in eyes affected by various degrees of myopia. Subjects were divided into group A (18-30 years) and group B (31-60 years). Subjects in both age groups were further divided based on refractive status: (1) high myopia (spherical equivalent, SE<-6.00 D); (2) low to moderate myopia (-6.00 D

High-salt loading exacerbates increased retinal content of aquaporins AQP1 and AQP4 in rats with diabetic retinopathy.

In the neural retina, glial cells control formation of ionic gradients by mediating transmembrane water fluxes through aquaporin (AQP) water channels. Retinal content and immunolocalization of two water channels, AQP1 and AQP4, in the diabetic rat retinas during high-salt loading were examined in this study. Diabetes was induced by an intraperitoneal injection of streptozotocin. Diabetic and control animals were observed after varying lengths of exposure to normal- and high-salt conditions. Ultrathin sections of retinal tissue, stained with uranyl acetate and lead citrate, were photographed using a transmission electron microscope (TEM). Retinal wholemounts were immunostained with AQP1 and AQP4 antibody to detect the immunolocalization changes by confocal microscopy. AQP1 and AQP4 content were evaluated by Western blot analysis. In the retinas of high-salt loading diabetic animals, obviously increased intracellular edema was observed by TEM in ganglion cells and mitochondrial swelling was observed in glial cells. Immunolocalization of AQP1 increased from the posterior to peripheral retina. Western blot results indicated that a high-salt diet may cause increased retinal content of AQP4 and may exacerbate increased retinal content of AQP1 caused by diabetic retinopathy. High-salt loading may increase neural retinal edema in rats with diabetic retinopathy, and altered glial cell mediated water transport via AQP channels in the retina may play an important role in the neural retinal edema formation and resolution.

High salt loading alters the expression and localization of glial aquaporins in rat retina.

In the neural retina, glial cells control the ionic concentrations in part by mediation of transmembrane water fluxes through aquaporin (AQP) water channels. The expression and immunolocalization of two water channels, AQP1 and AQP4, in the rat retina during experimental high salt loading were investigated in this study. Six-week-old Wistar rats were allowed free access to rat chow with 8% NaCl concentration. Of these rats, 6 were killed after 2, 6, 10 and 20 weeks. Twelve-week-old and 26-week-old Wistar rats with a normal diet (0.5% NaCl concentration) were used as controls. Retinal tissues were collected. Ultrathin sections stained with uranyl acetate and lead citrate were photographed using a transmission electron microscope (TEM). Retinal whole mounts and cryosections were immunostained with AQP1 and AQP4 antibodies to detect the immunolocalization changes by confocal microscopy. The AQP1 and AQP4 contents were evaluated by western blot analysis. In control tissues, no intracellular edema and mitochondria swelling were observed by TEM. The immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina. In the retinas of high salt loading animals, obvious intracellular edema was observed by TEM in retinal ganglion cell (RGC) and mitochondria swelling was observed in glial cells. Strong expression of AQP1 was found in glial cells located in the innermost retinal layers, mainly in astrocytes. The superficial retinal vessels were surrounded by AQP4 in control retinas, but by both AQP4 and AQP1 in retina of high salt loading animals. A similar alteration in the localization of AQP1 has been described in the rat retina after transient ischemia and diabetes. Western blot results supported the conclusion that the AQP1 expression increased during high salt diet. Our findings indicate that high salt loading may induce neural retina edema, and that altered glial cell-mediated water transport via AQP channels in the retina may be one of the reasons for intracellular edema in the neural retina.

Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation.

PURPOSE: To determine the role of microglial activation in light-induced photoreceptor degeneration and the neuroprotective effects of naloxone as a novel microglial inhibitor. METHODS: Sprague-Dawley rats were exposed to intense blue light for 24 hours. Daily intraperitoneal injection of naloxone or PBS as a control was given 2 days before exposure to light and was continued for 2 weeks. Apoptotic cells were detected by the TUNEL assay, and anti-OX42 antibody was used to label retinal microglia. Western blot was applied to evaluate the retinal interleukin (IL)-1beta protein levels. Retinal histologic examination and electroretinography (ERG) were also performed to evaluate the effects of naloxone on light-induced photoreceptor degeneration. RESULTS: TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina as early as 2 hours and peaked at 24 hours after exposure to light. OX42-positive microglia occurred in the ONL and subretinal space at 6 hours, peaked at 3 days, and changed morphologically from the resting ramified to the activated amoeboid. Expression of IL-1beta protein was also significantly increased at 3 days. Compared with the control, the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3 days, and the thickness of ONL and the amplitudes of dark-adapted a- and b-waves were also well preserved at 14 days. CONCLUSIONS: The activation and migration of microglia and the expression of neurotoxic factor (IL-1beta) coincide with photoreceptor apoptosis, suggesting that activated microglia play a major role in light-induced photoreceptor degeneration. Inhibiting microglial activation by naloxone significantly reduces this degeneration.

Dendritic abnormalities in retinal ganglion cells of three-month diabetic rats.

PURPOSE: To determine the effect of three-month diabetes on the retinal ganglion cell (RGC) morphology and density. METHODS: Experimental diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). Retinas from three-month diabetic and age-matched control rats were harvested, and immunohistochemistry with monoclonal anti-Thy-1 antibody was carried out for calculating RGC density. Random RGC labeling with gene gun propelled lipophilic fluorescent dye, DiI, coated particles (DiOlistic method) was done for detailed RGC classification, dendritic field, and soma size measurement. RESULTS: The number of Thy1-labeled RGCs in the three-month diabetic rats was significantly reduced compared with that in the age-matched control. Obvious RGC morphology changes were observed, and the number of RGCs that could not be classified was significantly greater in the diabetic retinas. Among those well-classified RGCs, cells with enlarged dendritic fields were more frequently seen in the RGA group (401+/-86 um, n = 59, P < 0.001), but the soma sizes were unchanged from the controls (P > 0.05). For cells in the groups of RGB and RGC, no significant changes in the dendritic fields and soma sizes were found (P > 0.05). CONCLUSIONS: In the three-month STZ-induced diabetic rat, retinal ganglion cell loss is associated with morphology change. The surviving RGCs in the diabetic retina, especially those in RGA group, show significant dendritic field enlargement. This plasticity of the surviving RGC dendrites may represent a compensatory response to the overall loss of RGCs in diabetes.