Molecular mechanism of wedelolactone inhibits high glucose-induced human retinal vascular endothelial cells injury through regulating miR-190 expression.

To investigate the effects and molecular mechanisms of wedelolactone (WEL) on high glucose-induced injury of human retinal vascular endothelial cells (HRECs). The cell injury model was established by incubating HRECs with 30 mmol/L glucose for 24 hour. HRECs were divided into control (Con) group, high glucose (HG) group, HG + WEL-low dose (L) group, HG + WEL-medium dose (M), HG + WEL-high dose (H) group, HG + miR-NC group, HG + miR-190 group, HG + WEL + antimiR-NC group, HG + WEL + antimiR-190 group. The kit detects cellular reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) content; cell apoptosis was analyzed by flow cytometry; miR-190 expression was detected by real-time quantitative PCR (RT-qPCR). Compared with Con group, the levels of ROS and MDA in the HG group were significantly increased (P < .01), the SOD activity and the expression of miR-190 expression were significantly decreased (P < .05), and the apoptosis rate was significantly increased (P < .01). Compared with HG group, the levels of ROS and MDA in HG + WEL-L group, HG + WEL-M group and HG + WEL-H group were significantly decreased (P < .05), SOD activity and miR-190 expression were significantly increased (P < .05), and apoptosis rate was significantly reduced (P < .05). Compared with the HG + miR-NC group, the levels of ROS and MDA in HG + miR-190 group were significantly reduced (P < .01), SOD activity was significantly increased (P < .01), and apoptosis rate was significantly reduced (P < .05). Compared with the HG + WEL + antimiR-NC group, the ROS level and MDA content in the HG + WEL + antimiR-190 group were significantly increased (P < .05), SOD activity was significantly decreased (P < .05), and apoptosis rate was significantly increased (P < .05). Wedelolactone can attenuate high glucose-induced HRECs apoptosis and oxidative stress by up-regulating miR-190 expression.

Notch1 and Notch4 core binding domain peptibodies exhibit distinct ligand-binding and anti-angiogenic properties.

The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.

Down-expression of the NLRP3 inflammasome delays the progression of diabetic retinopathy.

The investigation aimed to evaluate the effects of Mcc950, an inhibitor of the NLRP3 inflammasome, on diabetic retinopathy (DR) mice. The general physiological condition of each group of mice was recorded. Retinal blood vessels were stained for observation of the density of blood vessels, and retinas were used for further morphological examination and fluorescent staining after the intravitreal injection of Mcc950. Mcc950 partially reversed hyperglycemia-induced vascular damage and had reduced histological changes compared to DR mice. IL-1ß production in mice retinas in the diabetic model (DM) group increased, but pretreatment with Mcc950 significantly reversed these changes. Additionally, Mcc950 engineered reduced FITC dextran extravasation and vascular leakage. Therefore, it played an apparent protective role in DR and could be a new treatment strategy for DR.

Lycium barbarum polysaccharides attenuates high glucose-induced diabetic retinal angiogenesis by rescuing the expression of miR-15a-5p in RF/6A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum L., a classical traditional Chinese Medicine, has long been used to treat ocular diseases. Lycium barbarum polysaccharides (LBP) is an effective component of Lycium barbarum L. with a wide range of pharmacological activities. This research aims to investigate the inhibition of high glucose-induced angiogenesis by LBP in RF/6A cells. MATERIALS AND METHODS: A high-glucose-induced angiogenesis model was established using monkey retinal vascular endothelial (RF/6A) cells. Different dosages administration times of LBP and glucose concentrations were tested. Under the optimized conditions, RF/6A cells were treated with LBP for 48 h, followed by another 48-h culture in high glucose (25 mmol/L) medium. The effect and mechanism of LBP were investigated following the treatment. RESULTS: The expression of miR-15a-5p and miR-15a-3p in RF/6A cells decreased significantly after 48 h of 25 or 50 mmol/L high glucose treatment. The expression of miR-15a-5p was higher than that of miR-15a-3p. Mimic-miR-15a-5p or 600 mg/L LBP could increase the apoptosis of cells and the total length of vascular branches. The expression of VEGFA, VEGFR2, and ANG2 proteins was reduced, while the expression of ANG1 protein was elevated. Expression of ASM mRNA and protein was also inhibited. CONCLUSIONS: LBP attenuates diabetic retinal angiogenesis by rescuing the expression of miR-15a-5p in RF/6A cells.

Habitual intake of dietary advanced glycation end products is not associated with generalized microvascular function-the Maastricht Study.

BACKGROUND: Endogenously formed advanced glycation end products (AGEs) may be important drivers of microvascular dysfunction and the microvascular complications of diabetes. AGEs are also formed in food products, especially during preparation methods involving dry heat. OBJECTIVES: We aimed to assess cross-sectional associations between dietary AGE intake and generalized microvascular function in a population-based cohort. METHODS: In 3144 participants of the Maastricht Study (mean ± SD age: 60 ± 8 y, 51% men) the dietary AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were estimated using the combination of our ultra-performance LC-tandem MS dietary AGE database and an FFQ. Microvascular function was determined in the retina as flicker light-induced arteriolar and venular dilation and as central retinal arteriolar and venular equivalents, in plasma as a z score of endothelial dysfunction biomarkers (soluble vascular adhesion molecule 1 and soluble intracellular adhesion molecule 1, soluble E-selectin, and von Willebrand factor), in skin as the heat-induced skin hyperemic response, and in urine as 24-h albuminuria. Associations were evaluated using multiple linear regression adjusting for demographic, cardiovascular, lifestyle, and dietary factors. RESULTS: Overall, intakes of CML, CEL, and MG-H1 were not associated with the microvascular outcomes. Although higher intake of CEL was associated with higher flicker light-induced venular dilation (ß percentage change over baseline: 0.14; 95% CI: 0.02, 0.26) and lower plasma biomarker z score (ß: -0.04 SD; 95% CI: -0.08, -0.00 SD), the effect sizes were small and their biological relevance can be questioned. CONCLUSIONS: We did not show any strong association between habitual intake of dietary AGEs and generalized microvascular function. The contribution of dietary AGEs to generalized microvascular function should be further assessed in randomized controlled trials using specifically designed dietary interventions.

Knockdown of Long Non-coding RNA TUG1 Suppresses Migration and Tube Formation in High Glucose-Stimulated Human Retinal Microvascular Endothelial Cells by Sponging miRNA-145.

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus. The purpose of this study was to investigate the potential functional role of long non-coding RNA TUG1 in high glucose (HG)-stimulated human retinal microvascular endothelial cells (hRMECs). The results demonstrated that following 72 h of HG stimulation, enhanced proliferation, migration, and tube formation process were observed in hRMECs. Moreover, HG treatment markedly increased TUG1 expression in hRMECs, and knockdown of TUG1 notably restrained the aberrant phenotypes of hRMECs induced by HG. Mechanistically, TUG1 may serve as a competing endogenous RNA (ceRNA) for miR-145, thereby blocking the repression on VEGF-A in hRMECs. Rescue experiments further indicated that inhibition of miR-145 abolished the beneficial role of TUG1 knockdown in HG-treated hRMECs. Our data suggested that knockdown of TUG1 protects hRMECs against HG stimulation partly by regulating miR-145/VEGF-A axis.

ATP induced calcium signaling activity in perivascular cells differ at different vascular branch levels in the porcine retina.

BACKGROUND: The purine adenosine triphosphate (ATP) plays a significant role in retinal blood flow regulation and recent evidence suggests that the vasoactive effect of the compound differs in vessels at different branching level. However, the cellular basis for the regulation of retinal blood flow mediated by ATP has only been scarcely studied. METHODS: Perfused porcine hemiretinas (n = 60) were loaded with the calcium-sensitive fluorophore Oregon Green ex vivo. Spontaneous oscillations in fluorescence were studied in perivascular cells at five different vascular branching levels ranging from the main arteriole to the capillaries, before and after the addition of intra- and extravascular ATP alone or in the presence of a P2-purinergic receptor antagonist. RESULTS: Intravascular ATP induced an overall significant (p < 0.01) constriction of (mean ± SD) -9.79 ± 13.40% and extravascular ATP an overall significant (p < 0.01) dilatation of (mean ± SD) 19.62 ± 13.47%. Spontaneous oscillations of fluorescence in perivascular cells were significantly more intense around third order arterioles than around vessels at both lower and higher branching levels (p < 0.05 for all comparisons). ATP increased intracellular fluorescence in perivascular cells of first and second order arterioles after extravascular application, and the increase correlated with the accompanying vasodilatation (p < 0.03). Blocking of P2-receptors reduced oscillating fluorescence in pre-capillary arterioles secondary to intravascular ATP (p = 0.03). CONCLUSIONS: Spontaneous oscillations of calcium-sensitive fluorescence in perivascular retinal cells differ at different vascular branching levels. Extravascular ATP increases fluorescence in cells around the larger retinal arterioles exposed to the retinal surface. Future studies should investigate calcium signaling activity in perivascular retinal cells during interventions that simulate retinal pathology such as hypoxia.

Short-term changes in retinal and choroidal relative flow volume after anti-VEGF treatment for neovascular age-related macular degeneration.

The effects of anti-vascular endothelial growth factor (anti-VEGF) agents on the native ocular vasculature are poorly understood. This pilot study aimed to assess short-term changes in retinal and choroidal perfusion after anti-VEGF treatment for neovascular exudative age-related macular degeneration (nAMD) using the relative flow volume (RFV) parameter derived from laser speckle flowgraphy. Ten treatment-naïve nAMD patients underwent measurements of mean, maximum, minimum, and differential RFV within a retinal arteriolar segment and a choroidal vessel segment outside the neovascular area. Measurement of retinal RFV (rRFV), choroidal RFV (cRFV), and subfoveal choroidal thickness (SCT) was repeated 9 and 35 days after a single anti-VEGF injection. The treatment caused a statistically significant decrease in the mean rRFV, mean cRFV, and SCT during the follow-up (p < 0.05). At the intermediate visit, the mean cRFV and SCT were - 17.6% and - 6.4% compared to baseline, respectively. However, at the final measurement, the mean cRFV was not different from the baseline value, which indicated waning of the anti-VEGF effect. In conclusion, a single anti-VEGF injection in treatment-naïve nAMD resulted in a decrease in retinal arteriolar and choroidal perfusion, according to the RFV parameter, which is a promising tool to simultaneously assess retinal and choroidal perfusion changes in response to anti-VEGF therapy.

Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy.

Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

Involvement of Gap Junctions in Acetylcholine-Induced Endothelium-Derived Hyperpolarization-Type Dilation of Retinal Arterioles in Rats.

An electrical communication between the endothelial and smooth muscle cells via gap junctions, which provides the signaling pathway known as endothelium-dependent hyperpolarization (EDH), plays a crucial role in controlling the vascular tone. In this study, we investigated the role of gap junctions in the acetylcholine (ACh)-induced EDH-type dilation of rat retinal arterioles in vivo. The dilator response was evaluated by measuring the diameter of retinal arterioles. Intravitreal injection of gap junction blockers (18ß-glycyrrhetinic acid and carbenoxolone) reduced the ACh-induced dilation of retinal arterioles. Moreover, the retinal arteriolar response to ACh was attenuated by 18ß-glycyrrhetinic acid under treatment with a combination of NG-nitro-L-arginine methyl ester (a nitric oxide (NO) synthase inhibitor; 30 mg/kg) and indomethacin (a cyclooxygenase inhibitor; 5 mg/kg). The NO- and prostaglandin-independent, EDH-related component of ACh-induced dilation of retinal arterioles was prevented by intravitreal injection of iberiotoxin, which inhibits large-conductance Ca2+-activated K+ channels. Furthermore, the combination of 18ß-glycyrrhetinic acid and iberiotoxin produced greater attenuation in the EDH-related response than that by the individual agent. Treatment with 18ß-glycyrrhetinic acid revealed no significant effect on NOR3 (an NO donor)-induced retinal vasodilator response. These results suggest that gap junctions contribute to the ACh-induced, EDH-type dilation of rat retinal arterioles in vivo.

An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration.

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunction model and a comprehensive downstream validation system. METHODS: Human retinal vascular endothelial cells (HRMECs) and retinal ganglion cells (RGCs) were treated with different glucose concentrations with mannitol as matched osmotic controls. Cell proliferation and viability were evaluated by the Cell Counting Kit-8. Cell migration was measured using a transwell migration assay. Cell sprouting was assessed by a tube formation assay. The VEGF expression was assessed by ELISA. RGCs were labeled by neurons and RGC markers TUJ1 and BRN3A for quantitative and morphological analysis. Apoptosis was detected using PI/Hoechst staining and TUNEL assay and quantified by ImageJ. RESULTS: Cell proliferation and migration in HRMECs were significantly higher in the 25 mM glucose-treated group (p < 0.001) but lower in the 50 mM and 100 mM groups (p < 0.001). The permeability and the apoptotic index in HRMECs were statistically higher in the 25 mM, 50 mM, and 100 mM groups (p < 0.05). The tube formation assay found that all the parameters were significantly higher in the 25 mM and 50 mM groups (p < 0.001) concomitant with the elevated VEGFA expression in HRMECs (p = 0.016). Cell viability was significantly lower in the 50 mM, 100 mM, and 150 mM groups in RGCs (p 50mM = 0.013, p 100mM = 0.019, and p 150mM = 0.002). Apoptosis was significantly elevated, but the proportion of RGCs with neurite extension was significantly lower in the 50 mM, 100 mM, and 150 mM groups (p 50mM < 0.001, p 100mM < 0.001, and p 150mM < 0.001). CONCLUSIONS: We have optimized glucose concentrations to model diabetic retinal endothelial (25-50 mM) or neuronal (50-100 mM) dysfunction in vitro, which have a wide range of downstream applications.

Regulation of Adrenergic, Serotonin, and Dopamine Receptors to Inhibit Diabetic Retinopathy: Monotherapies versus Combination Therapies.

We compared monotherapies and combinations of therapies that regulate G-protein-coupled receptors (GPCRs) with respect to their abilities to inhibit early stages of diabetic retinopathy (DR) in streptozotocin-diabetic mice. Metoprolol (MTP; 0.04-1.0 mg/kg b.wt./day), bromocriptine (BRM; 0.01-0.1 mg/kg b.wt./day), doxazosin (DOX; 0.01-1.0 mg/kg b.wt./day), or tamsulosin (TAM; 0.05-0.25 mg/kg b.wt./day) were injected individually daily for 2 months in dose-response studies to assess their effects on the diabetes-induced increases in retinal superoxide and leukocyte-mediated cytotoxicity against vascular endothelial cells, both of which abnormalities have been implicated in the development of DR. Each of the individual drugs inhibited the diabetes-induced increase in retinal superoxide at the higher concentrations tested, but the inhibition was lost at lower doses. To determine whether combination therapies had superior effects over individual drugs, we intentionally selected for each drug a low dose that had little or no effect on the diabetes-induced retinal superoxide for use separately or in combinations in 8-month studies of retinal function, vascular permeability, and capillary degeneration in diabetes. At the low doses used, combinations of the drugs generally were more effective than individual drugs, but the low-dose MTP alone totally inhibited diabetes-induced reduction in a vision task, BRM or DOX alone totally inhibited the vascular permeability defect, and DOX alone totally inhibited diabetes-induced degeneration of retinal capillaries. Although low-dose MTP, BRM, DOX, or TAM individually had beneficial effects on some endpoints, combination of the therapies better inhibited the spectrum of DR lesions evaluated. SIGNIFICANCE STATEMENT: The pathogenesis of early stages of diabetic retinopathy remains incompletely understood, but multiple different cell types are believed to be involved in the pathogenic process. We have compared the effects of monotherapies to those of combinations of drugs that regulate GPCR signaling pathways with respect to their relative abilities to inhibit the development of early diabetic retinopathy.

The possible effect of pentoxifylline on development and severity of retinopathy of prematurity.

BACKGROUND AND AIM: Retinopathy of prematurity (ROP) is the major ocular problem of preterm infants that occurs with abnormal proliferation of immature retinal vessels. Although pentoxifylline (PTX) was reported to inhibit vasculogenesis and neovascularization in experimental studies, there is no clinical data about the effects of PTX treatment on the development and severity of ROP. This clinical study aimed to investigate the possible effects of PTX on the development of ROP. MATERIALS AND METHODS: A single-centre retrospective study was conducted including preterm infants who were hospitalised in the neonatal intensive care unit between 2015-2017 years. Infants were divided into two groups in terms of PTX administration for adjuvant therapy, as PTX and non-PTX groups. RESULTS: A total of 211 infants were included in the study [gestational age 29 (27-31) weeks, birth weight 1140 (960-1340) g]. From these, 97 infants (46%) were given PTX treatment. The two groups were similar in terms of demographic data and baseline clinical characteristics. Any stage of ROP was detected in 47.4% of infants in the PTX group, which was significantly higher than those in the non-PTX group (27.2%) (p = 0.002). The incidence of advanced-stage ROP in the PTX group (10.3%) was also higher than in the non-PTX group (2.6%) (p = 0.021). Repeated usage of PTX was not found to be related to the development of ROP (p = 0.059). The time of PTX administration was similar between the ROP and no-ROP groups (median; one vs one week, p = 0.825). Surfactant therapy, duration of hospital stay, and PTX treatment were found as significant risk factors for ROP in the logistic regression analysis. CONCLUSIONS: In contrast to the experimental studies and also promising results of PTX treatment in some neonatal morbidities, it may be associated with increased incidence and stage of ROP.

Nuclear factor of activated T-cells (NFAT) regulation of IL-1ß-induced retinal vascular inflammation.

Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1ß has the reputation of being a major driver of cytokine-induced inflammation. IL-1ß and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1ß-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1ß-induced expression of IL-1ß and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1ß-induced increases of IL-1ß, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1ß-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1ß-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.

Pericyte Insulin Receptors Modulate Retinal Vascular Remodeling and Endothelial Angiopoietin Signaling.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRß-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Adenosine A2A receptor antagonism protects against hyperoxia-induced retinal vascular loss via cellular proliferation.

Retinopathy of prematurity (ROP) remains one of the major causes of blindness in children worldwide. While current ROP treatments are mostly disruptive to reduce proliferative neovascularization by targeting the hypoxic phase, protection against early hyperoxia-induced retinal vascular loss represents an effective therapeutic window, but no such therapeutic strategy is available. Built upon our recent demonstration that the protection against oxygen-induced retinopathy by adenosine A2A receptor (A2A R) antagonists is most effective when administered at the hyperoxia (not hypoxic) phase, we here uncovered the cellular mechanism underlying the A2A R-mediated protection against early hyperoxia-induced retinal vascular loss by reversing the inhibition of cellular proliferation via possibly multiple signaling pathways. Specifically, we revealed two distinct stages of the hyperoxia phase with greater cellular proliferation and apoptosis activities and upregulation of adenosine signaling at postnatal 9 day (P9) but reduced cellular activities and adenosine-A2A R signaling at P12. Importantly, the A2A R-mediated protection at P9 was associated with the reversal of hyperoxia-induced inhibition of progenitor cells at the peripheral retina at P9 and of retinal endothelial proliferation at P9 and P12. The critical role of cellular proliferation in the hyperoxia-induced retinal vascular loss was validated by the increased avascular areas by siRNA knockdown of the multiple signaling molecules involved in modulation of cellular proliferation, including activin receptor-like kinase 1, DNA-binding protein inhibitor 1, and vascular endothelial growth factor-A.

MG53 inhibits angiogenesis through regulating focal adhesion kinase signalling.

Mitsugumin 53 (MG53), which is expressed predominantly in striated muscle, has been demonstrated to be a myokine/cardiokine secreted from striated muscle under specific conditions. The important roles of MG53 in non-striated muscle tissues have also been examined in multiple disease models. However, no previous study has implicated MG53 in the control of endothelial cell function. In order to explore the effects of MG53 on endothelial cells, human umbilical vein endothelial cells (HUVECs) were stimulated with recombinant human MG53 (rhMG53). Then, rhMG53 uptake, focal adhesion kinase (FAK)/Src/Akt/ERK1/2 signalling pathway activation, cell migration and tube formation were determined in vitro. The efficacy of rhMG53 in regulating angiogenesis was also detected in postnatal mouse retinas. The results demonstrated that rhMG53 directly entered into endothelial cells in a cholesterol-dependent manner. The uptake of rhMG53 directly bound to FAK in endothelial cells, which resulted in a significant decrease in FAK phosphorylation at Y397. Accompanied by the dephosphorylation of FAK, rhMG53 uncoupled FAK-Src interaction and reduced the phosphorylation of Src at Y416. Consequently, the activation of FAK/Src downstream signalling pathways, such as Akt and ERK1/2, was also significantly inhibited by rhMG53. Furthermore, rhMG53 remarkably decreased HUVEC migration and tube formation in vitro and postnatal mouse retinal angiogenesis in vivo. Taken together, these data indicate that rhMG53 inhibits angiogenesis through regulating FAK/Src/Akt/ERK1/2 signalling pathways. This may provide a novel molecular mechanism for the impaired angiogenesis in ischaemic diseases.

Evaluation of the Effect of a Novel ß3-Adrenergic Agonist on Choroidal Vascularity.

Purpose: To determine the effect of the new ß3-agonist (mirabegron), which is used for overactive bladder (OAB) treatment, on central retinal thickness (CRT) and choroidal vascularity. Material and Methods: The 26 eyes of 26 cases using 50 mg tablet mirabegron once per day for OAB were included in this prospective case control study. The CRT, choroidal thickness (ChT), and choroidal vascularity were measured at baseline, week 1 (W1), month 1 (M1), month 2 (M2), and month 3 (M3). Subfoveal ChT measurement included the total subfoveal choroidal thickness (SFCT), and the small and large choroidal vessel layer (SCVL and LCVL) thickness. The total choroidal area (TCA), lumen area (LA), stromal area (SA), stroma/lumen ratio, and choroidal vascularity index (CVI) were measured with the Image-J software. Results: The largest SFCT increase compared to baseline was at M1 (26.8 ± 40.8 µm, P = 0.001). The subfoveal SCVL thickness showed a significant decrease at M2 and M3 (-6.0 ± 8.9 µm, P = 0.002; -7.8 ± 13.4 µm, P = 0.046, respectively). LCVL thickness showed a significant increase at W1, M1, and M2, with the largest at M1. CVI showed a significant increase at M1, M2, and M3 (P < 0.05 for all). The TCA, LA, and SA showed a significant increasing trend at all follow-up periods. LA/SA decreased at W1 because of stromal expansion but increased at M3 with more prominent vascular dilatation. CRT values showed no significant change. Conclusions: Mirabegron had a significant effect on choroidal thickness. Choroidal vascular response is in the form of narrowing in the choriocapillaris and enlargement in the Haller's layer.

Neuroprotective effect of minocycline on rat retinal ischemia-reperfusion injury.

Purpose: To examine the neuroprotective effect of minocycline on retinal ischemia-reperfusion (IR) injury in rats and investigate its possible mechanism of action. Methods: Retinal IR injury was established by increasing the intraocular pressure in rats up to 110 mmHg for 60 min. The animals with retinal IR injury were intraperitoneally injected with 22.5 mg/kg minocycline twice a day for 14 days. The control group received the same amount of saline. Subsequently, funduscopic examination, retinal thickness measurement, retinal microvascular morphology, full-field electroretinography (ERG), retinal apoptotic cell count, and remaining retinal ganglion cell (RGC) count were performed. The expression of iNOS, Bax, Bcl2, IL-1α, IL-6, TNF-α, caspase-3, GFAP, Iba-1, Hif-1α, and Nrf2 was examined with real-time PCR and western blotting. Results: Minocycline treatment prevented IR-induced rat retinal edema and retinal cells apoptosis at the early stage and alleviated retina atrophy, blood vessel tortuosity, functional photoreceptor damage, and RGC degeneration at the late stage of the IR injury. At the molecular level, minocycline affected retinal gene and protein expression induced by IR. Conclusions: The results suggested that minocycline has a neuroprotective effect on rat retinal IR injury, possibly through anti-inflammation, antiapoptosis, antioxidation, and inhibition of microglial activation.

Retinal Pigment Epithelium Remodeling in Mouse Models of Retinitis Pigmentosa.

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.