Retinal Permeability Research Articles

Макулярный отек на фоне окклюзий ретинальных вен. Особенности морфометрии макулы и хориоретинальной гемодинамики

This review article summarizes current data on the pathogenesis of macular edema after retinal vein occlusions (RVOs) and patterns of macular morphometry and chorioretinal hemodynamics. RVOs account for 60% of acute vascular eye diseases. The major cause of vision loss in RVOs is macular edema which results from subclinical inflammation characterized by leukostasis and enhanced expression of adhesion molecules and production of cytokines increasing retinal capillary permeability. The association between the severity of macular edema and vitreous levels of pro-inflammatory mediators (IL-1β, IL-6, TNF-α, MCP-1, and VEGF) is established. Cystic lesions and detachment of the neuroepithelium (75%) are typical for macular edema after RVOs. Impaired chorioretinal hemodynamics (as demonstrated by a significant reduction of vascular density in the superficial and deep vascular plexus, significant decrease in peak diastolic flow velocity, and increase in resistivity index in the ophthalmic artery and short posterior ciliary arteries) is also reported. The paper addresses etiological factors of RVOs and pathogenesis of macular edema, technical tools for vital assessment of the retina, and choroidal/retinal hemodynamics. This provides great opportunities for a complex in-depth study of the development and course of macular edema to identify potential predictors of its development and persistence. Keywords: retinal vein occlusions, macular edema, morphometry, chorioretinal hemodynamics. For citation: Fil' A.A., Sorokin E.L., Kolenko O.V. Macular edema after retinal vein occlusions. Macular morphometry and chorioretinal hemodynamics. Russian Journal of Clinical Ophthalmology. 2021;21(3):164–168 (in Russ.). DOI: 10.32364/2311-7729-2021-21-3- 164-168.

Dual-Acting Antiangiogenic Gene Therapy Reduces Inflammation and Regresses Neovascularization in Diabetic Mouse Retina

Intravitreal injections of anti-vascular endothelial growth factor drugs have become the gold standard treatment for diabetic retinopathy (DR). However, several patients are classified as non-responders or poor responders to treatment. Therefore, it is essential to study alternative target molecules. We have previously shown that the progression of DR in the Ins2Akita mouse reflects the imbalance between pro- and anti-angiogenic molecules found in the human retina. We report, for the first time, the therapeutic potential of a dual-acting antiangiogenic non-viral gene therapy. We have used an expressing vector encoding both the pigment epithelium-derived factor gene and a short hairpin RNA (shRNA) targeted to the placental growth factor to restore the balance between these factors in the retina. Twenty-one days after a single subretinal injection, we observed a marked decrease in the inflammatory response in the neural retina and in the retinal pigment epithelium, together with reduced vascular retinal permeability in the treated diabetic mouse. These results were accompanied by the restoration of the retinal capillary network and regression of neovascularization, with significant improvement of DR hallmarks. Concomitant with the favorable therapeutic effects, this approach did not affect retinal ganglion cells. Hence our results provide evidence toward the use of this approach in DR treatment.

Non-invasive measurement of retinal permeability in a diabetic rat model.

The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P<.01, P<.001, P<.0001). This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.

Apigenin and Ethaverine Hydrochloride Enhance Retinal Vascular Barrier In Vitro and In Vivo.

PurposeThis study aims to develop an impedance-based drug screening platform that will help identify drugs that can enhance the vascular barrier function by stabilizing vascular endothelial cell junctions.MethodsChanges in permeability of cultured human retinal microvascular endothelial cells (HRMECs) monolayer were monitored in real-time with the xCELLigence RTCA system. Using this platform, we performed a primary screen of 2100 known drugs and confirmed hits using two additional secondary permeability assays: the transwell permeability assay and the XPerT assay. The cellular and molecular mechanisms of action and in vivo therapeutic efficacy were also assessed.ResultsEleven compounds blocked interleukin 1 beta (IL-1β) induced hyperpermeability in the primary screen. Two of 11 compounds, apigenin and ethaverine hydrochloride, reproducibly blocked multiple cytokines induced hyperpermeability. In addition to HRMEC monolayers, the two compounds stabilized three other types of primary vascular endothelial cell monolayers. Preliminary mechanistic studies suggest that the two compounds stabilize the endothelium by blocking ADP-ribosylation factor 6 (ARF6) activation, which results in enhanced VE-cadherin membrane localization. The two compounds showed in vivo efficacy in an animal model of retinal permeability.ConclusionsWe developed an impedance-based cellular phenotypic drug screening platform that can identify drugs that enhance vascular barrier function. We found apigenin and ethaverine hydrochloride stabilize endothelial cell junctions and enhance the vascular barrier by blocking ARF6 activation and increasing VE-cadherin membrane localization.Translational RelevanceThe drugs identified from the phenotypic screen would have potential therapeutic efficacy in retinal vascular diseases regardless of the underlying mechanisms that promote vascular leak.

Methamphetamine‐induced retinal hyperpermeability, hypoxia, and vascularization

PurposeMethamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation.Materials and methodsC57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot.ResultsThe level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P&lt;0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P&lt;0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P&lt;0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment.ConclusionOur present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation.Support or Funding InformationThis work was supported by funding from the National Institute of Health (NIH) EY025632.

Relationship between miR-375 regulating Ndrg2/IL-6/STAT3 signaling pathway and diabetic retinopathy in rats.

To explore the relationship between micro ribonucleic acid (miR)-375 in regulating the N-Myc downstream-regulated gene 2 (Ndrg2)/interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway and diabetic retinopathy (DR) in rats. Thirty Sprague- Dawley rats were randomly divided into Control group (n=10), Model group (n=10), and miR-375 inhibitor group [miR-375 small interfering RNA (siRNA) group, n=10]. The rats in Model group were injected with streptozotocin (STZ) via the tail vein to prepare into rat models of diabetes. The body weight, fasting blood glucose, and retinal barrier permeability of rats in each group were detected. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in rat serum were measured using kits. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of optic ganglion cells in rat retinal tissues. Additionally, the messenger RNA (mRNA) and protein levels of Ndrg2, IL-6 and STAT3 in rat retinal tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Compared with Control group, Model group had reduced body weight of rats, increased blood glucose and retinal permeability of rats, raised serum MDA content, decreased SOD activity, up-regulated apoptotic rate of optic ganglia, and notably elevated mRNA and protein levels of Ndrg2, IL-6 and STAT3 in retinal tissues. Compared with those in Model group, the body weight of rats declined, the blood glucose of rats rose, the retinal permeability of rats was decreased significantly, the serum MDA content was reduced, the SOD activity was raised, the apoptotic rate of optic ganglia was decreased, and the mRNA and protein levels of Ndrg2, IL-6 and STAT3 in retinal tissues were also decreased significantly in miR-375 siRNA group. MiR-375 inhibitors are able to reduce blood glucose, retinal permeability, and optic ganglion apoptosis in rats with DR, and the mechanism of action may be related to the regulation on the Ndrg2/IL-6/STAT3 signaling pathway.

Ginsenoside Rg1 prevents early diabetic retinopathy via reducing retinal ganglion cell layer and inner nuclear layer cell apoptosis in db/db mice.

BackgroundDiabetic retinopathy (DR), a diabetic vascular complication, is prone to developing into blindness. Ginsenoside Rg1 (GRg1), a major saponin in ginseng, exerts high anti-apoptotic activity.MethodsThis study aimed to explore the protective effects of GRg1 against diabetes-induced retinal damage. Measurements of blood glucose, blood lipids and vascular permeability were performed, as well as assessments of pathological changes, and the retinal thickness of each layer. Retinal cell apoptosis related protein expression levels were measured by immunofluorescence and western blot assays.ResultsOur data demonstrated that GRg1 effectively reduced blood glucose and triglyceride levels and maintained normal retinal permeability and physiological structure. GRg1 maintained the thickness of the ganglion cell layer (GCL) and the inner nuclear layer (INL) by reducing cell apoptosis.ConclusionsThese data strongly indicate that GRg1 prevents diabetic retinal changes by decreasing GCL and INL cell apoptosis. GRg1 may be a promising drug for early DR treatment.

The potent integrin antagonist THR‐687 is a promising next‐generation drug candidate for the treatment of multifactorial retinal diseases

AbstractPurposeIntegrins are implicated in eye diseases such as diabetic retinopathy (DR) and wet age‐related macular degeneration (AMD). Targeting integrins can attenuate the main pathologic hallmarks like vascular leakage, inflammation, gliosis, angiogenesis and fibrosis. Previously, we showed that the integrin antagonist, THR‐687, has potent anti‐angiogenic properties in the cynomolgus choroidal neovascularization (CNV) model. The goal of this study was to further investigate the therapeutic effect of THR‐687 in the diabetic streptozotocin (STZ)‐induced rat model and the cynomolgus CNV model.MethodsIn STZ rats, 3 intravitreal (IVT) injections of THR‐687 (75 µg/eye) or vehicle were given with a weekly interval. Four weeks after diabetes onset vascular leakage, retinal inflammation and gliosis were analyzed by means of respectively FITC‐BSA perfusion, CD11b and vimentin staining. THR‐687 was investigated in the cynomolgus CNV model, by administering 3 IVT injections of THR‐687 (4.5 mg/eye, 2.25 mg/eye or 0.45 mg/eye) or vehicle with a weekly interval. Three weeks after laser induction the anti‐fibrotic and anti‐inflammatory potential of THR‐687 was studied by Sirius red and CD11b staining, respectively.ResultsIVT administration of THR‐687 in diabetic rats significantly decreased retinal permeability, inflammation and gliosis as compared to vehicle‐treated controls. In the cynomolgus CNV model, THR‐687 exhibited a dose‐dependent inhibition of collagen deposition in the laser spots, whereas only a trend towards decreased inflammation levels could be discerned with the highest dose level 3 weeks post‐laser.ConclusionTHR‐687 potently antagonizes integrins implicated in vascular leakage, inflammation, gliosis, angiogenesis and fibrosis as demonstrated in the diabetic STZ rat and cynomolgus CNV models. Given its multifaceted mode of action and broad therapeutic potential, THR‐687 is a promising drug candidate for the treatment of vision‐threatening retinal pathologies such as DR and wet AMD.

Asymmetric dimethylarginine aggravates blood-retinal barrier breakdown of diabetic retinopathy via inhibition of intercellular communication in retinal pericytes.

Blood-retinal barrier breakdown is the main pathological characteristics of diabetic retinopathy (DR). Asymmetric dimethylarginine (ADMA) was reported to be elevated in DR patients. In this study, we observed the dynamic profile of ADMA, retinal morphology and permeability of BRB at 2, 4 or 8week of diabetic rats induced by a single intraperitoneal injection of streptozocin (60mg/kg) and in cultured rat retinal pericytes pretreated with D-glucose (30mM) for 1, 3, 5 and 7days or ADMA (3, 10, 30 μM) for 24, 48 and 72h, trying to explore the effects of ADMA on blood-retinal barrier in DR. Gap junction intercellular communication (GJIC) and the expression of blood-retinal barrier-specific component connexin 43 (Cx43) were examined in diabetic rats or cultured retinal pericytes to elucidate whether ADMA impacted blood-retinal barrier function via damaging Cx43-GJIC. The results showed that with increasing duration of diabetes, the ultrastructure of blood-retinal barrier of diabetic rats appeared cell junction damage, apoptosis of retinal pericytes and breakdown of barrier successively. The increases in retinal permeability, ADMA levels and Cx43 expression, and abnormal GJIC were observed in diabetic rats and retinal pericytes exposed to D-glucose (30mM). A glucose-like effect was seen using ADMA or another L-arginine analogue NG-monomethyl-L-arginine or dimethylarginine dimethylaminohydrolases (DDAHs) siRNA, implicating that ADMA aggravated the breakdown of blood-retinal barrier via damaging Cx43-GJIC.

Glycyrrhizin Protects the Diabetic Retina against Permeability, Neuronal, and Vascular Damage through Anti-Inflammatory Mechanisms.

Damage associated molecular pattern (DAMPs), such as high mobility group box 1 (HMGB1), may be involved in retinal inflammation in response to high glucose. To test whether HMGB1 inhibition could protect the diabetic retina, C57BL/6J mice were made diabetic and treated with glycyrrhizin, a HMGB1 inhibitor, for up to six months. Measurements of permeability, neuronal, and vascular changes were done, as well as assessments of HMGB1, tumor necrosis factor alpha (TNFα), and interleukin-1-beta (IL1β) levels. Retinal endothelial cells (REC) treated with glycyrrhizin had reduced IL1β and cleaved caspase 3 levels. Data also demonstrate that glycyrrhizin effectively reduced HMGB1 levels throughout the retina, as well as maintained normal retinal permeability and retinal capillary coverage. Glycyrrhizin maintained normal cell numbers in the ganglion cell layer and prevented thinning of the retina at two months. These histological changes were associated with reduced reactive oxygen species, as well as reduced HMGB1, TNFα, and IL1β levels. The data strongly imply that HMGB1 inhibition prevented diabetic retinal changes through anti-inflammatory pathways.

A Novel Methodology to Assess Retinal Vascular Permeability in a Rodent Model of Diabetes

PurposeDiabetic retinopathy (DR) is a major complication of diabetes and is one of the leading causes of blindness worldwide. Treatment of DR can only be achieved through an enhanced understanding of disease pathogenesis. Structural, functional and biochemical studies cannot be carried out in human subjects and chemically induced diabetic rodent models that mimic the early developmental changes in DR and have become the gold standard. In combination with fundus fluorescein angiography (FFA) retinal abnormalities, non‐perfusion and vascular permeability can be assessed and in response to treatments over time.MethodsFFA was carried out in non‐diabetic and diabetic Norway Brown rats (250–300g) on days 0, 14, 21 and 28 using the Micron IV retinal imaging microscope (Phoenix Research Labs, CA). Animals received a single dose of PBS (n=6) or streptozotocin (STZ) i.p., 50mg/kg (n=6) and blood glucose levels were measured throughout. Video angiograms were quantitatively analysed for suboptical resolution microvascular permeability by using the arterioles as the measure for luminal concentration. In a separate subset of experiments primary human retinal endothelial cells (pHREC) were exposed to normo‐(D‐Mannitol, 30mM) or hyperglycaemia (D‐Glucose, 30mM) and probed for glycosaminoglycans (GAGs) heparan sulfate (HS), chondroitin sulfate (CS), and the tight junction marker ZO‐1.ResultsDiabetes was successfully induced in Norway Brown rats following a single dose of STZ (Blood glucose 28.4±2.6 mmol/L, day 28). By adapting Fick's Law retinal permeability (Ps, um.s^−1) was calculated and shown to significantly increase (p&lt;0.05) for each time point, over the 28‐day period. To understand the mechanisms that may account for this increase in solute permeability across the retinal vessel wall we observed a significant decrease in sulphated GAG expression, more specifically HS (p&lt;0.05) in pHREC exposed to hyperglycaemia versus normoglycaemia. In addition, the expression of the tight junction protein ZO‐1 significantly decreased in pHREC when exposed to hyperglycaemic conditions.ConclusionsRetinal vascular solute permeability can be accurately quantified in a rodent model of diabetes using the outlined methodology and mathematical quantification. Advanced quantitative assessments such as this could substantially contribute to a better understanding of the pathogenesis of DR and identify potential drugs for treatment.Support or Funding InformationMRCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NF‐kB p65 Subunit Inhibitor: JSH‐23 Mitigates Diabetic Retinopathy via Reducing Oxidative Stress

Recently we demonstrated that chronic inflammation led to the loss of ocular homeostasis during diabetic retinopathy (DR). However; whether it was propagated via the induction of oxidative stress in the eyes is not known. Oxidative insults can lead to retinal blood vessels damage that in turn may predispose them to leakage of the fluids into the retina along with subsequent development of the retinal hypoxia. These patho‐physiological changes can also result into vision impairment and overtime loss of the vision in DR patients. DR has long been considered as an outcome of neuro degeneration stemming from the excessive inflammation. Studies have pointed out that retinal cells affected by the hyperglycemic environment might exhibit an increase in the levels of oxidative stress markers that are related with NF‐κB activation. We hypothesized that sustained expression of NF‐κB activity leads to an increase in the oxidative stress environment for the retinal capillary and the glial cells in the ocular compartment. These physiological perturbations then induce occlusion and degeneration of the retinal vessels. We set out to investigate the effects of 4‐methyl‐N1 ‐ (3‐phenylpropyl) benzene‐1, 2‐diamine (also referred as JSH‐23); a cell permeable inhibitor for the NF‐κB p65 subunit in a diabetic Akita mouse model. JSH‐23 was injected intraperitoneally; IP @ 5mg/Kg body weight to both Akita and the wild type (WT) mice strains on alternate days for a period of two weeks. Hyperpermeability of retinal vessels and the intraocular pressure (IOP) were assessed via fluorescence angiography and tonometry. Visual functions were measured via the electroretinograms (ERG), and the movement of mice was recorded employing a vision guided behavioral analysis via the light‐dark box test (LDBT). Also, the systemic blood pressure (BP) and the blood glucose levels were monitored using the tail‐cuff coda instrument and glucose meter. The expressions of anti‐oxidative stress enzymes/markers and cellular‐junctional proteins were measured via Western blotting experimentations. The JSH‐23 downregulated total NF‐κB expression levels in retinae but upregulated the expression of SOD1, and catalase to fight excessive oxidative stress in eyes of mice. It also upregulated the junctional proteins: connexin‐43, occludin, and caveolin‐1. Further JSH‐23 downregulated expression of inflammasome marker; such as NLRP3. On the other hand, intervention with JSH‐23 did not change BP however; blood glucose levels were successfully lowered. Additionally, JSH‐23 treatment decreased retinal permeability, and IOP. ERG recording in the light‐adapted settings showed increased amplitude for the A and B‐waves. The vision guided behavior analysis depicted a time decrease for the treated Akita mice. Thus, considering these results we conclude that alleviating oxidative stress via administration of JSH‐23 in the diabetic mice model could improve vision.Support or Funding InformationHL‐74815, HL‐107640, HL‐139047, AR071789, and NS‐084823This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The activation of retinal HCA2 receptors by systemic beta-hydroxybutyrate inhibits diabetic retinal damage through reduction of endoplasmic reticulum stress and the NLRP3 inflammasome.

ObjectiveThe role of the hydroxycarboxylic acid receptor 2 (HCA2) in the retinal damage induced by diabetes has never been explored. In this context, the present study highlights an upregulation of retinal HCA2 receptors in diabetic C57BL6J mice. Moreover, we illustrate that HCA2 receptors exert an anti-inflammatory effect on the retinal damage induced by diabetes when activated by the endogenous ligand β-hydroxybutyrate,MethodologySeven-to-10-week-old C57BL6J mice were rendered diabetic by a single intraperitoneal injection of streptozotocin (75 mg/kg of body weight) and monitored intermittently over a 10-week period extending from the initial diabetes assessment. Mice with a fasting blood glucose level higher than 250 mg/dl for 2 consecutive weeks after streptozotocin injection were treated twice a week with intraperitoneal injections of 25-50-100 mg/kg β-hydroxybutyrate.ResultsInterestingly, while the retinal endoplasmic reticulum stress markers (pPERK, pIRE1, ATF-6α) were elevated in diabetic C57BL6J mice, their levels were significantly reduced by the systemic intraperitoneal treatment with 50 mg/kg and 100 mg/kg β-hydroxybutyrate. These mice also exhibited high NLRP3 inflammasome activity and proinflammatory cytokine levels. In fact, the elevated levels of retinal NLRP3 inflammasome activation markers (NLRP3, ASC, caspase-1) and of the relative proinflammatory cytokines (IL-1β, IL-18) were significantly reduced by 50 mg/kg and 100 mg/kg β-hydroxybutyrate treatment. These doses also reduced the high apoptotic cell number exhibited by the diabetic mice in the retinal outer nuclear layer (ONL) and increased the ONL low connexin 43 expression, leading to an improvement in retinal permeability and homeostasis.ConclusionsThese data suggest that the systemic treatment of diabetic C57BL6J mice with BHB activates retinal HCA2 and inhibits local damage.

Inhibition of Atypical Protein Kinase C Reduces Inflammation-Induced Retinal Vascular Permeability

Changes in permeability of retinal blood vessels contribute to macular edema and the pathophysiology of numerous ocular diseases, including diabetic retinopathy, retinal vein occlusions, and macular degeneration. Vascular endothelial growth factor (VEGF) induces retinal permeability and macular thickening in these diseases. However, inflammatory agents, such as tumor necrosis factor-α (TNF-α), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-α induce permeability through distinct mechanisms; however, both require the activation of atypical protein kinase C (aPKC). We provide evidence, using genetic mouse models and therapeutic intervention with small molecules, that inhibition of aPKC prevented or reduced vascular permeability in animal models of retinal inflammation. Expression of a kinase-dead aPKC transgene, driven by a vascular and hematopoietic restricted promoter, reduced retinal vascular permeability in an ischemia-reperfusion model of retinal injury. This effect was recapitulated with a small-molecule inhibitor of aPKC. Expression of the kinase-dead aPKC transgene dramatically reduced the expression of inflammatory factors and blocked the attraction of inflammatory monocytes and granulocytes after ischemic injury. Coinjection of VEGF with TNF-α was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-α-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.

Diabetic Retinopathy Reversed by Intravitreous Application of Retinol Binding Protein 3

The Joslin Medalists study, enrolling people with type 1 diabetes of &amp;gt;50 years, suggested protective factors exist against diabetic retinopathy (DR), even in the presence of hyperglycemia. Proteomic analysis of retina and vitreous suggested that Retinol Binding Protein 3 (RBP3), secreted by photoreceptors, to be elevated in the Medalists without vs. those with advanced DR. This finding was confirmed by ELISA RBP3 assays in replicating vitreous samples of Medalists and other diabetic patients. In contrast, vitreous VEGF levels increased with DR severity; resulting in decreasing RBP3/VEGF with worsening DR. Transgenic mice overexpressing RBP3 targeted to the photoreceptors prevented diabetes induced retinal capillary permeability and formation of acellular capillaries. To determine whether RBP3 can inhibit DR progression, recombinant human RBP3 (rhRBP3) was injected intravitreously (IVT) in rats with VEGF or after 2 months of diabetes. IVT injection of rhRBP3 inhibited VEGF induced increases in retinal vascular permeability both after a few min or 1 day post-VEGF injections (p=0.009 or p=0.01), respectively. After 2 months of diabetes, IVT rhRBP3 reversed capillary permeability to levels of nondiabetic rats (p=0.007), whereas boiled hRBP3 was ineffective. Surprisingly, IVT rhRBP3 also reduced retinal expression of VEGF and IL-6 in diabetic rats and normalized retinal function, as measured by B wave amplitude of electroretinography. Using cultured retinal endothelial cells and Muller cells, the addition of rhRBP3 inhibited VEGF- or high glucose-induced cell migration, possibly by binding to VEGF receptors. Surprisingly, rhRBP3 also inhibited high glucose-induced expression of VEGF and IL-6 mRNA levels (p=0.05) similar to in vivo studies. Thus, IVT application of RBP3 can intervene to delay, stop and reverse diabetes-induced neuro- and vascular-retinal functional and inflammatory abnormalities, strongly supporting a therapeutic potential for the use of RBP3 for DR. Disclosure H. Yokomizo: None. K. Park: None. A. Clermont: Consultant; Self; Kalvista Pharmaceuticals. Y. Maeda: None. W. Fickweiler: None. A. Ishikado: Employee; Self; Sunstar Inc.. Employee; Spouse/Partner; Sunstar Inc.. Q. Li: None. L.J. Tinsley: None. D.M. Pober: None. I. Wu: None. L.P. Aiello: Consultant; Self; Novo Nordisk Inc., Sanofi Genzyme. Stock/Shareholder; Self; Kalvista Pharmaceuticals, Inc.. Consultant; Self; Kalvista Pharmaceuticals, Inc. H.A. Keenan: Research Support; Self; Sanofi. Employee; Self; Sanofi Genzyme. J. Sun: Other Relationship; Self; Novartis Pharmaceuticals Corporation. Research Support; Self; Genentech, Inc., Optovue, Incorporated, Boston Micromachines Corporation, Adaptive Sensory Technology, Kalvista Pharmaceuticals, Inc.. Other Relationship; Self; Novo Nordisk Inc. G.L. King: Research Support; Self; Sanofi-Aventis.

Extended Pharmacokinetic Model of the Rabbit Eye for Intravitreal and Intracameral Injections of Macromolecules: Quantitative Analysis of Anterior and Posterior Elimination Pathways

To extend the physiological features of the anatomically accurate model of the rabbit eye for intravitreal (IVT) and intracameral (IC) injections of macromolecules. The computational fluid dynamic model of the rabbit eye by Missel (2012) was extended by enhancing the mixing in the anterior chamber with thermal gradient, heat transfer and gravity, and studying its effect on IC injections of hyaluronic acids. In IVT injections of FITC-dextrans (MW 10-157kDa) the diffusion though retina was defined based on published in vitro data. Systematic changes in retinal permeability and convective transport were made, and the percentages of anterior and posterior elimination pathways were quantified. Simulations were compared with published in vivo data. With the enhanced mixing the elimination half-lives of hyaluronic acids after IC injection were 62-100 min that are similar to in vivo data and close to the theoretical value for the well-stirred anterior chamber (57min). In IVT injections of FITC-dextrans a good match between simulations and in vivo data was obtained when the percentage of anterior elimination pathway was over 80%. The simulations with the extended model closely resemble in vivo pharmacokinetics, and the model is a valuable tool for data interpretation and predictions.

Stable and Long-Lasting, Novel Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema.

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.

An automated exudates detection in diabetic retinopathy fundus images using multi kernel spatial fuzzy c means clustering method

Microvasculature change associated with tenacious Hyperglycemia are the hostile effects accompanying to diabetes mellitus. Diabetic Retinopathy (DR) is a progressive complication, which leads to retinal permeability, ischemia, neovascularization and macular edema. The pathology is characterized by variation in capillary diameter, size of microaneurysm, hemorrhage exudates. Thus it stimulates the growth of new abnormal blood vessels so as to nourish the eye muscles. But these newly grown blood vessels are subtle, and may get burst. Therefore it leads to leakage of blood, protein based particles named as exudates. Early determination of the DR signs will help the diabetic patient to eradicate austere vision damage. Medical image processing methods helps the ophthalmologists in easy diagnosis, and to estimate the severity of the pathology. Fuzzy based clustering methods are simple and effective methods that will classify the pathos. This work furnish an improved fuzzy clustering method with induced multi kernel and spatial constraint. Statistical evaluation is done to evaluate the performance of the proposed method.

Toll-Like Receptor 4 Reduces Occludin and Zonula Occludens 1 to Increase Retinal Permeability Both in vitro and in vivo

We reported that β-adrenergic receptors regulate toll-like receptor 4 (TLR4) signaling in the retina of diabetic mice and in retinal endothelial cells (REC) and Müller cells. We hypothesized that TLR4 regulates retinal permeability both in vitro and in vivo in the retinal vasculature. We used REC cultured in normal and high-glucose conditions and TLR4 siRNA treatments for cell culture studies of permeability and protein analyses of tumor necrosis factor α, occludin, and zonula occludens 1 (ZO-1). We used endothelial cell (EC)-specific Cre-Lox TLR4 knockout mice to study retinal permeability and neuronal and vascular changes following exposure to ocular ischemia/reperfusion (I/R) used as a retinal stressor. We found that the loss of TLR4 in the EC led to the reduced permeability following I/R and fewer degenerate capillaries. Retinal permeability was increased in REC grown in high-glucose conditions but was inhibited by TLR4 siRNA treatment. High-glucose culture conditions significantly reduced occludin and ZO-1 levels in REC, and TLR4 siRNA treatment restored levels to baseline. In conclusion, these studies demonstrate that TLR4 in EC strongly regulates retinal permeability and neuronal and vascular changes following exposure to stressors such as I/R.

The SRPK1 inhibitor SPHINX31 prevents increased retinal permeability in a rodent model of diabetes

PurposeIn diabetic retinopathy (DR) microvascular damage results from ischaemia driven production of pro‐angiogenic vascular endothelial growth factor (VEGF) inducing angiogenesis and increased permeability in the retina. Small molecule inhibitors of serine‐rich protein kinase‐1 (SRPK1), with good penetration properties to the retina, have been shown to inhibit choroidal neovascularisation in mice as eye drops by decreasing pro‐angiogenic and increasing anti‐angiogenic VEGF isoforms. SRPK1 inhibitors such as SPHINX31 may therefore switch splicing in DR and prevent increased vascular permeability.MethodsFFA was performed in Norway Brown rats on day 0 and 7, using the Micron IV retinal microscope (Phoenix Research). Animals received twice daily topical eye drops with eye formulation only control buffer (n = 5) or SPHINX31 (200 μg/ml, n = 6). On day 1 animals received a single dose of streptozotocin (50 mg/kg, i.p.) to induce type I diabetes and maintained for 1 week without insulin. The ratio of interstitial to vascular fluorescence was calculated and plotted against time to determine an estimate of permeability.ResultsRetinal permeability (12.67 ± 1.09 × 10−4 cms−1) was shown to significantly increase (p &lt; 0.01) in diabetics compared to before diabetes (8.85 ± 1.29 × 10−4 cms−1) in the eye formulation control group. Following a weekly regimen of twice daily topical eye drop treatment with SPHINX31 retinal permeability was no greater in the diabetics than (7.92 ± 1.65 × 10−4 cms−1) than before induction of diabetes, (8.15 ± 2.33 × 10−4 cms−1) and in the control group.ConclusionsSPHINX31 protected the retinal endothelial permeability barrier from diabetes‐associated loss of integrity. SPHINX31 may therefore be a potential alternative and more specific topical therapeutic for DR.