IL-33trap-mediated IL-33 neutralization does not exacerbate choroidal neovascularization, but fails to protect against retinal degeneration in a dry age-related macular degeneration model.

The progressive and sight-threatening disease, age-related macular degeneration (AMD), is a growing public health concern due to ageing demographics, with the highest unmet medical need for the advanced stage of dry AMD, geographic atrophy. The pathogenesis underlying AMD is driven by a complex interplay of genetic and environmental factors. There is ample evidence that inflammation is strongly involved in AMD development. Interleukin-33 (IL-33) has been proposed to be critically involved in retinal degeneration, but a protective role in eye pathophysiology was also demonstrated. The current study investigated the therapeutic potential of IL-33trap, a novel IL-33-neutralizing biologic, in dry AMD/geographic atrophy and, based on controversial data regarding the protective versus detrimental functions of IL-33 in neovascularization, evaluated the risk of progression to wet AMD by IL-33 neutralization. Repeated intravitreal (IVT) injections of IL-33trap in the mouse laser-induced choroidal neovascularization model did not exacerbate neovascularization or leakage, while it significantly inhibited inflammatory cell infiltration in the retinal pigment epithelium and choroid. On the contrary, IVT treatment with IL-33trap significantly induced retinal inflammation and could not prevent retinopathy induction in the mouse sodium iodate (NaIO3) model. Overall, these data suggest a complex and dichotomous role of IL-33 in eye pathology and indicate that IL-33 neutralization is not able to prevent onset and progression of dry AMD pathogenesis.

Single-cell transcriptome analysis of the Akimba mouse retina reveals cell-type-specific insights into the pathobiology of diabetic retinopathy.

AIMS/HYPOTHESIS: Diabetic retinopathy is a common complication of diabetes and a leading cause of visual impairment and blindness. Despite recent advances, our understanding of its pathophysiology remains incomplete. The aim of this study was to provide deeper insight into the complex network of molecular and cellular changes that underlie diabetic retinopathy by systematically mapping the transcriptional changes that occur in the different cellular compartments of the degenerating diabetic mouse retina. METHODS: Single-cell RNA sequencing was performed on retinal tissue from 12-week-old wild-type and Akimba (Ins2Akita×Vegfa+/-) mice, which are known to replicate features of clinical diabetic retinopathy. This resulted in transcriptome data for 9474 retinal cells, which could be annotated to eight distinct retinal cell types. Using STRING analysis, we studied differentially expressed gene networks in neuronal, glial and immune cell compartments to create a comprehensive view on the pathological changes that occur in the Akimba retina. Using subclustering analysis, we further characterised macroglial and inflammatory cell subpopulations. Prominent findings were confirmed at the protein level using immunohistochemistry, western blotting and ELISA. RESULTS: At 12 weeks, the Akimba retina was found to display degeneration of rod photoreceptors and presence of inflammatory cells, identified by subclustering analysis as monocyte, macrophage and microglial populations. Analysis of differentially expressed genes in the rod, cone, bipolar cell and macroglial compartments indicated changes in cell metabolism and ribosomal gene expression, gliosis, activation of immune system pathways and redox and metal ion dyshomeostasis. Experiments at the protein level supported a metabolic shift from glycolysis to oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase), activation of microglia/macrophages (isolectin-B4), metal ion and oxidative stress response (metallothionein and haem oxygenase-1) and reactive macroglia (glial fibrillary acidic protein and S100) in the Akimba retina, compared with wild-type mice. Our single-cell approach also indicates macroglial subpopulations with distinct fibrotic, inflammatory and gliotic profiles. CONCLUSIONS/INTERPRETATION: Our study identifies molecular pathways underlying inflammatory, metabolic and oxidative stress-mediated changes in the Akimba mouse model of diabetic retinopathy and distinguishes distinct functional subtypes of inflammatory and macroglial cells. DATA AVAILABILITY: RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI ( www.ebi.ac.uk/arrayexpress ) under accession number E-MTAB-9061. Graphical abstract.

The retinal tyrosine kinome of diabetic Akimba mice highlights potential for specific Src family kinase inhibition in retinal vascular disease.

Although anti-VEGF therapies have radically changed clinical practice, there is still an urgent demand for novel, integrative approaches for sight-threatening retinal vascular diseases. As we hypothesize that protein tyrosine kinases are key signaling mediators in retinal vascular disease, we performed a comprehensive activity-based tyrosine kinome profiling on retinal tissue of 12-week-old Akimba mice, a translational model displaying hallmarks of early and advanced diabetic retinopathy. Western blotting was used to confirm retinal tyrosine kinase activity in Akimba mice. HUVEC tube formation and murine organotypic choroidal sprouting assays were applied to compare tyrosine kinase inhibitors with different specificity profiles. HUVEC toxicity and proliferation were evaluated using the CellTox™ Green Cytotoxicity and PrestoBlue™ Assays. Our results indicate a shift of the Akimba retinal tyrosine kinome towards a hyperactive state. Functional network analysis of significantly hyperphosphorylated peptides and upstream kinase prediction revealed a central role for Src-FAK family kinases. Western blotting confirmed hyperactivity of this signaling node in the retina of Akimba mice. We demonstrated that not only Src but also FAK family kinase inhibitors with different selectivity profiles were able to suppress angiogenesis in vitro and ex vivo. In the latter model, the novel selective Src family kinase inhibitor eCF506 was able to achieve potent reduction of angiogenesis, comparable to the less specific inhibitor Dasatinib. None of the tested compounds demonstrated acute endothelial cell toxicity. Overall, the collected findings provide the first comprehensive overview of retinal tyrosine kinome changes in the Akimba model of diabetic retinopathy and for the first time highlight Src family kinase inhibition using highly specific inhibitors as an attractive therapeutic intervention for retinal vascular pathology.

The potent small molecule integrin antagonist THR-687 is a promising next-generation therapy for retinal vascular disorders.

Integrins are associated with various eye diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) and implicated in main pathologic disease hallmarks like neovascularization, inflammation, fibrosis and vascular leakage. Targeting integrins has the potential to attenuate these vision-threatening processes, independent of anti-vascular endothelial growth factor (VEGF) responsiveness. The current investigation characterized THR-687 as a novel pan RGD (arginylglycylaspartic acid) integrin receptor antagonist able to compete for binding with the natural ligand with nanomolar potency (e.g. αvß3 (IC50 of 4.4 ±â€¯2.7 nM), αvß5 (IC50 of 1.3 ±â€¯0.5 nM) and α5ß1 (IC50 of 6.8 ±â€¯3.2 nM)). THR-687 prevented the migration of human umbilical vein endothelial cells (HUVECs) into a cell-free area (IC50 of 258 ±â€¯113 nM) as well as vessel sprouting in an ex vivo mouse choroidal explant model (IC50 of 236 ±â€¯173 nM), and was able to induce the regression of pre-existing vascular sprouts. Moreover, combined intravitreal and intraperitoneal administration of THR-687 potently inhibited VEGF-induced leakage in the mouse retina. In addition, THR-687 injected intravitreally at 3 different dose levels (0.45 mg, 2.25 mg or 4.5 mg/eye) potently inhibited neovascularization-induced leakage in the cynomolgus laser-induced choroidal neovascularization (CNV) model. These data suggest that THR-687 is a promising drug candidate for the treatment of vision-threatening retinal vascular eye diseases such as DR and wet AMD.

Neutralization of placental growth factor as a novel treatment option in diabetic retinopathy.

The current standard of care in clinical practice for diabetic retinopathy (DR), anti-vascular endothelial growth factor (VEGF) therapy, has shown a significant improvement in visual acuity. However, treatment response can be variable and might be associated with potential side effects. This study was designed to investigate inhibition of placental growth factor (PlGF) as a possible alternative therapy for DR. The effect of the anti-PlGF antibody (PL5D11D4) was preclinically evaluated in various animal models by investigating different DR hallmarks, including inflammation, neurodegeneration, vascular leakage and fibrosis. The in vivo efficacy was tested in diabetic streptozotocin (STZ) and Akimba models and in the laser induced choroidal neovascularization (CNV) mouse model. Intravitreal (IVT) administration of the anti-PlGF antibody was compared to anti-VEGFR-2 antibody (DC101), anti-VEGF antibody (B20), VEGF-Trap (aflibercept) and triamcinolone acetonide (TAAC). Vascular leakage was investigated in the mouse STZ model by fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) perfusion and in the Akimba model by fluorescein angiography (FA). Repeated IVT administration of the anti-PlGF antibody reduced vascular leakage, which was comparable to a single administration of VEGFR-2 inhibition in the mouse STZ model. PL5D11D4 treatment did not alter retinal ganglion cell (RGC) density, as demonstrated by Brn3a staining, whereas DC101 significantly reduced RGC number with 20%. Immunohistological stainings were performed to investigate inflammation (CD45, F4/80) and fibrosis (collagen type 1a). In the CNV model, IVT injection(s) of PL5D11D4 dose-dependently reduced inflammation and fibrosis, as compared to PBS treatment. Equimolar single administration of the anti-PlGF antibody and aflibercept (21 nM) and TAAC decreased leukocyte and macrophage infiltration with 50%, whereas DC101 and B20 (21 nM) had no effect on the inflammatory response. Similar results were observed in the mouse STZ model on the number of microglia and macrophages in the retina. Repeated administration of PL5D11D4 (21 nM) and TAAC similarly reduced fibrosis, while no effect was observed after equimolar DC101, B20 nor aflibercept administration (21 nM). In summary, the anti-PlGF antibody showed comparable efficacy as well-characterized VEGF-inhibitor on the process of vascular leakage, but differentiates itself by also reducing inflammation and fibrosis, without triggering a neurodegenerative response.

Preclinical characterization of GLPG0634, a selective inhibitor of JAK1, for the treatment of inflammatory diseases.

The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors. In a search for JAK small molecule inhibitors, GLPG0634 was identified as a lead compound belonging to a novel class of JAK inhibitors. It displayed a JAK1/JAK2 inhibitor profile in biochemical assays, but subsequent studies in cellular and whole blood assays revealed a selectivity of ∼30-fold for JAK1- over JAK2-dependent signaling. GLPG0634 dose-dependently inhibited Th1 and Th2 differentiation and to a lesser extent the differentiation of Th17 cells in vitro. GLPG0634 was well exposed in rodents upon oral dosing, and exposure levels correlated with repression of Mx2 expression in leukocytes. Oral dosing of GLPG0634 in a therapeutic set-up in a collagen-induced arthritis model in rodents resulted in a significant dose-dependent reduction of the disease progression. Paw swelling, bone and cartilage degradation, and levels of inflammatory cytokines were reduced by GLPG0634 treatment. Efficacy of GLPG0634 in the collagen-induced arthritis models was comparable to the results obtained with etanercept. In conclusion, the JAK1 selective inhibitor GLPG0634 is a promising novel therapeutic with potential for oral treatment of rheumatoid arthritis and possibly other immune-inflammatory diseases.

Common pathways in dementia and diabetic retinopathy: understanding the mechanisms of diabetes-related cognitive decline.

Type 2 diabetes (T2D) is associated with multiple comorbidities, including diabetic retinopathy (DR) and cognitive decline, and T2D patients have a significantly higher risk of developing Alzheimer's disease (AD). Both DR and AD are characterized by a number of pathological mechanisms that coalesce around the neurovascular unit, including neuroinflammation and degeneration, vascular degeneration, and glial activation. Chronic hyperglycemia and insulin resistance also play a significant role, leading to activation of pathological mechanisms such as increased oxidative stress and the accumulation of advanced glycation end-products (AGEs). Understanding these common pathways and the degree to which they occur simultaneously in the brain and retina during diabetes will provide avenues to identify T2D patients at risk of cognitive decline.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

Targeting Plasma Kallikrein With a Novel Bicyclic Peptide Inhibitor (THR-149) Reduces Retinal Thickening in a Diabetic Rat Model.

Purpose: To investigate the effect of plasma kallikrein (PKal)-inhibition by THR-149 on preventing key pathologies associated with diabetic macular edema (DME) in a rat model. Methods: Following streptozotocin-induced diabetes, THR-149 or its vehicle was administered in the rat via either a single intravitreal injection or three consecutive intravitreal injections (with a 1-week interval; both, 12.5 µg/eye). At 4 weeks post-diabetes, the effect of all groups was compared by histological analysis of Iba1-positive retinal inflammatory cells, inflammatory cytokines, vimentin-positive Müller cells, inwardly rectifying potassium and water homeostasis-related channels (Kir4.1 and AQP4, respectively), vascular leakage (fluorescein isothiocyanate-labeled bovine serum albumin), and retinal thickness. Results: Single or repeated THR-149 injections resulted in reduced inflammation, as depicted by decreasing numbers and activation state of immune cells and IL-6 cytokine levels in the diabetic retina. The processes of reactive gliosis, vessel leakage, and retinal thickening were only significantly reduced after multiple THR-149 administrations. Individual retinal layer analysis showed that repeated THR-149 injections significantly decreased diabetes-induced thickening of the inner plexiform, inner nuclear, outer nuclear, and photoreceptor layers. At the glial-vascular interface, reduced Kir4.1-channel levels in the diabetic retina were restored to control non-diabetic levels in the presence of THR-149. In contrast, little or no effect of THR-149 was observed on the AQP4-channel levels. Conclusions: These data demonstrate that repeated THR-149 administration reduces several DME-related key pathologies such as retinal thickening and neuropil disruption in the diabetic rat. These observations indicate that modulation of the PKal pathway using THR-149 has clinical potential to treat patients with DME.

Targeting RGD-binding integrins as an integrative therapy for diabetic retinopathy and neovascular age-related macular degeneration.

Integrins are a class of transmembrane receptors that are involved in a wide range of biological functions. Dysregulation of integrins has been implicated in many pathological processes and consequently, they are attractive therapeutic targets. In the ophthalmology arena, there is extensive evidence suggesting that integrins play an important role in diabetic retinopathy (DR), age-related macular degeneration (AMD), glaucoma, dry eye disease and retinal vein occlusion. For example, there is extensive evidence that arginyl-glycyl-aspartic acid (Arg-Gly-Asp; RGD)-binding integrins are involved in key disease hallmarks of DR and neovascular AMD (nvAMD), specifically inflammation, vascular leakage, angiogenesis and fibrosis. Based on such evidence, drugs that engage integrin-linked pathways have received attention for their potential to block all these vision-threatening pathways. This review focuses on the pathophysiological role that RGD-binding integrins can have in complex multifactorial retinal disorders like DR, diabetic macular edema (DME) and nvAMD, which are leading causes of blindness in developed countries. Special emphasis will be given on how RGD-binding integrins can modulate the intricate molecular pathways and regulate the underlying pathological mechanisms. For instance, the interplay between integrins and key molecular players such as growth factors, cytokines and enzymes will be summarized. In addition, recent clinical advances linked to targeting RGD-binding integrins in the context of DME and nvAMD will be discussed alongside future potential for limiting progression of these diseases.

Heparanase Deficiency Is Associated with Disruption, Detachment, and Folding of the Retinal Pigment Epithelium.

PURPOSE: Pentosan polysulfate sodium (PPS; Elmiron) is a FDA-approved heparanase inhibitor for the treatment of bladder pain and interstitial cystitis. The chronic use of PPS has been associated with a novel pigmentary maculopathy, associated with discrete vitelliform deposits that exhibit hyperfluorescence, macular hyper-pigmentary spots, and foci of nodular RPE enlargement. Therefore, this study aimed to investigate the retinal morphology of heparanase knockout mice. MATERIAL AND METHODS: The retinal morphology of heparanase knock-out and age-matched control wild type mice of 3-, 9- and 15-weeks old was characterized by means of histological evaluation. Immuno-histological stains for RPE65, F4/80 and Ki67 were performed for investigating the RPE, inflammatory and proliferating cells, respectively. RESULTS: Histological analysis showed no changes in age-matched wild-type controls, whereas the eyes of heparanase null mice were characterized by alterations in RPE and neural retina, as manifest by RPE folds and choroidal thickening, detached RPE cells, thickening of the photoreceptor layer and retinal disorganization. The presence of discrete hyperfluorescent foci, however, was absent. The prevalence of the RPE/choroidal changes or protrusions seemed to progress over time and were correlated with more RPE65 signal rather than influx of F4/80- or Ki67-positive cells. These results indicate that the subretinal alterations were mostly RPE driven, without influx of inflammatory or proliferating cells. CONCLUSIONS: Our results indicate that heparanase deficiency in the mice leads to RPE folds, choroidal thickening, and retinal disorganization. The presence of discrete hyperfluorescent foci, a key characteristic of the human disease, was not observed. However, it can be concluded that some of the observations in mice are similar to those seen after chronic use of PPS in humans. These findings indicate that the toxicity observed in the presence of heparanase inhibitors is target-related and will preclude the clinical use of heparanase inhibition as a therapeutic intervention.

Phase I trial on robot assisted retinal vein cannulation with ocriplasmin infusion for central retinal vein occlusion.

PURPOSE: To evaluate the safety and feasibility of robot-assisted retinal vein cannulation with Ocriplasmin infusion for central retinal vein occlusion. METHODS: Prospective phase I trial including four patients suffering from central retinal vein occlusion (CRVO). Diagnosis was confirmed by preoperative fluo-angiography and followed by a standard three-port pars plana vitrectomy. Afterwards, a custom-built microneedle was inserted into a branch retinal vein with robotic assistance and infusion of Ocriplasmin started. Primary outcomes were the occurrence of intra-operative complications and success of cannulation. Secondary outcomes were change in visual acuity, central macular thickness (CMT) and venous filling times (VFT) during fluo-angiography two weeks after the intervention. RESULTS: Cannulation with infusion of ocriplasmin was successful in all four eyes with a mean total infusion time of 355 ± 204 seconds (range 120-600 seconds). Best corrected visual acuity (BCVA) remained counting fingers (CF) in case 3 and 4, increased in case 1 from CF to 0.9LogMAR and decreased in case 2 from 0.4 to 1.3 LogMAR. CMT and VFT both showed a trend towards significant decrease comparing preoperative measurements with two weeks postintervention (1061 ± 541 µm versus 477 ± 376 µm, p = 0.068) and 24 ll 4 seconds versus 15 ± 1 seconds, p = 0.068, respectively). In one eye a needle tip broke and could be removed with an endoforceps. There were no other intervention-related complications. CONCLUSION: Robot-assisted retinal vein cannulation is feasible and safe. Local intravenous infusion with Ocriplasmin led to an improved retinal circulation.

The Placental Growth Factor Pathway and Its Potential Role in Macular Degenerative Disease.

There is growing evidence that placental growth factor (PlGF) is an important player in multiple pathologies, including tumorigenesis, inflammatory disorders and degenerative retinopathies. PlGF is a member of the vascular endothelial growth factor (VEGF) family and in the retina, binding of this growth factor to specific receptors is associated with pathological angiogenesis, vascular leakage, neurodegeneration and inflammation. Although they share some receptor signalling pathways, many of the actions of PlGF are distinct from VEGF and this has revealed the enticing prospect that it could be a useful therapeutic target for treating early and late stages of diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD). Recent research suggests that modulation of PlGF could also be important in the geographic atrophy (GA) form of late AMD by protecting the outer retina and the retinal pigment epithelium (RPE). This review discusses PlGF and its signalling pathways and highlights the potential of blocking the bioactivity of this growth factor to treat irreversible visual loss due to the two main forms of AMD.

The role of placental growth factor (PlGF) and its receptor system in retinal vascular diseases.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Upon binding to VEGF- and neuropilin-receptor sub-types, PlGF modulates a range of neural, glial and vascular cell responses that are distinct from VEGF-A. As PlGF expression is selectively associated with pathological angiogenesis and inflammation, its blockade does not affect the healthy vasculature. PlGF actions have been extensively described in tumor biology but more recently there has been accumulating preclinical evidence that indicates that this growth factor could have an important role in retinal diseases. High levels of PlGF have been found in aqueous humor, vitreous and/or retina of patients exhibiting retinopathies, especially those with diabetic retinopathy (DR) and neovascular age-related macular degeneration (nvAMD). Expression of this growth factor seems to correlate closely with many of the key pathogenic features of early and late retinopathy in preclinical models. For example, studies using genetic modification and/or pharmacological treatment to block PlGF in the laser-induced choroidal neovascularization (CNV) model, oxygen-induced retinopathy model, as well as various murine diabetic models, have shown that PlGF deletion or inhibition can reduce neovascularization, retinal leakage, inflammation and gliosis, without affecting vascular development or inducing neuronal degeneration. Moreover, an inhibitory effect of PlGF blockade on retinal scarring in the mouse CNV model has also been recently demonstrated and was found to be unique for PlGF inhibition, as compared to various VEGF inhibition strategies. Together, these preclinical results suggest that anti-PlGF therapy might have advantages over anti-VEGF treatment, and that it may have clinical applications as a standalone treatment or in combination with anti-VEGF. Additional clinical studies are clearly needed to further elucidate the role of PlGF and its potential as a therapeutic target in ocular diseases.

Longitudinal In Vivo Characterization of the Streptozotocin-Induced Diabetic Mouse Model: Focus on Early Inner Retinal Responses.

Purpose: The goal of this study was to perform an extensive temporal characterization of the early pathologic processes in the streptozotocin (STZ)-induced diabetic retinopathy (DR) mouse model, beyond the vascular phenotype, and to investigate the potential of clinically relevant compounds in attenuating these processes. Methods: Visual acuity and contrast sensitivity (CS) were studied in the mouse STZ model until 24 weeks postdiabetes onset. ERG, spectral domain optical coherence tomography (SD-OCT), leukostasis, and immunohistochemistry were applied to investigate neurodegeneration, inflammation, and gliosis during early-, mid- and late-phase diabetes. Aflibercept or triamcinolone acetonide (TAAC) was administered to investigate their efficacy on the aforementioned processes. Results: Visual acuity and CS loss started at 4 and 18 weeks postdiabetes onset, respectively, and progressively declined over time. ERG amplitudes were diminished and OP latencies increased after 6 weeks, whereas SD-OCT revealed retinal thinning from 4 weeks postdiabetes. Immunohistochemical analyses linked these findings to retinal ganglion and cholinergic amacrine cell loss at 4 and 8 weeks postdiabetes onset, respectively, which was further decreased after aflibercept administration. The number of adherent leukocytes was augmented after 2 weeks, whereas increased micro- and macroglia reactivity was present from 4 weeks postdiabetes. Aflibercept or TAAC showed improved efficacy on inflammation and gliosis. Conclusions: STZ-induced diabetic mice developed early pathologic DR hallmarks, from which inflammation seemed the initial trigger, leading to further development of functional and morphologic retinal changes. These findings indicate that the mouse STZ model is suitable to study novel integrative non-vascular therapies to treat early DR.

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.

Is exogenous tissue plasminogen activator necessary for antithrombotic efficacy of an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI) in rats?

INTRODUCTION: TAFI indirectly reduces the action of tPA on plasminogen. Whether exogenous tPA is necessary for TAFI inhibitor efficacy is unclear. Potato carboxypeptidase inhibitor (PCI), a TAFI inhibitor, has shown variable tPA dependence in rat models of arteriovenous shunt thrombosis (required) and microthrombosis (not required). This study was designed to further explore the importance of exogenous tPA in revealing PCI activity in rat models of venous and arterial thrombosis and provoked bleeding. METHODS: PCI was given as a bolus (5, 10 mg/kg) +/- infusion (5, 10 mg/kg/h) and with or without low dose tPA (5, 10, 25 microg/kg/min). In each instance tPA was adjusted to produce subthreshold thrombus reduction. Arterial thrombosis was induced by FeCl2; venous thrombosis by tissue factor or FeCl2. Bleeding was induced by kidney incision with PCI given (5 mg + 5 mg/kg/h) in the presence or absence of tPA (10, 150, 200 microg/kg/min). RESULTS: PCI was ineffective without exogenous tPA in all tested thrombosis models. With exogenous tPA, PCI decreased thrombus weight 85% in tissue factor thrombosis, 59% in FeCl2 thrombosis, and 46% in arterial thrombosis. PCI prolonged bleeding only when combined with a relatively high tPA dose (200 microg/kg/min) that increased bleeding alone. CONCLUSIONS: If the current results predict clinical efficacy, the need for exogenous tPA in combination with TAFI inhibition is a potential problem. However, in acute settings where intravenous fibrinolytics are administered, or indications in which tPA production increases, TAFI inhibitors may prove to be safe and moderately effective profibrinolytic agents.

Assessment of Ocriplasmin Effects on the Vitreoretinal Compartment in Porcine and Human Model Systems.

Ocriplasmin (Jetrea®) is a recombinant protease used to treat vitreomacular traction. To gain insight into vitreoretinal observations reported after ocriplasmin treatment, we have developed an in vivo porcine ocriplasmin-induced posterior vitreous detachment (PVD) model in which we investigated vitreoretinal tissues by optical coherence tomography, histology, and cytokine profiling. Eight weeks postinjection, ocriplasmin yielded PVD in 82% of eyes. Subretinal fluid (85%) and vitreous hyperreflective spots (45%) were resolved by week 3. Histological analysis of extracellular matrix (ECM) proteins such as laminin, fibronectin, and collagen IV indicated no retinal ocriplasmin-induced ECM distribution changes. Retinal morphology was unaffected in all eyes. Cytokine profiles of ocriplasmin-treated eyes were not different from vehicle. In cell-based electrical resistance assays, blood-retinal barrier permeability was altered by ocriplasmin concentrations of 6 µg/mL and higher, with all effects being nontoxic, cell-type specific, and reversible. Ocriplasmin was actively taken up by RPE and Müller cells, and our data suggest both lysosomal and transcellular clearance routes for ocriplasmin. In conclusion, transient hyperreflective spots and fluid in a porcine ocriplasmin-induced PVD model did not correlate with retinal ECM rearrangement nor inflammation. Reversible in vitro effects on blood-retinal barrier permeability provide grounds for a hypothesis on the mechanisms behind transient subretinal fluid observed in ocriplasmin-treated patients.

Intravitreally Injected Fluid Dispersion: Importance of Injection Technique.

Purpose: The purpose of this study was to evaluate the dispersion of intravitreally injected solutions and investigate the influence of varying injection techniques. Methods: This was a prospective study using enucleated porcine eyes and ultra-high-resolution computed tomography (UHRCT) scanning to visualize iomeprol intravitreal dispersion. Sixty eyes were divided over 12 different groups according to the injection procedure: fast (2 seconds) or slow (10 seconds) injection speed and needle tip location (6- and 12-mm needle shaft insertion or premacular tip placement verified by indirect ophthalmoscopy). For each of these combinations, eyes were either injected with the combination of V20I (which is an analogue of ocriplasmin) and iomeprol or iomeprol alone. Distance to the macula and volume measurements were performed at 1, 2, 3, and 5 hours after injection. Results: The measured contrast bolus volume increases slowly over time to an average of 0.70 (P = 0.03), 1.04 (P = 0.006), and 0.79 (P = 0.0001) cm3 5 hours after the injection for the 6-mm needle shaft insertion, 12-mm needle shaft insertion, and premacular needle tip placement, respectively. The distance to the macular marker was significantly lower for premacular needle tip placement injections compared with 6- and 12-mm needle shaft insertion depths. Conclusions: Ultra-high-resolution computed tomography with three-dimensional reconstruction offers the possibility to study the dispersion of intravitreally injected solutions in a noninvasive manner. Intravitreal premacular solution delivery is possible with an indirect ophthalmoscope-guided injection technique and significantly reduces the time to reach the posterior pole in respect to 6- and 12-mm needle insertion depths. The speed of injection does not influence dispersion significantly.

Robot-assisted retinal vein cannulation in an in vivo porcine retinal vein occlusion model.

PURPOSE: To evaluate the feasibility of robot-assisted retinal vein cannulation for retinal vein occlusion. METHODS: Prospective experimental study performed in in vivo porcine eyes. A standard three port pars plana vitrectomy was followed by laser-induced branch retinal vein occlusion. Consequently, a retinal vein cannulation with the help of a surgical robot and a microneedle was performed. Complete success was defined as a stable intravenous position of the needle tip confirmed by blood washout for at least 3 min. Secondary outcomes were the occurrence of intra-operative complications and technical failures. RESULTS: Cannulation was successful in 15 of 18 eyes with a complete success rate (duration of infusion of more than 3 min) of 73% after exclusion of two eyes from analysis due to failure in establishing a blood clot. There were no technical failures regarding the robotic device. The intravessel injections of ocriplasmin in two of two eyes led to a clot dissolution. In a subset of five eyes, a second cannulation attempt at the border of the optic disc resulted in a stable intravessel position and infusion during 362 (±138) seconds. CONCLUSION: Robot-assisted retinal vein cannulation with prolonged infusion time is technically feasible. Human experiments are required to analyse the clinical benefit of this new therapy.