The age factor in optic nerve regeneration: Intrinsic and extrinsic barriers hinder successful recovery in the short-living killifish.

As the mammalian central nervous system matures, its regenerative ability decreases, leading to incomplete or non-recovery from the neurodegenerative diseases and central nervous system insults that we are increasingly facing in our aging world population. Current neuroregenerative research is largely directed toward identifying the molecular and cellular players that underlie central nervous system repair, yet it repeatedly ignores the aging context in which many of these diseases appear. Using an optic nerve crush model in a novel biogerontology model, that is, the short-living African turquoise killifish, the impact of aging on injury-induced optic nerve repair was investigated. This work reveals an age-related decline in axonal regeneration in female killifish, with different phases of the repair process being affected depending on the age. Interestingly, as in mammals, both a reduced intrinsic growth potential and a non-supportive cellular environment seem to lie at the basis of this impairment. Overall, we introduce the killifish visual system and its age-dependent regenerative ability as a model to identify new targets for neurorepair in non-regenerating individuals, thereby also considering the effects of aging on neurorepair.

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.

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.

Systemic exposure following intravitreal administration of therapeutic agents: an integrated pharmacokinetic approach. 1. THR-149.

Intravitreal (IVT) injection of pharmacological agents is an established and widely used procedure for the treatment of many posterior segment of the eye diseases. IVT injections permit drugs to reach high concentrations in the retina whilst limiting systemic exposure. Beyond the risk of secondary complications such as intraocular infection, the potential of systemic adverse events cannot be neglected. Therefore, a detailed understanding of the rules governing systemic exposure following IVT drug administration remains a prerequisite for the evaluation and development of new pharmacological agents intended for eye delivery. We present here a novel mathematical model to describe and predict circulating drug levels following IVT in the rabbit eye, a species which is widely used for drug delivery, pharmacokinetic, and pharmacodynamic studies. The mathematical expression was derived from a pharmacokinetic model that assumes the existence of a compartment between the vitreous humor compartment itself and the systemic compartment. We show that the model accurately describes circulating levels of THR-149, a plasma kallikrein inhibitor in development for the treatment of diabetic macular edema. We hypothesize that the model based on the rabbit eye has broader relevance to the human eye and can be used to analyze systemic exposure of a variety of drugs delivered in the eye.

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.

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.

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.

Tightening the retinal glia limitans attenuates neuroinflammation after optic nerve injury.

Increasing evidence suggests that functional impairments at the level of the neurovascular unit (NVU) underlie many neurodegenerative and neuroinflammatory diseases. While being part of the NVU, astrocytes have been largely overlooked in this context and only recently, tightening of the glia limitans has been put forward as an important neuroprotective response to limit these injurious processes. In this study, using the retina as a central nervous system (CNS) model organ, we investigated the structure and function of the glia limitans, and reveal that the blood-retina barrier and glia limitans function as a coordinated double barrier to limit infiltration of leukocytes and immune molecules. We provide in vitro and in vivo evidence for a protective response at the NVU upon CNS injury, which evokes inflammation-induced glia limitans tightening. Matrix metalloproteinase-3 (MMP-3) was found to be a crucial regulator of this process, thereby revealing its beneficial and immunomodulatory role in the CNS. in vivo experiments in which MMP-3 activity was deleted via genetic and pharmacological approaches, combined with a comprehensive study of tight junction molecules, glial end feet markers, myeloid cell infiltration, cytokine expression and neurodegeneration, show that MMP-3 attenuates neuroinflammation and neurodegeneration by tightening the glia limitans, thereby pointing to a prominent role of MMP-3 in preserving the integrity of the NVU upon injury. Finally, we gathered promising evidence to suggest that IL1b, which is also regulated by MMP-3, is at least one of the molecular messengers that induces glia limitans tightening in the injured CNS.

Single-Cell RNA Sequencing Maps Endothelial Metabolic Plasticity in Pathological Angiogenesis.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets.

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.

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.

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.

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.

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.

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.

Integrin α5ß1 Inhibition by CLT-28643 Reduces Postoperative Wound Healing in a Mouse Model of Glaucoma Filtration Surgery.

Purpose: To evaluate the therapeutic potential of the small molecule integrin α5ß1 inhibitor, CLT-28643, to improve the filtering surgery outcome in a mouse model. Different dose regimens and administration routes of the inhibitor were compared with mitomycin C (MMC), the gold standard in clin ical practice. Methods: The efficacy of CLT-28643 on surgical outcome was studied in a mouse model for filtering surgery (n = 40 eyes from 20 mice per group). Single and repeated subconjunctival (SCJ) injections (1 or 2 µg) and topical eye drops (10 µg) of the integrin inhibitor were compared with 2-minute administration of MMC 0.02%. Bleb size, survival, and signs of toxicity were examined until 28 days after surgery. Immunohistochemical analysis of angiogenesis, inflammation, collagen deposition, and integrin α5ß1 expression were performed on postoperative days 3, 8, 14, and 28. A masked observer performed all the assessments. Results: Immunostaining showed that integrin α5ß1 was highly expressed in the bleb at early time-points after surgery and that CLT-28643 inhibited this upregulation. Efficacy was shown to be dose-dependent for the integrin inhibitor CLT-28643 for bleb area and survival, and the wound healing process. While 2-µg single injection of CLT-28643 improved bleb characteristics in a similar way as 10-µg administered by eye drops and MMC, repeated injections of 2 µg showed superior efficacy compared to MMC, with no corneal toxicity. Conclusions: Administration of the integrin α5ß1 inhibitor CLT-28643 has therapeutic potential as an adjunct to glaucoma surgery, possibly with a superior efficacy and tolerability compared with MMC when used at the optimal dose.

The Combination of PlGF Inhibition and MMC as a Novel Anti-Scarring Strategy for Glaucoma Filtration Surgery.

PURPOSE: The complementary effects of mitomycin-C (MMC) and anti-placental growth factor (PlGF) therapy were explored and compared to the combined administration of MMC and aflibercept. Additionally, the effect of PlGF (inhibition) on IOP was investigated, since aqueous PlGF is known to be upregulated in glaucoma patients. METHODS: In the trabeculectomy mouse model, intracameral injection(s) of the PlGF inhibitor (5D11D4) were compared to MMC or aflibercept and to the combination of both compounds. Treatment outcome was studied by bleb investigation and by Sirius Red staining. The effect of subconjunctival PlGF administration and topical 5D11D4 on IOP was investigated in normotensive mice and was compared to topical administration of latanoprost, the gold standard for IOP-lowering. RESULTS: Combination of MMC and 5D11D4 was able to significantly improve surgical outcome compared to monotherapy of MMC or 5D11D4 (n = 20; P < 0.001). Compared to combined treatment of MMC with aflibercept, the simultaneous administration of MMC and 5D11D4 was equally efficacious in improving surgical outcome (n = 15; P = 0.88). In normotensive mice, 5D11D4 was able to significantly reduce the IOP-elevation induced by PlGF (n = 10; P < 0.05), whereas no effect of 5D11D4 was seen in naive mice, which was in contrast to latanoprost. CONCLUSIONS: The current data suggest that application of MMC together with PlGF inhibition may have complementary effects in the improvement of surgical outcome and is equally efficacious as the combined treatment of MMC and aflibercept. Inhibition of PlGF also might open alternative perspectives as IOP-lowering strategy for glaucoma patients with increased aqueous PlGF levels.

Modulation of wound healing in glaucoma surgery.

Glaucoma is a neurodegenerative disease and is the second most important cause of irreversible blindness. Filtration surgery remains the most effective therapy to reduce intraocular pressure in glaucoma patients. The main determinant of long-term surgical success is the healing response. Excessive postoperative wound healing with subsequent fibrosis may lead to obstruction of the created channel which frequently results in early surgical failure and consequent progression of visual field loss. Preoperative use of antimitotics, such as mitomycin-C and 5-fluorouracyl, effectively improves surgery outcome. However, the use of these nonspecific antiproliferative agents can be associated with severe side effects. This review provides an overview of the most important efforts that have been made to explore novel, more specific, and safer agents to prevent glaucoma filtration failure and improve surgery outcome.

Rho kinase inhibitor AMA0526 improves surgical outcome in a rabbit model of glaucoma filtration surgery.

PURPOSE: First, to elucidate the effect of Rho kinase inhibitor, AMA0526, on Human Tenon Fibroblast (HTF) proliferation and transdifferentiation to myofibroblasts. Second, the effects of ROCK inhibition on the wound healing process and surgical outcome were investigated in a rabbit model of glaucoma filtration surgery. METHODS: After exposure of HTF to AMA0526 (0.1-25 µM), a water-soluble tetrazolium salt-1 assay and caspase 3/7 activity assay were used to assess its effect on cell proliferation and to elucidate any toxic effects, respectively. Immunohistochemistry of α-smooth muscle actin expression was used to investigate fibroblast-to-myofibroblast differentiation induced by transforming growth factor-beta 1 (TGF-ß1) in the presence or absence of the ROCK inhibitor. The effect of topical treatment was studied in a rabbit model of glaucoma filtration surgery. Treatment outcome was studied by performing intraocular pressure (IOP) measurements and clinical investigation of the bleb area and survival. Immunohistological analysis for inflammation (CD45), angiogenesis (CD31), and collagen I was performed at day 8, 14, and 30 after surgery (n=5/time point). Separate control groups treated with vehicle were used as control. RESULTS: In vitro results showed that AMA0526 dose dependently inhibited proliferation of HTF (P<0.05) without the induction of caspase 3/7 activity. Incubation of HTF with the AMA0526 inhibited TGF-ß1 induced fibroblast-to-myofibroblast differentiation. In the rabbit model, topical treatment significantly improved surgical outcome. Compared to vehicle-treated eyes, AMA0526 resulted in increased bleb area (P<0.0001) and prolonged survival (P=0.0025). IOP remained significantly lower throughout the course of the experiment in the AMA0526 group (P<0.0001). Histological evaluation revealed that blebs treated with the ROCK inhibitor were characterized by reduced inflammation, angiogenesis, and collagen deposition at the site of filtration surgery (P<0.05). CONCLUSIONS: AMA0526 had profound effects on HTF proliferation and myofibroblast transition and improved glaucoma filtration surgery outcome by interfering at different levels of the wound healing process. Therefore, these data indicate that ROCK inhibitors may be considered as more physiological agents which specifically target the wound healing process to improve the outcome of glaucoma surgery.