Search Results (33)

Retinal ischemia is implicated in ocular diseases involving aberrant neovessel proliferation that characterizes proliferative retinopathies. Their therapy still remains confined to the intravitreal administration of anti-vascular endothelial growth factor (VEGF) medication, which is limited by side effects and progressive reduction in efficacy. Mimicking neovascular diseases in rodents, although of great help for translating fundamental mechanistic findings and assessing therapeutic potential in humans, is limited by the rodent’s short life span, which prevents retinal vessel proliferation over time. However, the oxygen-induced retinopathy (OIR) model, which mimics retinopathy of prematurity, seems to meet some criteria that are common to proliferative retinopathies. The present review provides insight into preclinical models and their suitability to mimic proliferative retinopathies. Further considerations will be applied to emerging approaches and advanced methodologies for the management of proliferative retinopathies, leading to the identification of new therapeutic targets, including our contribution in the field. Major emphasis is given to the possibility of using systemic therapies either alone or in combination with intravitreal anti-VEGF administration to maximize clinical benefits by combining drugs with different modes of action. This review is concluded by an in-depth discussion on future advancements and a critical view of preclinical finding translatability. Despite the major effort of preclinical and clinical research to develop novel therapies, the blockade of VEGF activity still remains the only treatment for proliferative retinopathies for more than twenty years since its first therapeutic application. Full article

Background/Objectives: Exudative age-related macular degeneration (AMD) is a disease of choroidal neovascularization that causes blindness. Current treatments to preserve vision in this prevalent and blinding condition are repeat intraocular injections of anti-vascular endothelial growth factor medicines for a patient’s lifetime to preserve and prevent vision loss leading to blindness. Rifampicin, a small-molecule antibiotic, has previously been reported to exhibit anti-angiogenic properties and a topical safety profile that is well-tolerated. Based on this evidence, we investigated the feasibility of formulating rifamycin as an ophthalmic drop capable of delivering therapeutic concentrations to the posterior segment of the eye. Methods: Inhibition of neovascularization by administration of rifampicin was analyzed in the rat oxygen-induced retinopathy (OIR) and mouse laser-induced choroidal neovascularization (CNV) models. Pharmacokinetic (PK) studies were conducted in mice, rats, and rabbits by dosing various formulations containing rifampicin, and the compound was quantified by LC/MS analysis. Results: Results from dose escalation studies in the mouse laser-induced CNV model suggested the minimum effective dose of rifampicin required for inhibiting neovascularization in subretinal tissues to be 0.7 mg/kg, which is substantially lower than the 20 mg/kg dosage approved for infectious disease treatments. The previous studies did not report the minimum effective dose in the anti-angiogenesis effects. The effective area under the concentration-time curve (AUC) in the sub-retina was evaluated as 0.27 h·ng/mg. In rabbits, rifampicin was delivered to the sub-retina by a single topical application of various formulations in a dose-dependent manner. The topical application of the formulations containing 1% rifampicin, which was well-tolerated in clinical trials previously reported for ocular trachoma, achieved subretinal delivery approximately 2–32 times greater than the effective AUC. Plasma exposure of the compound by the topical application was evaluated to range approximately 0.5–10 ng/mL. Conclusions: Rifampicin was delivered to the sub-retina in rabbits with an efficiency greater than the effective dose required for inhibiting neovascularization. Limited amounts of plasma exposure by the topical application were detected. These results suggested the therapeutic potential of the rifampicin formulations for the topical treatment of exudative macular degeneration. Full article

Premature infants with retinopathy of prematurity (ROP) have neovascularization of the retina, potentially resulting in low vision and even blindness. Some of these infants still have visual impairment, even if ROP resolves as they age. However, the mechanisms underlying the visual problems post–ROP are poorly understood. Because the pathological neovascularization in ROP infants can be mimicked in a mouse model with oxygen–induced retinopathy (OIR), we recapitulated post–ROP with post–OIR mice a few months after spontaneous regression of retinal neovascularization. Our pattern electroretinogram test demonstrates that post–OIR mice exhibit reduced P1–N2 responses, suggesting the impairment of retinal ganglion cells, the retina’s output neurons. However, immunohistochemistry reveals that the density of retinal ganglion cells remains unchanged in post–OIR mice, indicating that the aforementioned pattern electroretinogram changes are functional. Our data further demonstrate that both light–adapted ex vivo electroretinogram a–waves (cone responses) and in vivo electroretinogram b–waves (ON cone bipolar cell responses) were significantly impaired in post–OIR mice. These results suggest that post–OIR impairment of the retinal cone pathway appears to result in the dysfunction of retinal ganglion cells, contributing to visual problems. A similar cellular mechanism could occur in post–ROP children, which is responsible for their visual impairment. Full article

Background: Retinopathy of prematurity (ROP) is the major cause of blindness in children. It is a biphasic disease with retinal vessel growth cessation and loss (Phase I) followed by uncontrolled retinal vessel growth (Phase II). Folate is an essential nutrient for fetal development and growth. Premature infants have a high risk for folate deficiency. However, the contribution of folate to ROP risk remains unknown. Methods: In mouse oxygen-induced retinopathy (OIR), the nursing dams were fed with a folic acid-deficient or control diet after delivery until the end of hyperoxia. Alternatively, pups received direct injection of either folic acid or vehicle during Phase I hyperoxia. Genes involved in the folate cycle and angiogenic responses were examined using real-time PCR. Total retinal folate levels were measured with the Lactobacillus casei assay. Results: Maternal folic acid deficiency in early life exacerbated pathological retinal vessel growth, while supplementation with folic acid suppressed it. Genes involved in the folate cycle were downregulated in Phase I OIR retinas and were highly expressed in Müller glia. Folic acid reduced pro-angiogenic signaling in cultured rat retinal Müller glia in vitro. Conclusions: Appropriate supplementation of folic acid might be a new and safe treatment for ROP at an early stage. Full article

Retinopathy of prematurity (ROP) is primarily caused by the exposure of preterm infants with underdeveloped blood vessels to high oxygen concentrations. This damages the astrocytes that promote normal vascular development, leading to avascularity, pathological neovascularization, and retinal detachment, and even blindness as the disease progresses. In this study, the aim was to investigate the differences in the characteristics of astrocytes and blood vessels between wild-type (WT) and genetically modified mice overexpressing platelet-derived growth factor subunit A (PDGF-A) in the retina immediately after high oxygen exposure, a protocol in the oxygen-induced retinopathy (OIR) model of ROP. Our results showed that PDGF-A mice exhibited an increased population of astrocytes and higher vascular density than WT mice and that PDGF-A strengthened the resistance to hyperoxic conditions. In the OIR model, PDGF-A mice had reduced avascular zone areas following hyperoxia exposure. Furthermore, immunostaining for NG2 and CD31 showed that pericytes tended to regress earlier than endothelial cells, particularly at the vessel edges in both WT and transgenic mice, indicating relatively higher susceptibility to hyperoxia-induced damage. These findings suggest that PDGF-A plays a crucial role in stabilizing retinal vessels and may serve as a novel therapeutic target for ROP, highlighting the potential significance of PDGF-A in the pathological mechanisms of retinal diseases. Full article

Retinopathy of prematurity (ROP) is a primary cause of visual impairment and blindness in premature newborns, characterized by vascular abnormalities in the developing retina, with microvascular alteration, neovascularization, and in the most severe cases retinal detachment. To elucidate the pathophysiology and develop therapeutics for ROP, several pre-clinical experimental models of ROP were developed in different species. Among them, the oxygen-induced retinopathy (OIR) mouse model has gained the most popularity and critically contributed to our current understanding of pathological retinal angiogenesis and the discovery of potential anti-angiogenic therapies. A deeper comprehension of molecular regulators of OIR such as hypoxia-inducible growth factors including vascular endothelial growth factors as primary perpetrators and other new metabolic modulators such as lipids and amino acids influencing pathological retinal angiogenesis is also emerging, indicating possible targets for treatment strategies. This review delves into the historical progressions that gave rise to the modern OIR models with a focus on the mouse model. It also reviews the fundamental principles of OIR, recent advances in its automated assessment, and a selected summary of metabolic investigation enabled by OIR models including amino acid transport and metabolism. Full article

Retinopathy of prematurity (ROP) has a dual-phase disease pathology; in phase 1, hyperoxia-induced vaso-obliteration occurs in the retinal vasculature due to increased oxidative stress (OS) and inflammation, followed by phase 2, where hypoxia increases the overproduction of growth factors, inducing retinal neovascularization. Toll-like receptor 2 and -4 (TLR2 and TLR4) overactivation, hyper-inflammation, macrophages, and neutrophil infiltration contribute to the developing ROP. AVR-121 and AVR-123 are novel classes of small-molecule dual inhibitors of TLR2/4 tested in a human leukemia monocytic cell line (THP-1) and cord-blood-derived mononuclear cells (CBMCs). Both compounds inhibited TLR2/4 signaling-related inflammatory cytokines in THP-1 cells and inhibited VEGF-induced neovascularization in human retinal endothelial cells (HRECs), which are hallmarks of ROP. In an oxygen-induced retinopathy (OIR) murine model, the intraperitoneal injection of AVR-123 in the hyperoxia phase (P7–P12) or a nanosuspension eyedrop of AVR-123 in the hypoxic phase (P12–P17) significantly reduced vaso-obliteration, angiogenesis, and inflammatory cytokine profiles while not inhibiting the necessary growth factor VEGF in the juvenile mouse eyes. The results are consistent with our hypothesis that targeting the dual TLR2/4 pathway will reduce inflammation, angiogenesis, and vaso-obliteration in vitro and in vivo and reduce cytotoxic immune cells. AVR-123 has the potential to be developed as a therapy for ROP. Full article

Notch ligands and receptors are important for cell specification and angiogenesis, but their role in oxygen-induced retinopathy (OIR) is not well studied. Delta-like ligand (DLL)-4/Notch inhibits angiogenesis, while Jagged-1/Notch promotes angiogenesis. We tested the hypothesis that early supplementation with antioxidants and/or fish oil curtails severe OIR by inducing DLL-4/Notch and reducing Jagged-1/Notch. Newborn rats were exposed to brief intermittent hypoxia (IH) during hyperoxia, during which they received daily oral supplements of (1) fish oil, (2) coenzyme Q10 (CoQ10) in olive oil (OO), (3) glutathione nanoparticles (nGSH), (4) fish oil + CoQ10, or (5) OO (controls) from birth (P0) to P14. At P14, the pups were placed in room air (RA) until P21, with no further treatment. Oxidative stress, apoptosis, ocular histopathology, and Notch signaling were assessed. Neonatal IH resulted in severe retinal damage consistent with retinopathy of prematurity (ROP). Retinal damage was associated with induced oxidative stress and Jagged-1/Notch signaling, as well as reduced DLL-4/Notch signaling. All treatments reversed these outcomes, but nGSH produced the most beneficial outcomes. Severe OIR promoted the induction of Jagged-1/Notch and curtailed DLL-4/Notch, which was an effect that could be reversed with nGSH supplementation. These findings may indicate a potential alternate pathway for ROP treatment and/or prevention. Full article

Retinal inflammation is a central feature of ocular neovascular diseases such as diabetic retinopathy and retinopathy of prematurity, but the contribution of neutrophils to this process is not fully understood. We studied oxygen-induced retinopathy (OIR) which develops in two phases, featuring hyperoxia-induced retinal vaso-obliteration in phase I, followed by retinal neovascularization in phase II. As neutrophils are acute responders to tissue damage, we evaluated whether neutrophil depletion with an anti-Ly6G mAb administered in phase I OIR influenced retinal inflammation and vascular injury. Neutrophils were measured in blood and spleen via flow cytometry, and myeloperoxidase, an indicator of neutrophil activity, was evaluated in the retina using Western blotting. Retinal vasculopathy was assessed by quantitating vaso-obliteration, neovascularization, vascular leakage, and VEGF levels. The inflammatory factors, TNF, MCP-1, and ICAM-1 were measured in retina. In the OIR controls, neutrophils were increased in the blood and spleen in phase I but not phase II OIR. In OIR, the anti-Ly6G mAb reduced neutrophils in the blood and spleen, and myeloperoxidase, inflammation, and vasculopathy in the retina. Our findings revealed that the early rise in neutrophils in OIR primes the retina for an inflammatory and angiogenic response that promotes severe damage to the retinal vasculature. Full article

Hypoxia-induced retinal neovascularization is a leading cause of blindness worldwide. Oxygen-induced retinopathy (OIR) mouse, a well-established angiogenesis model, has been extensively used to evaluate the effect of anti-angiogenic agents through intravitreal injection. Here, we serendipitously found that the needles used for intravitreal injection caused an unexpected “anti-angiogenic” effect in the OIR mice. To evaluate the effects of various intravitreal puncture sizes on retinal neovascularization and explore the potential underlying mechanism, intravitreal punctures using 0.5 mm (25 G), 0.3 mm (30 G), or 0.21 mm (33 G) needles were performed in OIR mice. Compared with 0.3 mm and 0.21 mm puncture, the 0.5 mm puncture remarkably suppressed the formation of pathological angiogenesis, inhibited vascular leakage, and remodeled the retinal vasculature. Mechanistically, the 0.5 mm puncture induced a substantial reduction in intraocular pressure (IOP), leading to an improvement in oxygen partial pressure (pO₂) and significant reduction in Hif1a expression, resulting in resolution of angiogenic and inflammatory responses. Furthermore, IOP-lowering drugs, Travatan or Azarga, also promoted the alleviation of hypoxia and exhibited a potent anti-angiogenesis efficacy. Our study revealed an acute and significant reduction in IOP caused by a large puncture, which could remarkably suppress HIF-1α-mediated retinal neovascularization, indicating that lowering IOP may be a promising therapeutic avenue for treating retinal neovascular diseases. Full article

Endothelial Progenitor Cells (EPCs) can actively participate in revascularization in oxygen-induced retinopathy (OIR). Yet the mechanisms responsible for their dysfunction is unclear. Nogo-A, whose function is traditionally related to the inhibition of neurite function in the central nervous system, has recently been documented to display anti-angiogenic pro-repellent properties. Based on the significant impact of EPCs in retinal vascularization, we surmised that Nogo-A affects EPC function, and proceeded to investigate the role of Nogo-A on EPC function in OIR. The expression of Nogo-A and its specific receptor NgR1 was significantly increased in isolated EPCs exposed to hyperoxia, as well as in EPCs isolated from rats subjected to OIR compared with respective controls (EPCs exposed to normoxia). EPCs exposed to hyperoxia displayed reduced migratory and tubulogenic activity, associated with the suppressed expression of prominent EPC-recruitment factors SDF-1/CXCR4. The inhibition of Nogo-A (using a Nogo-66 neutralizing antagonist peptide) or siRNA-NGR1 in hyperoxia-exposed EPCs restored SDF-1/CXCR4 expression and, in turn, rescued the curtailed neovascular functions of EPCs in hyperoxia. The in vivo intraperitoneal injection of engineered EPCs (Nogo-A-inhibited or NgR1-suppressed) in OIR rats at P5 (prior to exposure to hyperoxia) prevented retinal and choroidal vaso-obliteration upon localization adjacent to vasculature; coherently, the inhibition of Nogo-A/NgR1 in EPCs enhanced the expression of key angiogenic factors VEGF, SDF-1, PDGF, and EPO in retina; CXCR4 knock-down abrogated suppressed NgR1 pro-angiogenic effects. The findings revealed that hyperoxia-induced EPC malfunction is mediated to a significant extent by Nogo-A/NgR1 signaling via CXCR4 suppression; the inhibition of Nogo-A in EPCs restores specific angiogenic growth factors in retina and the ensuing vascularization of the retina in an OIR model. Full article

MicroRNA (miRNA) is a non-coding RNA that can regulate the expression of many target genes, and it is widely involved in various important physiological activities. MiR-124-3p was found to associate with the normal development of retinal vessels in our previous study, but the mechanism of its anti-angiogenic effect on pathological retinal neovascularization still needed to be explored. Therefore, this study aimed to investigate the effect and mechanism of miR-124-3p on retinal neovascularization in mice with oxygen-induced retinopathy (OIR). Here, we found that intravitreal injection of miR-124-3p agomir attenuated pathological retinal neovascularization in OIR mice. Moreover, miR-124-3p preserved the astrocytic template, inhibited reactive gliosis, and reduced the inflammatory response as well as necroptosis. Furthermore, miR-124-3p inhibited the signal transducer and activator of transcription 3 (STAT3) pathway and decreased the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor. Taken together, our results revealed that miR-124-3p inhibited retinal neovascularization and neuroglial dysfunction by targeting STAT3 in OIR mice. Full article

Small GTPase R-Ras regulates vascular permeability in angiogenesis. In the eye, abnormal angiogenesis and hyperpermeability are the leading causes of vision loss in several ischemic retinal diseases such as proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Oxygen-induced retinopathy (OIR) is the most widely used experimental model for these ischemic retinopathies. To shed more light on how the R-Ras regulates vascular permeability in pathological angiogenesis, we performed a comprehensive (>2900 proteins) characterization of OIR in R-Ras knockout (KO) and wild-type (WT) mice by sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. OIR and age-matched normoxic control retinas were collected at P13, P17, and P42 from R-Ras KO and WT mice and were subjected to SWATH-MS and data analysis. The most significant difference between the R-Ras KO and WT retinas was an accumulation of plasma proteins. The pathological vascular hyperpermeability during OIR in the R-Ras KO retina took place very early, P13. This led to simultaneous hypoxic cell injury/death (ferroptosis), glycolytic metabolism as well compensatory mechanisms to counter the pathological leakage from angiogenic blood vessels in the OIR retina of R-Ras deficient mice. Full article

Angiogenesis, neovascularization, and vascular remodeling are highly dynamic processes, where endothelial cell–cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity, and barrier function. The cadherin–catenin adhesion complex is a key contributor to inner blood–retinal barrier (iBRB) integrity and dynamic cell movements. However, the pre-eminent role of cadherins and their associated catenins in iBRB structure and function is not fully understood. Using a murine model of oxygen-induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we try to understand the significance of IL-33 on retinal endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability. Using electric cell-substrate impedance sensing (ECIS) analysis and FITC-dextran permeability assay, we observed that IL-33 at a 20 ng/mL concentration induced endothelial-barrier disruption in HRMVECs. The adherens junction (AJs) proteins play a prominent role in the selective diffusion of molecules from the blood to the retina and in maintaining retinal homeostasis. Therefore, we looked for the involvement of adherens junction proteins in IL-33-mediated endothelial dysfunction. We observed that IL-33 induces α-catenin phosphorylation at serine/threonine (Ser/Thr) residues in HRMVECs. Furthermore, mass-spectroscopy (MS) analysis revealed that IL-33 induces the phosphorylation of α-catenin at Thr⁶⁵⁴ residue in HRMVECs. We also observed that PKCμ/PRKD1-p38 MAPK signaling regulates IL-33-induced α-catenin phosphorylation and retinal endothelial cell-barrier integrity. Our OIR studies revealed that genetic deletion of IL-33 resulted in reduced vascular leakage in the hypoxic retina. We also observed that the genetic deletion of IL-33 reduced OIR-induced PKCμ/PRKD1-p38 MAPK-α-catenin signaling in the hypoxic retina. Therefore, we conclude that IL-33-induced PKCμ/PRKD1-p38 MAPK-α-catenin signaling plays a significant role in endothelial permeability and iBRB integrity. Full article

Vision loss in diabetic retinopathy features damage to the blood–retinal barrier and neovascularization, with hypertension and the renin–angiotensin system (RAS) having causal roles. We evaluated if finerenone, a non-steroidal mineralocorticoid receptor (MR) antagonist, reduced vascular pathology and inflammation in diabetic and neovascular retinopathy. Diabetic and hypertensive transgenic (mRen-2)27 rats overexpressing the RAS received the MR antagonist finerenone (10 mg/kg/day, oral gavage) or the angiotensin-converting enzyme inhibitor perindopril (10 mg/kg/day, drinking water) for 12 weeks. As retinal neovascularization does not develop in diabetic rodents, finerenone (5 mg/kg/day, i.p.) was evaluated in murine oxygen-induced retinopathy (OIR). Retinal vasculopathy was assessed by measuring gliosis, vascular leakage, neovascularization, and VEGF. Inflammation was investigated by quantitating retinal microglia/macrophages, pro-inflammatory mediators, and anti-inflammatory regulatory T-cells (Tregs). In diabetes, both treatments reduced systolic blood pressure, gliosis, vascular leakage, and microglial/macrophage density, but only finerenone lowered VEGF, ICAM-1, and IL-1ß. In OIR, finerenone reduced neovascularization, vascular leakage, and microglial density, and increased Tregs in the blood, spleen, and retina. Our findings, in the context of the FIDELIO-DKD and FIGARO-DKD trials reporting the benefits of finerenone on renal and cardiovascular outcomes in diabetic kidney disease, indicate the potential of finerenone as an effective oral treatment for diabetic retinopathy. Full article