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Example photographs taken from retinal wholemounts stained with cresyl blue and optic nerve cross-sections stained with toluidine blue of wild-type (a, c) and ApoE-/- mice (b, d). Total cell number in the RGC layer (e) and axon number (f) in the optic nerve was similar in wild-type and ApoE-/- mice. Values are presented as mean±SE (n=8 per genotype). Scale bar=50 μm.
Source publication
Objective. Atherogenic lipoproteins may impair vascular reactivity consecutively causing tissue damage in multiple organs via abnormal perfusion and excessive reactive oxygen species generation. We tested the hypothesis that chronic hypercholesterolemia causes endothelial dysfunction and cell loss in the retina. Methods. Twelve-month-old apolipopro...
Citations
... levels were measured in frozen sections of unfixed retinas by staining with the fluorescent dye dihydroethidium (DHE) [19,[31][32][33]. Immediately after thawing, tissue sections were incubated with 1 µM of DHE for 30 min at 37 °C. ...
... nih.gov/ij/, NIH, Bethesda, MD, USA), and the average numbers were calculated as axon density per square millimeter of ON and then multiplied by cross-sectional area to calculate the total number of axons per ON [31]. ...
Background
NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat.
Methods
Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2−/− mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2⁻) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study.
Results
We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst.
Conclusions
Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.
... NOX2-generated ROS in bone marrow-derived cells and resident retinal cells were shown to significantly contribute to retinal vascular damage in the diabetic retina (Rojas et al., 2013). Moreover, upregulation of NOX2 and endothelial dysfunction were observed in retinal arterioles following intraocular pressure elevation, ischemia, lipopolysaccharide exposure and hypercholesterolemia Rojas et al., 2013;Wang et al., 2022;Zadeh et al., 2019aZadeh et al., , 2019b. Under conditions of oxidative stress, peroxynitrite, a potent reactive nitrogen species, oxidizes tetrahydrobiopterin, a fundamental cofactor of endothelial nitric oxide synthase (eNOS), thereby "uncoupling" eNOS and reducing the production of nitric oxide, an essential molecule required for proper endothelial function (Farmer and Kennedy, 2009b;Förstermann, 2006). ...
... Moreover, there was no hint that alternative signaling pathways had been activated in the ophthalmic artery from ApoE−/− mice, because a blockade of nNOS, iNOS, COX, lipoxygenase, CYP450, and gap junctions had similar effects on endothelium-dependent vasodilation as in wild-type mice. All together, these findings suggest that hypercholesterolemia did not exert marked effects on endothelial function in the ophthalmic artery, which contrasts with our previously published findings in retinal arterioles, where pronounced endothelial dysfunction was observed in ApoE−/− mice [51]. ...
Atherogenic lipoproteins may impair vascular reactivity, leading to tissue damage in various organs, including the eye. This study aimed to investigate whether ophthalmic artery reactivity is affected in mice lacking the apolipoprotein E gene (ApoE−/−), a model for hypercholesterolemia and atherosclerosis. Twelve-month-old male ApoE−/− mice and age-matched wild-type controls were used to assess vascular reactivity using videomicroscopy. Moreover, the vascular mechanics, lipid content, levels of reactive oxygen species (ROS), and expression of pro-oxidant redox enzymes and the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were determined in vascular tissue. Unlike the aorta, the ophthalmic artery of ApoE−/− mice developed no signs of endothelial dysfunction and no signs of excessive lipid deposition. Remarkably, the levels of ROS, nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1), NOX2, NOX4, and LOX-1 were increased in the aorta but not in the ophthalmic artery of ApoE−/− mice. Our findings suggest that ApoE−/− mice develop endothelial dysfunction in the aorta by increased oxidative stress via the involvement of LOX-1, NOX1, and NOX2, whereas NOX4 may participate in media remodeling. In contrast, the ophthalmic artery appears to be resistant to chronic apolipoprotein E deficiency. A lack of LOX-1 expression/overexpression in response to increased oxidized low-density lipoprotein levels may be a possible mechanism of action.
... Moreover, there was no hint that alternative signaling pathways had been activated in ophthalmic arteries from ApoE-/-mice, because blockade of nNOS, iNOS, COX, lipoxygenase, CYP450 and gap junctions had similar effects on endothelium-dependent vasodilation as in wild-type mice. All together, these findings suggest that hypercholesterolemia did not exert marked effects on endothelial function in the ophthalmic artery, which contrasts with our previously published findings in retinal arterioles, where pronounced endothelial dysfunction was observed in ApoE-/-mice [46]. ...
Atherogenic lipoproteins may impair vascular reactivity, leading to tissue damage in various organs, including the eye. This study aimed to investigate whether ophthalmic artery reactivity is affected in mice lacking the apolipoprotein E gene (ApoE-/-), a model for hypercholesterolemia and atherosclerosis. Twelve-month-old male ApoE-/- mice and age-matched wild-type controls were used to assess vascular reactivity using videomicroscopy. Moreover, vascular mechanics, lipid content, levels of reactive oxygen species (ROS), expression of pro-oxidant redox enzymes and of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were determined in vascular tissue. Unlike the aorta, the ophthalmic artery of ApoE-/- mice developed no signs of endothelial dysfunction and no signs of excessive lipid deposition. Remarkably, levels of ROS, nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1), NOX2, NOX4, and of LOX-1 were increased in the aorta but not in the ophthalmic artery of ApoE-/- mice. Our findings suggest that ApoE-/- mice develop endothelial dysfunction in the aorta by increased oxidative stress via involvement of LOX-1, NOX1 and NOX2, whereas NOX4 may participate in media remodeling. In contrast, the ophthalmic artery appears to be resistant to chronic apolipoprotein E deficiency. Lack of LOX-1 expression/overexpression in response to increased ox-LDL levels may be a possible mechanism of action.
... Likewise, moderately elevated IOP induced endothelial dysfunction in retinal arterioles together with RGC loss [140,141]. Zadeh et al. found in apolipoprotein E (ApoE)-deficient mice that hypercholesterolemia caused oxidative stress and endothelial dysfunction in retinal arterioles but did neither lead to increased ROS levels in the RGC layer nor to loss of RGCs, indicative of compensatory effects [142]. In contrast, a study in pigs reported that after only 12 min of ocular ischemia and 20 h of reperfusion, endothelial dysfunction, retinal edema, and RGC loss occurred [143]. ...
Optic nerve disorders encompass a wide spectrum of conditions characterized by the loss of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve. The etiology of these disorders can vary significantly, but emerging research highlights the crucial role of oxidative stress, an imbalance in the redox status characterized by an excess of reactive oxygen species (ROS), in driving cell death through apoptosis, autophagy, and inflammation. This review provides an overview of ROS-related processes underlying four extensively studied optic nerve diseases: glaucoma, Leber’s hereditary optic neuropathy (LHON), anterior ischemic optic neuropathy (AION), and optic neuritis (ON). Furthermore, we present preclinical findings on antioxidants, with the objective of evaluating the potential therapeutic benefits of targeting oxidative stress in the treatment of optic neuropathies.
... Levels of ROS were determined in situ by using the fluorescent dye, dihydroethidium (DHE), as described previously [39]. DHE is converted by ROS to highly fluorescent oxidized products (e.g., 2-hydroxyethidium, which is specific for superoxide as well as ethidium, which is the unspecific oxidation product formed by hydroxyl radicals via Fenton reaction or peroxide/peroxidase reactivity). ...
... Notably, NOX2 expression was shown to be upregulated in the vascular wall of retinal blood vessels under hypoxic and ischemic conditions [28,79]. Apart from I/R events, NOX2 upregulation has been associated with endothelial dysfunction in ocular blood vessels in a variety of other pathological conditions, such as elevated intraocular pressure, hypercholesterolemia and angiotensin II exposure [39,51,81,82]. Moreover, NOX2-derived ROS were reported to elevate arginase expression and activity, to decrease nitric oxide formation and to induce premature vascular endothelial cell senescence in diabetic retinopathy [83]. ...
Ischemia-reperfusion (I/R) events are involved in the development of various ocular pathologies, e.g., retinal artery or vein occlusion. We tested the hypothesis that resveratrol is protective against I/R injury in the murine retina. Intraocular pressure (IOP) was elevated in anaesthetized mice to 110 mm Hg for 45 min via a micropipette placed in the anterior chamber to induce ocular ischemia. In the fellow eye, which served as control, IOP was kept at a physiological level. One group received resveratrol (30 mg/kg/day p.o. once daily) starting one day before the I/R event, whereas the other group of mice received vehicle solution only. On day eight after the I/R event, mice were sacrificed and retinal wholemounts were prepared and immuno-stained using a Brn3a antibody to quantify retinal ganglion cells. Reactivity of retinal arterioles was measured in retinal vascular preparations using video microscopy. Reactive oxygen species (ROS) and nitrogen species (RNS) were quantified in ocular cryosections by dihydroethidium and anti-3-nitrotyrosine staining, respectively. Moreover, hypoxic, redox and nitric oxide synthase gene expression was quantified in retinal explants by PCR. I/R significantly diminished retinal ganglion cell number in vehicle-treated mice. Conversely, only a negligible reduction in retinal ganglion cell number was observed in resveratrol-treated mice following I/R. Endothelial function and autoregulation were markedly reduced, which was accompanied by increased ROS and RNS in retinal blood vessels of vehicle-exposed mice following I/R, whereas resveratrol preserved vascular endothelial function and autoregulation and blunted ROS and RNS formation. Moreover, resveratrol reduced I/R-induced mRNA expression for the prooxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2). Our data provide evidence that resveratrol protects from I/R-induced retinal ganglion cell loss and endothelial dysfunction in the murine retina by reducing nitro-oxidative stress possibly via suppression of NOX2 upregulation.
... Oxidative stress is a key mechanism in the development of endothelial dysfunction in a variety of retinal pathologies [24,45,46]. Under hyperglycemic conditions, elevated ROS levels were shown to contribute to endothelial dysfunction in bovine retinal endothelial cells and pericytes [47], human umbilical venous endothelial cells [32], and human retinal endothelial cells [6]. ...
... In the context of in vivo studies, authors usually observed a concordant change in NOX expression at the mRNA and protein levels [24,45,46]. An expression change at the transcriptional level seems to occur mainly in a prolonged diabetic milieu, as already shown in diabetic rat models, in which increased mRNA expression of the NOX4 and p22phox subunits was seen in the kidney after several weeks of exposure [50,55]. ...
Diabetes mellitus may cause severe damage to retinal blood vessels. The central aim of this study was to test the hypothesis that sulodexide, a mixture of glycosaminoglycans, has a protective effect against hyperglycemia-induced endothelial dysfunction in the retina. Functional studies were performed in isolated porcine retinal arterioles. Vessels were cannulated and incubated with highly concentrated glucose solution (HG, 25 mM D-glucose) +/− sulodexide (50/5/0.5 μg/mL) or normally concentrated glucose solution (NG, 5.5 mM D-glucose) +/− sulodexide for two hours. Endothelium-dependent and endothelium-independent vasodilatation were measured by videomicroscopy. Reactive oxygen species (ROS) were quantified by dihydroethidium (DHE) fluorescence. Using high-pressure liquid chromatography (HPLC), the intrinsic antioxidant properties of sulodexide were investigated. Quantitative PCR was used to determine mRNA expression of regulatory, inflammatory, and redox genes in retinal arterioles, some of which were subsequently quantified at the protein level by immunofluorescence microscopy. Incubation of retinal arterioles with HG caused significant impairment of endothelium-dependent vasodilation, whereas endothelium-independent responses were not affected. In the HG group, ROS formation was markedly increased in the vascular wall. Strikingly, sulodexide had a protective effect against hyperglycemia-induced ROS formation in the vascular wall and had a concentration-dependent protective effect against endothelial dysfunction. Although sulodexide itself had only negligible antioxidant properties, it prevented hyperglycemia-induced overexpression of the pro-oxidant redox enzymes, NOX4 and NOX5. The data of the present study provide evidence that sulodexide has a protective effect against hyperglycemia-induced oxidative stress and endothelial dysfunction in porcine retinal arterioles, possibly by modulation of redox enzyme expression.
... ROS levels were determined in DHE (1 µM)-stained retinal cryosections of 10 µm thickness, as previously reported [8,14]. The retinal cryosections were imaged using the Zeiss Imager M.2 equipped with an Apotome.2 (Carl Zeiss; Jena, Germany). ...
... In our previous study of mice subjected to chronic social defeat stress, retinal vascular dysfunction was also not associated with glaucomatous damage [19]. Similarly, retinal vascular dysfunction, which was observed in ApoE -/mice, did not affect the viability of RGCs and their axons [14]. However, some studies have reached different conclusions. ...
... The fluorescence intensity of the DHE-stained retinal cryosection and the retinal NOX2 immunoreactivities were increased in HP animals. Oxidative stress has been reported to promote RGC death and is a risk factor for endothelial dysfunction in the aorta, and the retinal and mesenteric vessels [8,14,[40][41][42][43][44][45]. Moreover, a similar trend was found regarding the proinflammatory cytokines of the TNF-α mRNA level. ...
Research has been conducted into vascular abnormalities in the pathogenesis of glaucoma, but conclusions remain controversial. Our aim was to test the hypothesis that retinal endothelial dysfunction induced by elevated intraocular pressure (IOP) persists after IOP normalization, further triggering retinal ganglion cell (RGC) loss. High intraocular pressure (HP) was induced in mice by episcleral vein occlusion (EVO). Retinal vascular function was measured via video microscopy in vitro. The IOP, RGC and their axons survival, levels of oxidative stress and inflammation as well as vascular pericytes coverage, were determined. EVO caused HP for two weeks, which returned to baseline afterwards. Mice with HP exhibited endothelial dysfunction in retinal arterioles, reduced density of RGC and their axons, and loss of pericytes in retinal arterioles. Notably, these values were similar to those of mice with recovered IOP (RP). Levels of oxidative stress and inflammation were increased in HP mice but went back to normal in the RP mice. Our data demonstrate that HP induces persistent endothelial dysfunction in retinal arterioles, which persists one month after RP. Oxidative stress, inflammation, and loss of pericytes appear to be involved in triggering vascular functional deficits. Our data also suggest that retinal endothelial dysfunction does not affect RGC and their axon survival.
... Згідно з цими даними, зниження кровотоку може бути викликане механічним стиском судинних стінок внаслідок підвищеного ВОТ. Цей процес впливає на кровопостачання ламінарних сегментів, пошкоджує аксони ГКС, викликає дизрегуляцію судин сітківки внаслідок надлишкової продукції АФК та збільшення експресії НАДФНоксидази-2 та окисленого рецептора ЛПНП1 (LOX1) лектинового типу та ендотеліальної дисфункції в артеріолах сітківки [64]. ...
... Ендотелій ТС може вивільняти ендотеліни, які здатні викликати вазоконстрикцію, змінювати рухливість ТС, проникність судин та зміни ВОТ [21]. Крім того, ендотелій ТС індукує ішемію, не пов'язану з конструкцією судин, шляхом зниження активності АТФ-залежного Na + / K + -насоса [21,64]. ...
Relevance. Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with loss of retinal ganglion cells (RGCs) and narrowing of the visual fields in the eyes with a gonioscopic open angle. The main mechanisms of this are increased intraocular pressure (IOP), circulatory disorders, trabecular meshwork (TM), ischemic metabolic disorders and chronic inflammation. However, questions about the role of POAG genetic predisposition remain open.
Objective: analysis of current data on the mechanisms of pathogenesis of progressive neuropathy in POAG and the role of genetic predisposition.
Methods. The analysis of scientific publications in open international electronic scientometric databases: Scopus, PubMed, Web of Science, Google Scholar, SID, MagIran, IranMedex, IranDoc, ScienceDirect, Embase by keywords (a total of 67 sources). Search depth – 10 years (2012-2022).
Results. There are more than 60 million glaucoma patients in the world, 20% of whom have an incurable stage. By 2040, the number of patients is projected to increase to 112 million, with POAG accounting for 75% of cases. Among the main mechanisms of glaucoma, an important role belongs to chronic inflammation and immune damage, which occur in response to ischemic injury. Prolonged inflammatory process leads to hypersecretion of inflammatory mediators and infiltration of inflammatory cells into ischemic tissue, which aggravates the effects of increased IOP and ischemia. It is known that mutations in the gene of Toll-like receptor 4 (TLR4) are associated with both infectious and non-infectious diseases, including POAG: activation of TLR4 initiates TM fibrosis, causes increased IOP, activates RGCs apoptosis in the model of acute glaucoma. TLR4 ligands, such as heat shock proteins and lipopolysaccharides are candidate antigens for glaucoma. TLR4 overexpression at retinal microglia and astrocytes induce an innate immune response through NF-κB activation, which enhances the expression of proinflammatory cytokines.
Conclusions. A promising direction is to study the contribution of TLR4 mutations to the POAG mechanisms, which will identify the mechanisms of immune disorders and establish the genetic risk of individual mutations in different ethnic groups.
... 27 It has been reported that ApoE is an endogenous antioxidant. 28 Previous research has demonstrated that in transgenic mice lacking ApoA-IV, exogenous expression of ApoE is effective against the oxidative stress of mouse vascular endothelial cells and thus protects the mice from atherosclerosis. 29 In addition, ApoE significantly inhibits the formation of conjugated dienes and reduces the aggregation state of LDLs, thereby further eliminating free radicals in cells and reducing apoptosis induced by oxidants. ...
Objective
To address the relationship between apolipoprotein E (ApoE) and oxidative stress in gestational diabetes mellitus (GDM).
Methods
Fifty pregnant women diagnosed with GDM and normal pregnant women were recruited separately and their blood and placental tissue were collected. Western blot assay, qRT‐PCR assay and ELISA were used to detect the expression levels of ApoE and other oxidative stress factors in these samples. ApoE−/− mice with a C57BL/6J background were used to evaluate the relationship between ApoE deficiency and oxidative stress during GDM.
Results
Serum and placental ApoE were both down‐regulated in GDM patients (serum: 45.25 ± 19.27 μg/ml for GDM and 96.34 ± 24.05 μg/ml for control; placental: 14.49 ± 6.52 ng/mg tissue for GDM and 48.76 ± 13.59 ng/mg tissue for control). There was a statistical correlation between placental ApoE level and oxidative stress in GDM (r = −0.4904 with MDA, −0.4258 with NO, 0.4476 with SOD, 0.6316 with GSH). ApoE deficiency exacerbated blood glucose, insulin anomaly and oxidative stress in placenta in GDM mouse models. Placental Apo E deficiency correlates to oxidative stress in GDM.
Conclusion
In conclusion, we innovatively revealed the relationship between ApoE and GDM oxidative stress among GDM patients in this study.
