Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells

© 2018 Royal Australian and New Zealand College of Ophthalmologists. This author accepted manuscript is made available following 12 month embargo from date of publication (January 2018) in accordance with the publisher's archiving policy.Background Over‐production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS‐generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Methods Differential Nox enzyme expression was studied by real‐time‐quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2′,7′‐dichlorofluorescein. DMOG‐stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Results Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG‐induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Conclusions Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases

NADPH oxidase, NOX1, mediates vascular injury in ischemic retinopathy

<b>Aims:</b> Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. <b>Results:</b> Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. <b>Innovation:</b> Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. <b>Conclusions:</b> Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathie

Aldosterone and the mineralocorticoid receptor’s role in inflammation and vascular pathology in retinal disease

Purpose: Retinopathy of prematurity (ROP) is a significant cause of severe vision loss and blindness in children, and can be replicated in rodent models of the disease where it is known as oxygen induced retinopathy (OIR). A distinct feature of OIR is retinal neovascularization, which is also present in patients with proliferative diabetic retinopathy. Blockade of the renin angiotensin-aldosterone system (RAAS) via inhibitors of the angiotensin II receptor type 1 (AT1-R) or angiotensin-converting enzyme (ACE) in diabetic patients are associated with reduced incidence and progression of retinopathy. However, these interventions do not completely prevent retinal pathology. One reason for this may be incomplete suppression of the RAAS. For instance, aldosterone may still be present in plasma and tissues due to the aldosterone escape phenomenon. Indeed, in patients, blockade of aldosterone via the mineralocorticoid receptor (MR) combined with angiotensin II blockade, provides superior cardiorenal protection than monotherapy. Another consideration is an underlying critical mechanism for ROP, which is the upregulation of reactive oxygen species (ROS), with studies showing that global inhibitors of ROS such as apocynin reduced both retinal ROS levels and neovascularization. However, these findings do not seamlessly translate to humans, with strategies such as antioxidants such as vitamin E being associated with a significant risk of infection, which has precluded the widespread use of this therapy. The explanation for the failure of such antioxidant therapies is not entirely known, but it may be due to their lack of efficacy, and nonspecific scavenging of all ROS might lead to untoward effects. Thus, there has been substantial interest in identifying a more suitable ROS-related target for ROP. A likely candidate is NADPH oxidase (NOX) enzymes which solely function to produce ROS and are involved in various pathological processes. Of relevance to this thesis is that angiotensin II and aldosterone are stimulators of NOX isoforms. Lastly, a Western diet typified by high sodium levels has been viewed as a risk factor for a number of pathological conditions, such as hypertension, cardioavascular disease and diabetic retinopathy. The reasons for this are not completely understood, but studies suggest that it is likely due to oxidative stress, inflammation and disturbances of the RAAS. At present, the role for aldosterone, NOX isoforms and dietary salt in ROP is unclear. The current study was aimed to elucidate (1) the role of aldosterone in OIR using an aldosterone synthase inhibitor, FAD286, (2) the role of NOX isoforms using mice lacking NOX1, NOX2 and NOX4, a novel inhibitor of NOX1 and NOX4, GKT137831, and the association between NOX isoforms and the RAAS as well as (3) the role of dietary salt in OIR. Methods: To address Aim 1, the rodent model of OIR was induced in Sprague-Dawley (SD) rats by exposure to 80% oxygen from postnatal days (P) 0 to 11, followed by 7 days in room air. Treatment with either FAD286 (30 mg/kg/day) and the AT1-R blocker, valsartan (10mg/kg/day) was administered from P12-P18. Retinal neovascularization was evaluated by counting pre-retinal vessels on paraffin sections and by measuring the area of neovascular tufts on FITC-lectin stained retinal wholemounts. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate retinal vascular endothelial growth factor (VEGF) levels. The density of microglia was evaluated by quantitating ionized calcium binding adaptor protein (Iba-1, a microglial marker) immunolabeling on retinal paraffin sections. Quantitative real-time PCR was employed to measure the expression of inflammatory factors, such as intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecular 1 (VCAM-1) and tumor necrosis factor-α (TNF-α) and RAAS components, such as aldosterone synthase. Finally, the cellular location of the aldosterone/system in the retina was determined by PCR using primary cultures of retinal cell populations, such as retinal ganglion cells (RGC), microglia, glial cells and vascular cells. To address Aim 2, the mouse model of OIR was induced in NOX1, NOX2, NOX4 knockout (KO) and their respective wild type (WT) control mice by exposure to 75% oxygen from P7 to P11, followed by 5 days in room air. Control animals were in room air from P0-P18. Retinal vasculopathy was evaluated by quantitating pre-retinal vessels on paraffin sections and neovascular tufts on retinal wholemounts. The extent of vaso-obliteration was quantitated by measuring the area of vessel loss on retinal wholemounts. Vascular leakage was quantitated by measuring levels of albumin in the retina using ELISA. A rhodamine-conjugated concanavalin A (ConA) perfusion technique was used to stain leukocytes adhering to retinal vasculature, and the total number of leukocytes was counted. Superoxide levels were measured by dihydroethidium (DHE) labeling on retinal cryosections. The density of microglia was evaluated by Iba-1 immunolabeling on retinal paraffin sections. Quantitative real-time PCR was used to measure VEGF, erythropoietin (EPO), angiopoetin-2 and ICAM-1. To examine if RAAS blockade can attenuate NOX derived ROS, primary cultures of rat retinal microglia were exposed to hypoxia to mimic the in vivo environment of OIR. The levels of superoxide in microglial lysates were evaluated by DHE staining. The expression of NOX isoforms, angiogenic and inflammatory factors were quantitated by quantitative real-time PCR and a protein cytokine array. Finally, to investigate if specific NOX isoform inhibition is a potential treatment for ROP, a subset of SD rats was employed to study the effect of GKT137831 in a rat model of ROP. To address Aim 3, a low-salt (LS, 0.03% sodium) and normal-salt (NS, 0.3% sodium) diet were given to the mothers from gestational day (G) 20 to P18. Retinal neovascularization and vaso-obliteration were quantitated on paraffin sections and retinal wholemounts. ELISA was used to measure retinal VEGF levels and vascular leakage. Western blotting was used to quantitate EPO and phosphorylated extracellular signal-regulated kinase-1 and 2 (pERK1/2). Immunohistochemistry was used to evaluate the density of microglia (Iba-1), TNF-α and the expression of water and ion channels such as aquaporin (APQ) 4, the inwardly rectifying potassium channel Kir4.1 on retinal paraffin sections. Glial fibrillary acidic protein (GFAP) immonofluorescence labeling on paraffin sections was used to evaluate retinal gliosis. Quantitative real-time PCR was used to quantitate the mRNA levels of RAAS elements, such as angiotensinogen, renin, AT1-R, aldosterone synthase and MR, and water and ion transport channels, such as AQP1, AQP4, Kir4.1 and the epithelial sodium channel (ENaC)α. Results: Firstly, in the rat model of OIR, treatment with FAD286 significantly reduced retinal neovascularization, microglial density, angiogenic and inflammatory factors, such as VEGF, ICAM-1, VCAM-1, MCP-1 and TNF-α. FAD286 effectively reduced aldosterone synthase mRNA in the retina. MR and 11βHSD2 were expressed in RGC, microglia, glial, endothelial cells and pericytes, but aldosterone synthase was only detected in RGC, microglia and glial. Secondly, deficiency in NOX1 but not NOX2 and NOX4 is protective against retinopathy. In addition, NOX1 but not NOX2 and NOX4 had reduced retinal neovascularization, decreased retinal avascular vaso-obliteration and leukostasis. These effects in NOX1 KO mice with OIR were accompanied by a reduction in the density of microglia, retina vascular leakage, the expression of angiogenic and inflammatory markers as well as ROS levels, such as VEGF, EPO, angiopoeitin-2, ICAM-1, superoxide and nitrotyrosine levels. In cultured microglia, ROS levels and the expression of NOX isoforms as well as angiogenic and inflammatory factors were elevated in response to hypoxia, and these increases were reduced with RAAS blockade. NOX1 was expressed in RGC, glia, microglia and pericytes, but was absence in endothelial cells. NOX2 was expressed in all retinal types, but was not detected in glial cells. NOX4 was presence in all retinal cell types. GKT137831 showed similar retinoprotective effects in rats with OIR as NOX1 KO mice with OIR. In vitro, GKT137831 effectively reduced hypoxia induced ROS in microglia. Lastly, the LS diet exerted protective effects in the rat OIR model with the benefits of reduced neovascularization, vaso-obliteration, angiogenic factors (VEGF, EPO, pERK1/2), inflammatory factors (TNF-α), microglial density, retinal RAAS elements (renin, aldosterone synthase, AT1-R and MR), retinal gliosis, vascular leakage. In addition, OIR rats fed with NS showed an increase in the expression of water and ion transport channels, such as AQP4, Kir4.1 and ENaCα, which remained unchanged with a LS diet. Conclusions: The results of the thesis suggest that FAD286, GKT137831 and a LS diet are anti-angiogenic and anti-inflammatory in OIR and that NOX1 is a key player in OIR. Therefore, FAD286, GKT137831 and a low-salt diet are potential treatments for ROP and other retinal diseases characterized by neovascularization, such as diabetic retinopathy which is the leading cause of vision impairment in the adult population

Finerenone, a Non-Steroidal Mineralocorticoid Receptor Antagonist, Reduces Vascular Injury and Increases Regulatory T-Cells: Studies in Rodents with Diabetic and Neovascular Retinopathy

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

FT011, a novel cardiorenal protective drug, reduces inflammation, gliosis and vascular injury in rats with diabetic retinopathy

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach

A potent Nrf2 activator, dh404, bolsters antioxidant capacity in glial cells and attenuates ischaemic retinopathy

Abstract An imbalance in oxidative stress and antioxidant defense mechanisms contributes to the development of ischaemic retinopathies such as diabetic retinopathy and retinopathy of prematurity (ROP). Currently, the therapeutic utility of targeting key transcription factors to restore this imbalance remains to be determined. We postulated that dh404, an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), the master regulator of oxidative stress responses, would attenuate retinal vasculopathy by mechanisms involving protection against oxidative stress-mediated damage to glia. Oxygen-induced retinopathy (OIR) was induced in neonatal C57BL/6J mice by exposure to hyperoxia (phase I) followed by room air (phase II). dh404 (1 mg/kg/every second day) reduced the vaso-obliteration of phase I OIR and neovascularization, vascular leakage and inflammation of phase II OIR. In phase I, the astrocytic template and vascular endothelial growth factor (VEGF) expression necessary for physiological angiogenesis are compromised resulting in vaso-obliteration. These events were attenuated by dh404 and related to dh404's ability to reduce the hyperoxia-induced increase in reactive oxygen species (ROS) and markers of cell damage as well as boost the Nrf2-responsive antioxidants in cultured astrocytes. In phase II, neovascularization and vascular leakage occurs following gliosis of Müller cells and their subsequent increased production of angiogenic factors. dh404 reduced Müller cell gliosis and vascular leakage in OIR as well as the hypoxia-induced increase in ROS and angiogenic factors with a concomitant increase in Nrf2-responsive antioxidants in cultured Müller cells. In conclusion, agents such as dh404 that reduce oxidative stress and promote antioxidant capacity offer a novel approach to lessen the vascular and glial cell damage that occurs in ischaemic retinopathies