Devy Deliyanti

NADPH oxidase, NOX1, mediates vascular injury in ischemic retinopathy.

AIMS 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. RESULTS 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. INNOVATION Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. CONCLUSIONS Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies.

Neovascularization Is Attenuated With Aldosterone Synthase Inhibition in Rats With Retinopathy

Neovascularization is a hallmark feature of retinopathy of prematurity and diabetic retinopathy. Type 1 angiotensin receptor blockade reduces neovascularization in experimental retinopathy of prematurity, known as oxygen-induced retinopathy (OIR). We investigated in OIR whether inhibiting aldosterone with the aldosterone synthase inhibitor FAD286 reduced neovascularization as effectively as angiotensin receptor blockade (valsartan). OIR was induced in neonatal Sprague-Dawley rats, and they were treated with FAD286 (30 mg/kg per day), valsartan (10 mg/kg per day), or FAD286+valsartan. The cellular sources of aldosterone synthase, the mineralocorticoid receptor, and 11&bgr;-hydroxysteroid dehydrogenase 2 were evaluated in retinal cells involved in neovascularization (primary endothelial cells, pericytes, microglia, ganglion cells, and glia). In OIR, FAD286 reduced neovascularization and neovascular tufts by 89% and 67%, respectively, and normalized the increase in vascular endothelial growth factor mRNA (1.74-fold) and protein (4.74-fold) and was as effective as valsartan and FAD286+valsartan. In retina, aldosterone synthase mRNA was reduced with FAD286 but not valsartan. Aldosterone synthase was detected in microglia, ganglion cells, and glia, whereas mineralocorticoid receptor and 11&bgr;-hydroxysteroid dehydrogenase 2 were present in all of the cell types studied. Given the location of aldosterone synthase in microglia and their contribution to retinal inflammation and neovascularization in OIR, the effects of FAD286 on microglial density were studied. The increase in microglial density (ionized calcium binding adaptor protein 1 immunolabeling) in OIR was reduced with all of the treatments. In OIR, FAD286 reduced the increase in mRNA for tumor necrosis factor-&agr;, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and monocyte chemoattractant molecule 1. These findings indicate that aldosterone inhibition may be a potential treatment for retinal neovascularization.

The retinal renin-angiotensin system: roles of angiotensin II and aldosterone.

In the present review we examine the experimental and clinical evidence for the presence of a local renin-angiotensin system within the retina. Interest in a pathogenic role for the renin-angiotensin system in retinal disease originally stemmed from observations that components of the pathway were elevated in retina during the development of certain retinal pathologies. Since then, our knowledge about the contribution of the RAS to retinal disease has greatly expanded. We discuss the known functions of the renin-angiotensin system in retinopathy of prematurity and diabetic retinopathy. This includes the promotion of retinal neovascularization, inflammation, oxidative stress and neuronal and glial dysfunction. The contribution of specific components of the renin-angiotensin system is evaluated with a particular focus on angiotensin II and aldosterone and their cognate receptors. The therapeutic utility of inhibiting key components of the renin-angiotensin system is complex, but may hold promise for the prevention and improvement of vision threatening diseases.

Inhibition of NOX1/4 with GKT137831: a potential novel treatment to attenuate neuroglial cell inflammation in the retina

BackgroundInflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that GKT137831, a dual inhibitor of NADPH oxidases (NOX) 1 and NOX4, reduces inflammation in the ischemic retina by dampening the pro-inflammatory phenotype of retinal immune cells as well as macroglial Müller cells and neurons.MethodsIschemic retinopathy was induced in Sprague-Dawley rats by exposure to 80 % O2 cycled with 21 % O2 for 3 h per day from postnatal day (P) 0 to P11, followed by room air (P12 to P18). GKT137831 was administered P12 to P18 (60 mg/kg, subcutaneous) and comparisons were to room air controls. Retinal inflammation was examined by measuring leukocyte adherence to the retinal vasculature, ionized calcium-binding adaptor protein-1-positive microglia/macrophages, and the mRNA and protein levels of key inflammatory factors involved in retinal disease. Damage to Müller cells was evaluated by quantitating glial fibrillary acidic protein-positive cells and vascular leakage with an albumin ELISA. To verify the anti-inflammatory actions of GKT137831 on glia and neurons involved in ischemic retinopathy, primary cultures of rat retinal microglia, Müller cells, and ganglion cells were exposed to the in vitro counterpart of ischemia, hypoxia (0.5 %), and treated with GKT137831 for up to 72 h. ROS levels were evaluated with dihydroethidium and the protein and gene expression of inflammatory factors with quantitative PCR, ELISA, and a protein cytokine array.ResultsIn the ischemic retina, GKT137831 reduced the increased leukocyte adherence to the vasculature, the pro-inflammatory phenotype of microglia and macroglia, the increased gene and protein expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and leukocyte adhesion molecules as well as vascular leakage. In all cultured cell types, GKT137831 reduced the hypoxia-induced increase in ROS levels and protein expression of various inflammatory mediators.ConclusionsNOX1/4 enzyme inhibition with GKT137831 has potent anti-inflammatory effects in the retina, indicating its potential as a treatment for a variety of vision-threatening retinopathies.

Deleting the BAFF receptor TACI protects against systemic lupus erythematosus without extensive reduction of B cell numbers.

B cell-activating factor of the TNF family (BAFF) is an essential B cell survival factor. However, high levels of BAFF promote systemic lupus erythematosus (SLE) in mice and humans. Belimumab (anti-human BAFF) limits B cell survival and is approved for use in patients with SLE. Surprisingly, the efficacy of rituximab (anti-human CD20) in SLE remains controversial, despite depleting B cells more potently than belimumab. This raises the question of whether B cell depletion is really the mechanism of action of belimumab. In BAFF transgenic mice, SLE development is T cell-independent but relies on innate activation of B cells via TLRs, and TLR expression is modulated by the BAFF receptor TACI. Here, we show that loss of TACI on B cells protected against BAFF-mediated autoimmune manifestations while preserving B cells, suggesting that loss of BAFF signaling through TACI rather than loss of B cells may underpin the effect of belimumab in the clinic. Therefore, B cell-sparing blockade of TACI may offer a more specific and safer therapeutic alternative to broad B cell depletion in SLE.

Brain and retinal microglia in health and disease: An unrecognized target of the renin–angiotensin system

Microglia are the resident immune cells within the brain and retina, commonly known as the macrophages of the central nervous system (CNS). Microglia survey the surrounding milieu to eliminate invading microbes, clear cellular debris and enforce programmed cell death by removing apoptotic cells. Complementary to their ‘house‐keeping’ role, microglia are capable of releasing brain‐derived neurotrophic factor (BDNF), as well as various anti‐inflammatory cytokines that sustain and support neuronal survival. Although microglia are essential for maintaining a healthy CNS, paradoxically they may undergo phenotypic changes to influence numerous neurodegenerative disorders, including Parkinsons disease and Alzheimers disease. Understanding the underlying mechanisms that determine whether microglia are supportive or toxic could elucidate novel and more effective therapeutic targets to treat an array of neurological and retinal diseases. Although relatively little is known about the influences that evoke phenotypic changes in the microglial population, there is accumulating evidence illustrating an interaction with the renin‐angiotensin system (RAS). The angiotensin AT1 and AT2 receptors may have differential roles in mediating the activity of microglia. Understanding the actions of these angiotensin receptors will be important in defining whether microglia are an important therapeutic target for RAS blockade in brain and ocular diseases.

Retinal Vasculopathy Is Reduced by Dietary Salt Restriction Involvement of Glia, ENaCα, and the Renin–Angiotensin–Aldosterone System

Objective— Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin–angiotensin–aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin–angiotensin–aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin–angiotensin–aldosterone system in ischemic retinopathy. Approach and Results— Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-&agr;. Macroglial Müller cells maintain the integrity of the blood–retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor–sensitive epithelial sodium channel alpha (ENaC&agr;), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaC&agr;, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaC&agr;, angiotensin type 1 receptor, and mineralocorticoid receptor expression. Conclusions— An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin–angiotensin–aldosterone system.Objective— Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin–angiotensin–aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin–angiotensin–aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin–angiotensin–aldosterone system in ischemic retinopathy. Approach and Results— Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Muller cells maintain the integrity of the blood–retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor–sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Muller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Muller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression. Conclusions— An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin–angiotensin–aldosterone system. # Significance {#article-title-63}

Inhibition of the Nuclear Receptor RORγ and Interleukin-17A Suppresses Neovascular Retinopathy

Objective— Although inhibitors of vascular endothelial growth factor (VEGF) provide benefit for the management of neovascular retinopathies, their use is limited to end-stage disease and some eyes are resistant. We hypothesized that retinoic acid–related orphan nuclear receptor &ggr; (ROR&ggr;) and its downstream effector, interleukin (IL)-17A, upregulate VEGF and hence are important treatment targets for neovascular retinopathies. Approach and Results— Utilizing a model of oxygen-induced retinopathy, confocal microscopy and flow cytometry, we identified that retinal immunocompetent cells, microglia, express IL-17A. This was confirmed in primary cultures of rat retinal microglia, where hypoxia increased IL-17A protein as well as IL-17A, ROR&ggr;, and tumor necrosis factor-&agr; mRNA, which were reduced by the ROR&ggr; inhibitor, digoxin, and the ROR&agr;/ROR&ggr; inverse agonist, SR1001. By contrast, retinal macroglial Müller cells and ganglion cells, key sources of VEGF in oxygen-induced retinopathy, did not produce IL-17A when exposed to hypoxia and IL-1&bgr;. However, they expressed IL-17 receptors, and in response to IL-17A, secreted VEGF. This suggested that ROR&ggr; and IL-17A inhibition might attenuate neovascular retinopathy. Indeed, digoxin and SR1001 reduced retinal vaso-obliteration, neovascularization, and vascular leakage as well as VEGF and VEGF-related placental growth factor. Digoxin and SR1001 reduced microglial-derived IL-17A and Müller cell and ganglion cell damage. The importance of IL-17A in oxygen-induced retinopathy was confirmed by IL-17A neutralization reducing vasculopathy, VEGF, placental growth factor, tumor necrosis factor-&agr;, microglial density and Müller cell, and ganglion cell injury. Conclusions— Our findings indicate that an ROR&ggr;/IL-17A axis influences VEGF production and neovascular retinopathy by mechanisms involving neuroglia. Inhibition of ROR&ggr; and IL-17A may have potential for the improved treatment of neovascular retinopathies.

Prorenin stimulates a pro-angiogenic and pro-inflammatory response in retinal endothelial cells and an M1 phenotype in retinal microglia

Angiogenesis and inflammation are causative factors in the development of neovascular retinopathies. These processes involve the retinal endothelium and the retinal immune cells, microglia. The renin‐angiotensin system contributes to retinal injury via the actions of the type 1 angiotensin receptor (AT1R). However, it has been suggested that prorenin, the initiator of the renin‐angiotensin system cascade, influences retinal injury independently from the AT1R. We evaluated whether prorenin induced a pro‐angiogenic and pro‐inflammatory response in retinal endothelial cells and a pro‐inflammatory phenotype in retinal microglia. Primary cultures of retinal endothelial cells and microglia were studied. Rat recombinant prorenin (2 nmol/L) stimulated the proliferation and tubulogenesis of retinal endothelial cells; it increased the levels of pro‐angiogenic factors, vascular endothelial growth factor, angiopoietin‐1, and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains, and pro‐inflammatory factors, intercellular adhesion molecule‐1 and monocyte chemoattractant protein‐1, relative to the controls. The messenger RNA levels of the (pro)renin receptor were also increased. These effects occurred in the presence of the AT1R blocker candesartan (10 μmol/L) and the renin inhibitor aliskiren (10 μmol/L). Microglia, which express the (pro)renin receptor, elicited an activated phenotype when exposed to prorenin, which was characterized by increased levels of intercellular adhesion molecule‐1, monocyte chemoattractant protein‐1, tumour necrosis factor‐α, interleukin‐6, and interleukin‐1β and by decreased levels of interleukin‐10 and arginase‐1 relative to controls. Candesartan did not influence the effects of prorenin on retinal microglia. In conclusion, prorenin has distinct pro‐angiogenic and pro‐inflammatory effects on retinal cells that are independent of the AT1R, indicating the potential importance of prorenin in retinopathy.

Foxp3+ Tregs are recruited to the retina to repair pathological angiogenesis

Neovascular retinopathies are major causes of vision loss; yet treatments to prevent the condition are inadequate. The role of regulatory T cells in neovascular retinopathy is unknown. Here we show that in retinopathy regulatory T cells are transiently increased in lymphoid organs and the retina, but decline when neovascularization is established. The decline is prevented following regulatory T cells expansion with an IL-2/anti-IL-2 mAb complex or the adoptive transfer of regulatory T cells. Further, both approaches reduce vasculopathy (vaso-obliteration, neovascularization, vascular leakage) and alter the activation of Tmem119+ retinal microglia. Our in vitro studies complement these findings, showing that retinal microglia co-cultured with regulatory T cells exhibit a reduction in co-stimulatory molecules and pro-inflammatory mediators that is attenuated by CTLA-4 blockade. Collectively, we demonstrate that regulatory T cells are recruited to the retina and, when expanded in number, repair the vasculature. Manipulation of regulatory T cell numbers is a previously unrecognized, and promising avenue for therapies to prevent blinding neovascular retinopathies.The local immune responses in the eye are attenuated to preserve sight. Surprisingly, Deliyanti et al. show that regulatory T cells (Tregs) take an active role in protecting the eye from neovascularization in oxygen-induced retinopathy, and that interventions that augment the retinal Treg numbers reduce neovascular retinopathy in mice.