Lung and Eye Disease Develop Concurrently in Supplemental Oxygen-Exposed Neonatal Mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are two debilitating disorders that develop in preterm infants exposed to supplemental oxygen to prevent respiratory failure. Both can lead to lifelong disabilities, such as chronic obstructive pulmonary disease and vision loss. Due to the lack of a standard experimental model of coincident disease, the underlying associations between BPD and ROP are not well characterized. To address this gap, we used the robust mouse model of oxygen-induced retinopathy exposing C57BL/6 mice to 75% oxygen from postnatal day 7 to 12. The cardinal features of ROP were replicated by this strategy, and the lungs of the same mice were simultaneously examined for evidence of BPD-like lung injury, investigating both the short- and long-term effects of early-life supplemental oxygen exposure. At postnatal days 12 and 18, mild lung disease was evident by histopathologic analysis together with the expected vasculopathy in the inner retina. At later time points, the lung lesion had progressed to severe airspace enlargement and alveolar simplification, with concurrent thinning in the outer layer of the retina. In addition, critical angiogenic oxidative stress and inflammatory factors reported to be dysregulated in ROP were similarly impaired in the lungs. These data shed new light on the interconnectedness of these two neonatal disorders, holding potential for the discovery of novel targets to treat BPD and ROP.

Angiotensin II and aldosterone activate retinal microglia.

Microglial cells are important contributors to the neuroinflammation and blood vessel damage that occurs in ischemic retinopathies. We hypothesized that key effectors of the renin-angiotensin aldosterone system, angiotensin II (Ang II) and aldosterone, increase the density of microglia in the retina and stimulate their production of reactive oxygen species (ROS) as well as pro-angiogenic and pro-inflammatory factors. Two animal models were studied that featured up-regulation of Ang II or aldosterone and included transgenic Ren-2 rats which overexpress renin and Ang II in tissues including the retina, and Sprague Dawley rats with ischemic retinopathy and infused with aldosterone. Complementary studies were performed in primary cultures of retinal microglia from neonatal Sprague Dawley rats exposed to hypoxia (0.5% O2) and inhibitors of the angiotensin type 1 receptor (valsartan), the mineralocorticoid receptor (spironolactone) or aldosterone synthase (FAD286). In both in vivo models, the density of ionized calcium-binding adaptor protein-1 labelled microglia/macrophages was increased in retina compared to genetic or vehicle controls. In primary cultures of retinal microglia, hypoxia increased ROS (superoxide) levels as well as the expression of the NADPH oxidase (NOX) isoforms, NOX1, NOX2 and NOX4. The elevated levels of ROS as well as NOX2 and NOX4 were reduced by all of the treatments, and valsartan and FAD286 also reduced NOX1 mRNA levels. A protein cytokine array of retinal microglia revealed that valsartan, spironolactone and FAD286 reduced the hypoxia-induced increase in the potent pro-angiogenic and pro-inflammatory agent, vascular endothelial growth factor as well as the inflammatory factors, CCL5 and interferon γ. Valsartan also reduced the hypoxia-induced increase in IL-6 and TIMP-1 as well as the chemoattractants, CXCL2, CXCL3, CXCL5 and CXCL10. Spironolactone and FAD286 reduced the levels of CXCL2 and CXCL10, respectively. In conclusion, our findings that both Ang II and aldosterone influence the activation of retinal microglia implicates the renin-angiotensin aldosterone system in the pathogenesis of ischemic retinopathies.

Endothelin-2 Injures the Blood-Retinal Barrier and Macroglial Müller Cells: Interactions with Angiotensin II, Aldosterone, and NADPH Oxidase.

Although increasing evidence indicates that endothelin-2 (Edn2) has distinct roles in tissue pathology, including inflammation, glial cell dysfunction, and angiogenesis, its role in the retina and the factors that regulate its actions are not fully understood. We hypothesized that Edn2 damages the blood-retinal barrier (BRB) and that this is mediated by interactions with the renin-angiotensin-aldosterone system and reactive oxygen species derived from NADPH oxidase (Nox). C57BL/6J mice received an intravitreal injection of Edn2 or control vehicle to examine the blood pressure-independent effects of Edn2. Mice administered Edn2 were randomized to receive by intraperitoneal injection treatments that inhibited the Edn type a receptor, Edn type b receptor, angiotensin type 1 receptor, mineralocorticoid receptor, or Nox isoforms 1 to 4. One month later, mice administered Edn2 exhibited breakdown of the BRB with increased vascular leakage, vascular endothelial growth factor expression, and infiltrating macrophages (Ly6C+CD45highCD11b+). Further, macroglial Müller cells, which influence the integrity of the BRB and prevent retinal edema, became gliotic and expressed increased levels of water (aquaporin-4) and ion (Kir4.1) channels. This Edn2-mediated retinopathy was reduced by all treatments. Complementary in vitro studies in cultured Müller cells supported these findings and demonstrated the importance of reactive oxygen species in mediating these events. In conclusion, Edn2 has detrimental effects on the BRB and Müller cells that involve interactions with the renin-angiotensin aldosterone system and Nox1/4.

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.

Inhibition of the Nuclear Receptor RORγ and Interleukin-17A Suppresses Neovascular Retinopathy: Involvement of Immunocompetent Microglia.

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 γ (RORγ) 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, RORγ, and tumor necrosis factor-α mRNA, which were reduced by the RORγ inhibitor, digoxin, and the RORα/RORγ 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-1ß. However, they expressed IL-17 receptors, and in response to IL-17A, secreted VEGF. This suggested that RORγ 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-α, microglial density and Müller cell, and ganglion cell injury. CONCLUSIONS: Our findings indicate that an RORγ/IL-17A axis influences VEGF production and neovascular retinopathy by mechanisms involving neuroglia. Inhibition of RORγ and IL-17A may have potential for the improved treatment of neovascular retinopathies.

Ebselen by modulating oxidative stress improves hypoxia-induced macroglial Müller cell and vascular injury in the retina.

Oxidative stress is an important contributor to glial and vascular cell damage in ischemic retinopathies. We hypothesized that ebselen via its ability to reduce reactive oxygen species (ROS) and augment nuclear factor-like 2 (Nrf2) anti-oxidants would attenuate hypoxia-induced damage to macroglial Müller cells and also lessen retinal vasculopathy. Primary cultures of rat Müller cells were exposed to normoxia (21% O2), hypoxia (0.5% O2) and ebselen (2.5 µM) for up to 72 h. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice while control mice were housed in room air. Mice received vehicle (saline, 5% dimethyl sulfoxide) or ebselen (10 mg/kg) each day between postnatal days 6-18. In cultured Müller cells, flow cytometry for dihydroethidium revealed that ebselen reduced the hypoxia-induced increase in ROS levels, whilst increasing the expression of Nrf2-regulated anti-oxidant genes, heme oxygenase 1, glutathione peroxidase-1, NAD(P)H dehydrogenase quinone oxidoreductase 1 and glutamate-cysteine ligase. Moreover, in Müller cells, ebselen reduced the hypoxia-induced increase in protein levels of pro-angiogenic and pro-inflammatory factors including vascular endothelial growth factor, interleukin-6, monocyte chemoattractant-protein 1 and intercellular adhesion molecule-1, and the mRNA levels of glial fibrillary acidic protein (GFAP), a marker of Müller cell injury. Ebselen improved OIR by attenuating capillary vaso-obliteration and neovascularization and a concomitant reduction in Müller cell gliosis and GFAP. We conclude that ebselen protects against hypoxia-induced injury of retinal Müller cells and the microvasculature, which is linked to its ability to reduce oxidative stress, vascular damaging factors and inflammation. Agents such as ebselen may be potential treatments for retinopathies that feature oxidative stress-mediated damage to glia and the microvasculature.