Antonia Grace Miller

Identification of a Retinal Aldosterone System and the Protective Effects of Mineralocorticoid Receptor Antagonism on Retinal Vascular Pathology

Blockade of the renin–angiotensin–aldosterone system (RAAS) is being evaluated as a treatment for diabetic retinopathy; however, whether the mineralocorticoid receptor (MR) and aldosterone influence retinal vascular pathology is unknown. We examined the effect of MR antagonism on pathological angiogenesis in rats with oxygen-induced retinopathy (OIR). To determine the mechanisms by which the MR and aldosterone may influence retinal angiogenesis; inflammation and glucose-6-phosphate dehydrogenase (G6PD) were evaluated in OIR and cultured bovine retinal endothelial cells (BRECs) and bovine retinal pericytes (BRPs). In OIR, MR antagonism (spironolactone) was antiangiogenic. Aldosterone may mediate the pathogenic actions of MR in the retina, with 11&bgr;-hydroxysteroid dehydrogenase type 2 mRNA being detected and with aldosterone stimulating proliferation and tubulogenesis in BRECs and exacerbating angiogenesis in OIR, which was attenuated with spironolactone. The MR and aldosterone modulated retinal inflammation, with leukostasis and monocyte chemoattractant protein-1 mRNA and protein in OIR being reduced by spironolactone and increased by aldosterone. A reduction in G6PD may be an early response to aldosterone. In BRECs, BRPs, and early OIR, aldosterone reduced G6PD mRNA, and in late OIR, aldosterone increased mRNA for the NAD(P)H oxidase subunit Nox4. A functional retinal MR–aldosterone system was evident with MR expression, translocation of nuclear MR, and aldosterone synthase expression, which was modulated by RAAS blockade. We make the first report that MR and aldosterone influence retinal vasculopathy, which may involve inflammatory and G6PD mechanisms. MR antagonism may be relevant when developing treatments for retinopathies that target the RAAS.

Candesartan attenuates diabetic retinal vascular pathology by restoring glyoxalase-I function.

OBJECTIVE Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy. RESEARCH DESIGN AND METHODS Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats. RESULTS In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO•), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO•. Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO•, and restored GLO-I. CONCLUSIONS We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.

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.

RILLKKMPSV Influences the Vasculature, Neurons and Glia, and (Pro)Renin Receptor Expression in the Retina

The (pro)renin receptor [(P)RR] is implicated in organ pathology. We examined the cellular location of the (P)RR and whether a putative (P)RR antagonist, RILLKKMPSV, corresponding to the handle region of the prorenin prosegment (handle region peptide [HRP]) influences angiogenesis, inflammation, and neuronal and glial function in rat retina. The (P)RR was localized to retinal vessels, endothelial cells, and pericytes, but most immunolabeling was in ganglion cells and glia. HRP (1 mg/kg per day by IP injection) reduced physiological angiogenesis in developing retina. Moreover, HRP (0.1 mg/kg per day by subcutaneous minipump) reduced pathological retinal angiogenesis, inflammation, and vascular endothelial growth factor and intercellular adhesion molecule-1 mRNA in rats with oxygen-induced retinopathy (OIR) to an extent similar to valsartan (10 mg/kg per day, IP). In contrast to its effects on vasculature, HRP compromised the electroretinogram in shams and OIR and increased phosphorylated extracellular-signal–related protein kinase 1/2 immunolabeling in shams but not in OIR, whereas valsartan did not affect the electroretinogram and reduced extracellular-signal–related protein kinase 1/2 immunolabeling in OIR. Retinal (P)RR mRNA levels were increased in OIR; HRP, but not valsartan, increased (P)RR mRNA levels in shams, whereas both HRP and valsartan reduced (P)RR mRNA levels in OIR. A control peptide (VSPMKKLLIR, 0.1 mg/kg per day) did not influence retinal vasculopathy or function. Circulating HRP levels in rats administered 1 mg/kg per day HRP were undetectable (<3 pmol/L). We conclude that HRP had protective effects on the retinal vasculature similar to those of valsartan; however, unlike valsartan, HRP injured neuro-glia, which may involve the (P)RR, although the undetectable circulating HRP level makes a direct effect of HRP on retinal (P)RR function unlikely.

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.

Update on the treatment of diabetic retinopathy

Retinopathy is the most feared complication of diabetes, compromising quality of life in most sufferers. Almost all patients with type 1 diabetes will develop retinopathy over a 15- to 20-year period, and approximately 20–30% will advance to the blinding stage of the disease[1]. Greater than 60% of patients with type 2 diabetes will have retinopathy. This situation is highlighted by the frightening statistic that diabetic retinopathy (DR) remains the most common cause of vision impairment in people of working age in Western society. With the global epidemic of type 2 diabetes, this predicament is set to worsen as over 360 million people are projected to suffer from diabetes and its complications by 2030. Vision loss from diabetes is due to a number of factors, including haemorrhage from new and poorly formed blood vessels, retinal detachment due to contraction of deposited fibrous tissue, and neovascular glaucoma resulting in an increase in intraocular pressure. Diabetic macular oedema is now the principal cause of vision loss in diabetes and involves leakage from a disrupted blood-retinal barrier. In terms of treatment, there is clear evidence that strict metabolic and blood pressure control can lower the risk of developing DR and reduce disease progression. Laser photocoagulation and vitrectomy are effective in preventing severe vision loss in DR, particularly in the most advanced stages of the disease. However, both procedures have limitations. This review examines evidence from preclinical and clinical studies that shows that targeting inhibition of the renin-angiotensin system, vascular endothelial growth factor, corticosteroids, protein kinase C, growth hormone, and advanced glycation end-products are potential treatments for DR.

Prorenin and the (pro)renin receptor: recent advances and implications for retinal development and disease

Purpose of reviewThe renin–angiotensin system (RAS) is a treatment target for diabetic retinopathy and possibly other ocular diseases. However, angiotensin II blockade, though beneficial in diabetic retinopathy, is not completely retinoprotective. There is speculation that this shortfall is due to incomplete suppression of other RAS components. This review discusses the possibility that prorenin, which initiates the RAS, and the (pro)renin receptor [(P)RR] are potential candidates. Recent findingsDespite prorenin being elevated in diabetic retinopathy, it remains unclear whether it exerts any functional effects in tissues, including the eye. Of interest are newly identified functions for the (P)RR based on its homology with an accessory protein of vacuolar ATPase, ATP6AP2. These include roles in the viability of the central nervous system, including the retina, via the Wnt signaling pathway. Additionally, (P)RR/ATP6AP2 is implicated in other vacuolar ATPase-related events, including the regulation of cellular pH in the kidney and cell survival. Yet to be determined is whether the effects of (P)RR/ATP6AP2 are relevant to retinal cell function in health and disease and require the participation of its ligand prorenin. SummaryNew functions for the (P)RR highlight previously unrecognized roles for this receptor in cellular events that may have implications for both the developing and diseased retina.

The renin-angiotensin system and advanced glycation end-products in diabetic retinopathy: impacts and synergies.

Diabetic retinopathy is a major cause of vision impairment and blindness and represents a significant health burden throughout the world. There is considerable interest in developing new treatments that retard the progression of diabetic retinopathy from its early to proliferative stages. It could be argued that the absence of an ideal therapy for diabetic retinopathy comes from an incomplete understanding about the biochemical mechanisms that underlie this disease, and their precise impact on specific retinal cell populations. Findings from pre-clinical and clinical studies indicate that both the renin-angiotensin system (RAS) and advanced glycation end-products (AGEs) influence various aspects of diabetic retinopathy. Of interest is growing evidence of cross-talk between the RAS and AGEs pathways. This review will discuss the role of both the RAS and AGEs in diabetic retinopathy, and how the identification of interactions between the two pathways may have implications for the development of new treatment strategies.

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.

1030 ACTIVATION OF RETINAL MICROGLIA IS ATTENUATED WITH ANGIOTENSIN II AND ALDOSTERONE BLOCKADE

Objectives: Inflammation and oxidative stress contribute to the vascular and neuroglial dysfunction of diabetic retinopathy and retinopathy of prematurity. Microglia are the resident immune cells of the retina and emerging evidence indicates that their activation by hyperglycaemia and hypoxia contributes to retinopathy. The aim of this study was to determine if activation of retinal microglia with hypoxia stimulated angiogenic, inflammatory and oxidative stress factors, and whether these events were attenuated by renin-angiotensin system (RAS) blockade. Methods: Primary cultures of rat retinal microglia were exposed to 0.5% hypoxia for 2 hours and incubated with and without the type 1 angiotensin receptor (AT1R) blocker, valsartan (1 &mgr;M, 10 &mgr;M) or the aldosterone synthase inhibitor, FAD286 (100 nM, 1 &mgr;M). The expression of microglial activation markers, angiogenic and inflammatory factors, RAS components and NADPH oxidase (NOX) subunits were assessed with quantitative real-time PCR. Reactive oxygen species were measured with dihydroethidium fluorescence. Results: Microglia expressed the AT1R, the mineralocorticoid receptor and aldosterone synthase. When exposed to hypoxia, microglia became activated (elevated CD11b) and increased their expression of NOX1, NOX4 and ROS. Hypoxia also increased the levels of vascular endothelial growth factor (VEGF) and various cytokines (e.g. IL-1&agr;, IL-10, MIP-3&agr;, MIP-2) in supernatant. All these events were reduced with valsartan and FAD286. Conclusions: The increased expression of angiogenic, inflammatory and oxidative stress factors in activated retinal microglia, highlights the potential contribution of microglia to retinal pathology. The finding that valsartan and FAD286 reduced these events, demonstrates that microglia may play a key role in RAS-mediated pathological effects in retinopathy.