Antonia G. Miller

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 11beta-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.

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.

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.

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.