The deterioration of the inner blood-retinal barrier and consequent macular edema is a cardinal manifestation of diabetic retinopathy (DR) and the clinical feature most closely associated with loss of sight. We provide evidence from both human and animal studies for the critical role of the classical neuronal guidance cue, semaphorin 3A, in instigating pathological vascular permeability in diabetic retinas via its cognate receptor neuropilin-1. We reveal that semaphorin 3A is induced in early hyperglycemic phases of diabetes within the neuronal retina and precipitates initial breakdown of endothelial barrier function. We demonstrate, by a series of orthogonal approaches, that neutralization of semaphorin 3A efficiently prevents diabetes-induced retinal vascular leakage in a stage of the disease when vascular endothelial growth factor neutralization is inefficient. These observations were corroborated in Tg(Cre-Esr1)/Nrp1(flox/flox) conditional knockout mice. Our findings identify a therapeutic target for macular edema and provide further evidence for neurovascular crosstalk in the pathogenesis of DR.

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased O2MathClass-punc.MathClass-bin− formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces O2MathClass-punc.MathClass-bin− and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased O2MathClass-punc.MathClass-bin− formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

Rationale: Preterm infants exposed to mechanical ventilation and oxygen are at risk for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disorder characterized by arrested alveolar development. Studies have described disruption of microvascular development in BPD, characterized by primitive angioarchitectural patterns reminiscent of the canalicular/saccular stages of lung development. The molecular regulation of this BPD-associated dysangiogenesis remains undetermined. Objectives: Endoglin (CD105), a hypoxia-inducible transforming growth factor-b coreceptor, has been implicated as an important regulator of angiogenesis in various neoplastic and nonneoplastic conditions. The aim of this study was to investigate the expression of endoglin and other angiogenesis-related factors in ventilated preterm human lungs. Methods: We have studied endoglin protein and mRNA expression in postmortem lungs of short-term and long-term ventilated preterm infants. Control subjects were age-matched infants who had lived for less than 1 hour. Measurements and Main Results: Lungs of short-term ventilated preterm infants showed significant upregulation of endoglin mRNA and protein levels, immunolocalized to the microvasculature. Similar but more variable endoglin upregulation was noted in lungs of longterm ventilated infants with BPD. The mRNA levels of vascular endothelial growth factor, angiopoietin-1, and their respective receptors were significantly lower in ventilated lungs than in age-matched nonventilated control lungs. Conclusions: BPD is associated with a shift from traditional angiogenic growth factors (vascular endothelial growth factor, angiopoietin-1) to alternative regulators such as endoglin, which may contribute to BPD-associated microvascular dysangiogenesis.Keywords: chronic lung disease of prematurity; bronchopulmonary dysplasia; neonatal lung disease; angiogenesis Preterm newborns who require mechanical ventilation and supplemental oxygen are at risk for bronchopulmonary dysplasia (BPD), a chronic lung disease of newborn infants associated with significant mortality and morbidity (1). BPD in the postsurfactant era is seen mainly in very low birthweight infants and affects 30% of infants born between 24 and 28 weeks of gestation, many of whom will require long-term respiratory support (2, 3).The dominant pathological finding in postsurfactant BPD is an arrest in alveolar development, resulting in large and simplified airspaces that show varying degrees of interstitial fibrosis (2,(4)(5)(6)(7)(8). Studies have shown that, in addition to impaired alveolar development, there is also a disruption of pulmonary microvascular development in infants with BPD (8-10) or in BPD-like animal models such as chronically ventilated premature baboons (11,12). In view of the intimate relation between alveolar and microvascular development during pulmonary morphogenesis (13-16), disruption of microvascular development in premature lungs has been implicated as a critical factor in the arrest of alveolar development that i...