Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism

Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N′-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism.     

Beta‐lapachone inhibits pathological retinal neovascularization in oxygen‐induced retinopathy via regulation of HIF‐1α

Retinal neovascularization in retinopathy of prematurity (ROP) is the most common cause of blindness for children. Despite evidence that hypoxia inducible factor (HIF)‐1α ‐VEGF axis is associated with the pathogenesis of ROP, the inhibitors of HIF‐1α have not been established as a therapeutic target in the control of ROP pathophysiology. We investigated the hypothesis that degradation of HIF‐1α as a master regulator of angiogenesis in hypoxic condition, using β‐lapachone, would confer protection against hypoxia‐induced retinopathy without affecting physiological vascular development in mice with oxygen‐induced retinopathy (OIR), an animal model of ROP. The effects of β‐lapachone were examined after intraocular injection in mice with OIR. Intraocular administration of β‐lapachone resulted in significant reduction in hypoxia‐induced retinal neovascularization without retinal toxicity or perturbation of developmental retinal angiogenesis. Our results demonstrate that HIF‐1α–mediated VEGF expression in OIR is associated with pathological neovascularization, not physiological angiogenesis. Thus, strategies blocking HIF‐1α in the developing eye in the pathological hypoxia could serve as a novel therapeutic target for ROP.     

Morphological and functional changes in the retina after chronic oxygen-induced retinopathy

The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment.     

Sirtuin1 Over-Expression Does Not Impact Retinal Vascular and Neuronal Degeneration in a Mouse Model of Oxygen-Induced Retinopathy

Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy.     

Secretoneurin, substance P and neuropeptide Y in the oxygen-induced retinopathy in C57Bl/6N mice

In this study, we investigated whether the proangiogenic neuropeptides secretoneurin (SN), substance P (SP), and neuropeptide Y (NPY) contribute to the development of abnormal neovascularization in the oxygen-induced retinopathy (OIR) model in mice. By exposing litters of C57Bl/6N mice to 75% oxygen from postnatal day 7 (P7) until postnatal day 11 (P11) and then returning them to normoxic conditions, retinal ischemia and subsequent neovascularization on the retinal surface were induced. Retinae were dissected on P9, P11, P12-P14, P16 and P20, and the concentrations of SN, SP, NPY and VEGF determined by radioimmunoassay or ELISA. The levels of SN and SP increased in controls from P9 until P16 and from P9 until P14, respectively, whereas the levels of NPY were high at P9 and decreased thereafter until P20, suggesting that NPY may participate in the development of the retina. However, dipeptidyl peptidase IV (DPPIV) and the NPY-Y2 receptor were not detectable in the immature retina indicating that NPY is not involved in the physiological vascularization in the retina. Compared to controls, OIR had no effect on the levels of SN, whereas levels of both SP and NPY slightly decreased during hyperoxia. Normalization of the levels of SP, and to a more pronounced extent of NPY, was significantly delayed during relative hypoxia. This clearly indicates that these three neuropeptides are not involved in the pathogenesis of neovascularization in OIR. Moreover, since there were no differences in the expression of two vessel markers in the retina of NPY knockout mice versus controls at P14, NPY is also not involved in the delayed development of the intermediate and deep vascular plexus in the retina in this animal model.     

Adiponectin inhibits high glucose-induced angiogenesis via inhibiting autophagy in RF/6A cells

Adiponectin, one of the adipose-derived hormone with metabolic activity, has been reported to conversely affect angiogenesis of endothelial cells in vitro. The previous study in animal models has demonstrated that adiponectin has a protective role in retinal vascular injury following pathological stimuli. However, clinical research regarding the relationship between plasma adiponectin level and diabetic retinopathy (DR) are inconclusive. The aim of this study was to investigate the effect of adiponectin on high glucose-induced retinal angiogenesis and its association with autophagy by using rhesus choroid-retinal endothelial (RF-6A) cells as a model. We found that cell vitality decreased and cell migration and tube formation increased in the high-glucose group. Treatment with adiponectin or 3-methyladenine (3-MA, an autophagy inhibitor) increased cell viability and inhibited cell migration and tube formation. In the high-glucose group, the protein expression of Bax and apoptosis rate of cells increased and the expression of Bcl-2 decreased, whereas treatment with adiponectin or 3-MA reversed these results. Autophagy was activated in the high-glucose group to present as more LC3B fluorescent dots and higher expressions of LC3B, Atg5 proteins as well as lower expression of p62. Treatment with adiponectin or 3-MA inhibited autophagy by promoting the expression of p-PI3K, p-AKT, and p-mTOR when compared with the high-glucose group. The results of this study suggested that adiponectin inhibits high glucose-induced angiogenesis of RF/6A cells by inhibiting autophagy, and promotion of the PI3K/AKT/mTOR pathway might be involved in the anti-autophagy activities of adiponectin.     

Targeting proliferative retinopathy: Arginase 1 limits vitreoretinal neovascularization and promotes angiogenic repair

Current therapies for treatment of proliferative retinopathy focus on retinal neovascularization (RNV) during advanced disease and can trigger adverse side-effects. Here, we have tested a new strategy for limiting neurovascular injury and promoting repair during early-stage disease. We have recently shown that treatment with a stable, pegylated drug form of the ureohydrolase enzyme arginase 1 (A1) provides neuroprotection in acute models of ischemia/reperfusion injury, optic nerve crush, and ischemic stroke. Now, we have determined the effects of this treatment on RNV, vascular repair, and retinal function in the mouse oxygen-induced retinopathy (OIR) model of retinopathy of prematurity (ROP). Our studies in the OIR model show that treatment with pegylated A1 (PEG-A1), inhibits pathological RNV, promotes angiogenic repair, and improves retinal function by a mechanism involving decreased expression of TNF, iNOS, and VEGF and increased expression of FGF2 and A1. We further show that A1 is expressed in myeloid cells and areas of RNV in retinal sections from mice with OIR and human diabetic retinopathy (DR) patients and in blood samples from ROP patients. Moreover, studies using knockout mice with hemizygous deletion of A1 show worsened RNV and retinal injury, supporting the protective role of A1 in limiting the OIR-induced pathology. Collectively, A1 is critically involved in reparative angiogenesis and neuroprotection in OIR. Pegylated A1 may offer a novel therapy for limiting retinal injury and promoting repair during proliferative retinopathy.Subject terms: Translational research, Preclinical research, Neuro-vascular interactions     

The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.     

Gene expression microarray analysis of early oxygen-induced retinopathy in the rat

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague–Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague–Dawley list returned the term “response to hypoxia,” absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1α were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat.     

Macrophage Metalloelastase (MMP-12) Deficiency Mitigates Retinal Inflammation and Pathological Angiogenesis in Ischemic Retinopathy

Pathological angiogenesis is a major cause of vision loss in ischemic and inflammatory retinal diseases. Recent evidence implicates macrophage metalloelastase (MMP-12), a macrophage-derived elastinolytic protease in inflammation, tissue remodeling and angiogenesis. However, little is known about the role of MMP-12 in retinal pathophysiology. The present study aims to explore the enzyme’s contributions to retinal angiogenesis in oxygen-induced retinopathy (OIR) using MMP-12 knockout (KO) mice. We find that MMP-12 expression was upregulated in OIR, accompanied by elevated macrophage infiltration and increased inflammatory markers. Compared to wildtype mice, MMP-12 KO mice had decreased levels of adhesion molecule and inflammatory cytokines and reduced vascular leakage in OIR. Concomitantly, these mice had markedly reduced macrophage content in the retina with impaired macrophage migratory capacity. Significantly, loss of MMP-12 attenuated retinal capillary dropout in early OIR and mitigated pathological retinal neovascularization (NV). Similar results were observed in the study using MMP408, a pharmacological inhibitor of MMP-12. Intriguingly, in contrast to reducing pathological angiogenesis, lack of MMP-12 accelerated revascularization of avascular retina in OIR. Taken together, we conclude that MMP-12 is a key regulator of macrophage infiltration and inflammation, contributing to retinal vascular dysfunction and pathological angiogenesis.     

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

BACKGROUND: Ocular neovascular disorders, such as diabetic retinopathy and age-related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper-dependent adenovirus (HD-Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization. METHODS: We first analyzed efficiency and stability of intraretinal gene transfer following intravitreous injection in mice. A HD-Ad vector expressing green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (HD-Ad/GFP) was compared with a first-generation (E1/E3-deleted) Ad vector carrying an identical GFP expression cassette (FG-Ad/GFP). We also constructed HD-Ad vectors expressing a soluble form of the VEGF receptor (sFlt-1) in a constitutive (HD-Ad/sFlt-1) or doxycycline (dox)-inducible (HD-Ad/S-M2/sFlt-1) manner and tested their therapeutic efficacy upon intravitreous delivery in a rat model of oxygen-induced retinopathy (OIR). RESULTS: HD-Ad/GFP promoted long-lasting (up to 1 year) transgene expression in retinal Müller cells, in marked contrast with the short-term expression observed with FG-Ad/GFP. Intravitreous injection of HD-Ad vectors expressing sFlt-1 resulted in detectable levels of sFlt-1 and inhibited retinal neovascularization by more than 60% in a rat model of OIR. Notably, the therapeutic efficacy of the inducible vector HD-Ad/S-M2/sFlt-1 was strictly dox-dependent. CONCLUSIONS: HD-Ad vectors enable stable gene transfer and regulated expression of angiostatic factors following intravitreous injection and thus are attractive vehicles for the gene therapy of neovascular diseases of the retina.     

Fatty Acid Binding Protein 4 Deficiency Protects against Oxygen-Induced Retinopathy in Mice

Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4^−/− mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4^−/− OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies.     

Endoplasmic reticulum stress is implicated in retinal inflammation and diabetic retinopathy

Diabetic retinopathy is a chronic low-grade inflammatory disease; however, the mechanisms remain elusive. In the present study, we demonstrated that endoplasmic reticulum (ER) stress was activated in the retina in animal models of diabetes and oxygen-induced retinopathy (OIR). Induction of ER stress by tunicamycin resulted in significantly increased expression of inflammatory molecules in the retina. Inhibition of ER stress by chemical chaperone 4-phenyl butyric acid ameliorated inflammation in cultured human retinal endothelial cells exposed to hypoxia, and in the retinas of diabetic and OIR mice. These findings indicate that ER stress is a potential mediator of retinal inflammation in diabetic retinopathy.     

Arginase 2 Deficiency Prevents Oxidative Stress and Limits Hyperoxia-Induced Retinal Vascular Degeneration

Background

Hyperoxia exposure of premature infants causes obliteration of the immature retinal microvessels, leading to a condition of proliferative vitreoretinal neovascularization termed retinopathy of prematurity (ROP). Previous work has demonstrated that the hyperoxia-induced vascular injury is mediated by dysfunction of endothelial nitric oxide synthase resulting in peroxynitrite formation. This study was undertaken to determine the involvement of the ureahydrolase enzyme arginase in this pathology.

Methods and Findings

Studies were performed using hyperoxia-treated bovine retinal endothelial cells (BRE) and mice with oxygen-induced retinopathy (OIR) as experimental models of ROP. Treatment with the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) prevented hyperoxia-induced apoptosis of BRE cells and reduced vaso-obliteration in the OIR model. Furthermore, deletion of the arginase 2 gene protected against hyperoxia-induced vaso-obliteration, enhanced physiological vascular repair, and reduced retinal neovascularization in the OIR model. Additional deletion of one copy of arginase 1 did not improve the vascular pathology. Analyses of peroxynitrite by quantitation of its biomarker nitrotyrosine, superoxide by dihydroethidium imaging and NO formation by diaminofluoroscein imaging showed that the protective actions of arginase 2 deletion were associated with blockade of superoxide and peroxynitrite formation and normalization of NOS activity.

Conclusions

Our data demonstrate the involvement of arginase activity and arginase 2 expression in hyperoxia-induced vascular injury. Arginase 2 deletion prevents hyperoxia-induced retinal vascular injury by preventing NOS uncoupling resulting in decreased reactive oxygen species formation and increased nitric oxide bioavailability.     

670nm Photobiomodulation as a Novel Protection against Retinopathy of Prematurity: Evidence from Oxygen Induced Retinopathy Models

Introduction

To investigate the validity of using 670nm red light as a preventative treatment for Retinopathy of Prematurity in two animal models of oxygen-induced retinopathy (OIR).

Materials and Methods

During and post exposure to hyperoxia, C57BL/6J mice or Sprague-Dawley rats were exposed to 670nm light for 3 minutes a day (9J/cm²). Whole mounted retinas were investigated for evidence of vascular abnormalities, while sections of neural retina were used to quantify levels of cell death using the TUNEL technique. Organs were removed, weighed and independent histopathology examination performed.

Results

670nm light reduced neovascularisation, vaso-obliteration and abnormal peripheral branching patterns of retinal vessels in OIR. The neural retina was also protected against OIR by 670nm light exposure. OIR-exposed animals had severe lung pathology, including haemorrhage and oedema, that was significantly reduced in 670nm+OIR light-exposed animals. There were no significance differences in the organ weights of animals in the 670nm light-exposed animals, and no adverse effects of exposure to 670nm light were detected.

Discussion

Low levels of exposure to 670nm light protects against OIR and lung damage associated with exposure to high levels of oxygen, and may prove to be a non-invasive and inexpensive preventative treatment for ROP and chronic lung disease associated with prematurity.     

Blockade of VEGFR1 and 2 suppresses pathological angiogenesis and vascular leakage in the eye

Objective

VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.

Materials and Methods

Neutralizing antibodies specific for mouse VEGFR1 (MF1) and VEGFR2 (DC101) were administrated systemically. CNV was induced by laser photocoagulation and assessed 14d after laser treatment. Retinal NV was generated in oxygen-induced ischemic retinopathy (OIR) and assessed at p17. NV quantification was determined by measuring NV tufts and vascular leakage was quantified by measuring [³H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.

Results

VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.

Conclusion

Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents.     

Sodium Salicylate Reduced Insulin Resistance in the Retina of a Type 2 Diabetic Rat Model

Sodium salicylate has been reported to reduce markers of diabetic retinopathy in a type 1 rat model. Because rates of type 2 diabetes are on the rise, we wanted to determine whether salicylate could improve insulin resistance in a type 2 rat model, as well as improve retinal function. We treated lean and obese BBZDR/Wor type 2 diabetic rats with salicylate in their chow for 2 months. Prior to salicylate treatment, rats underwent an electroretinogram to measure retinal function. After 2 months of treatment, rats underwent an additional electroretinogram prior to sacrifice. In addition to the animal model, we also treated retinal endothelial cells (REC) and rat Müller cells with salicylate and performed the same analyses as done for the rat retinal lysates. To investigate the role of salicylate in insulin signaling, we measured TNFα and caspase 3 levels by ELISA, as well as performed Western blotting for insulin receptor substrate 1, insulin receptor, SOCS3, and pro- and anti-apoptotic markers. Data demonstrated that salicylate significantly improved retinal function, as well as reduced TNFα and SOCS3-induced insulin resistance in all samples. Overall, results suggest that salicylate is effective in reducing insulin resistance in the retina of type 2 diabetic rat models.     

血小板反应素1在STZ诱导大鼠糖尿病性视网膜病变的表达研究

探讨TSP1表达在糖尿病视网膜病变中的作用和机制,为治疗和预防糖尿病视网膜病变提供新的实验和理论依据。用链脲佐菌素(STZ)腹腔注射建立糖尿病模型8周后,采用免疫组织化学、RT-PCR及实时荧光定量PCR法,分析TSP1在早期链尿佐菌素诱导的糖尿病SD大鼠视网膜中的表达。结果显示在早期糖尿病大鼠的视网膜表面血管、神经节细胞层、内外核层中均有明显的TSP1表达,糖尿病视网膜组TSP1 mRNA表达要高于对照组,其中实时荧光定量PCR CT值的结果显示糖尿病组TSP1 mRNA表达量较对照组要高约3．48倍,二组间差别有显著性意义(P<0．01),提示TSP1在视网膜组织中的表达与糖尿病视网膜病变的发生密不可分,TSP1表达的增加可能在糖尿病视网膜病变的发生和发展中起重要作用。     

Nerve growth factor regulates endothelial cell survival and pathological retinal angiogenesis

The mechanism underlying vasoproliferative retinopathies like retinopathy of prematurity (ROP) is hypoxia‐triggered neovascularisation. Nerve growth factor (NGF), a neurotrophin supporting survival and differentiation of neuronal cells may also regulate endothelial cell functions. Here we studied the role of NGF in pathological retinal angiogenesis in the course of the ROP mouse model. Topical application of NGF enhanced while intraocular injections of anti‐NGF neutralizing antibody reduced pathological retinal vascularization in mice subjected to the ROP model. The pro‐angiogenic effect of NGF in the retina was mediated by inhibition of retinal endothelial cell apoptosis. In vitro, NGF decreased the intrinsic (mitochondria‐dependent) apoptosis in hypoxia‐treated human retinal microvascular endothelial cells and preserved the mitochondrial membrane potential. The anti‐apoptotic effect of NGF was associated with increased BCL2 and reduced BAX, as well as with enhanced ERK and AKT phosphorylation, and was abolished by inhibition of the AKT pathway. Our findings reveal an anti‐apoptotic role of NGF in the hypoxic retinal endothelium, which is involved in promoting pathological retinal vascularization, thereby pointing to NGF as a potential target for proliferative retinopathies.     

Autophagy activation attenuates renal ischemia-reperfusion injury in rats

Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future.