Therapeutic effects of PPARα agonists on diabetic retinopathy in type 1 diabetes models.

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARα dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARα agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-κB in the retinas of OIR and diabetic models. Fenofibrate's beneficial effects were blocked by a specific PPARα antagonist. Furthermore, Pparα knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARα-dependent mechanism.

Pathogenic role of the Wnt signaling pathway activation in laser-induced choroidal neovascularization.

PURPOSE: Choroidal neovascularization (CNV) is a severe complication of AMD. The Wnt signaling pathway has been shown to mediate angiogenesis. The purpose of this study was to investigate the pathogenic role of the Wnt pathway in CNV and explore the therapeutic potential of a novel Wnt signaling inhibitor in CNV. METHODS: Adult rats and mice were photocoagulated using diode laser to induce CNV. On the same day, the animals were intravitreally injected with a monoclonal antibody (Mab2F1) blocking LRP6 or nonspecific mouse IgG. The Wnt signaling activation and target gene expression in the eyecup were determined by Western blot analysis. Fundus angiography was used to examine leakage from the laser lesion. CNV areas were measured on choroidal flatmount using FITC-dextran. RESULTS: Levels of Wnt pathway components and Wnt target gene expression were elevated in both laser-induced CNV rat and mouse eyecups, suggesting activation of the Wnt pathway. Significant suppression of Wnt signaling was observed in the Mab2F1 treatment group. Mab2F1 decreased vascular leakage from CNV lesions and reduced the neovascular area in laser-induced CNV rats. Mab2F1 inhibited the hypoxia-induced activation of Wnt signaling in cultured RPE cells. Mab2F1 also ameliorated retinal inflammation and vascular leakage in the eyecups of very low-density lipoprotein receptor knockout mice, a model of subretinal neovascularization. CONCLUSIONS: The Wnt pathway is activated in the laser-induced CNV models and plays a pathogenic role in CNV. Blockade of Wnt signaling using an anti-LRP6 antibody has therapeutic potential in CNV.

Therapeutic potential of a monoclonal antibody blocking the Wnt pathway in diabetic retinopathy.

Dysregulation of Wnt/ß-catenin signaling contributes to the development of diabetic retinopathy by inducing retinal inflammation, vascular leakage, and neovascularization. Here, we evaluated the inhibitory effect of a monoclonal antibody (Mab) specific for the E1E2 domain of Wnt coreceptor low-density lipoprotein receptor-related protein 6, Mab2F1, on canonical Wnt signaling and its therapeutic potential for diabetic retinopathy. Mab2F1 displayed robust inhibition on Wnt signaling with a half-maximal inhibitory concentration (IC50) of 20 µg/mL in retinal pigment epithelial cells. In addition, Mab2F1 also attenuated the accumulation of ß-catenin and overexpression of vascular endothelial growth factor, intercellular adhesion molecule-1, and tumor necrosis factor-α induced by high-glucose medium in retinal endothelial cells. In vivo, an intravitreal injection of Mab2F1 significantly reduced retinal vascular leakage and decreased preretinal vascular cells in oxygen-induced retinopathy (OIR) rats, demonstrating its inhibitory effects on ischemia-induced retinal neovascularization. Moreover, Mab2F1 blocked the overexpression of the inflammatory/angiogenic factors, attenuated leukostasis, and reduced retinal vascular leakage in both early and late stages of streptozotocin-induced diabetes. In conclusion, Mab2F1 inhibits canonical Wnt signaling, vascular leakage, and inflammation in the retina of diabetic retinopathy models, suggesting its potential to be used as a therapeutic agent in combination with other antiangiogenic compounds.

Short communication: Investigation of incident HIV infections among U.S. army soldiers deployed to Afghanistan and Iraq, 2001-2007.

The U.S. Army initiated an investigation in response to observations of a possible increase in HIV incidence among soldiers deployed to combat. Human immunodeficiency virus (HIV)-infected U.S. Army soldiers are not eligible to deploy. Combat presents a health hazard to HIV-infected soldiers and they pose a threat to the safety of the battlefield blood supply and their contacts. All soldiers are routinely screened for HIV every 2 years and those who deploy are also screened both prior to and after deployment. Seroconversion rates were estimated for all soldiers who deployed to Afghanistan or Iraq in the period 2001-2007 and all active duty soldiers who did not. Seroconverters with an estimated date of infection, based on calculation of the midpoint between the last seronegative and first seropositive test date, that was either before or during deployment were eligible for inclusion. Confidential interviews and medical record reviews were conducted to determine the most likely time, geographic location, and mode of infection. Reposed predeployment samples were tested for HIV ribonucleic acid. The HIV seroconversion rate among all soldiers who deployed was less than the rate among those who did not deploy: 1.04 and 1.42 per 10,000 person-years, respectively. Among 48 cases, most were determined to have been infected in the United States or Germany and prior to deployment (n=20, 42%) or during rest and relaxation leave (n=13, 27%). Seven seronegative acute infections were identified in the predeployment period. Subtype was determined for 40 individuals; all were subtype B infections. All were acquired through sexual contact. These findings can inform development of preventive interventions and refinement of existing screening policy to further reduce HIV-infected deployed soldier person time.

Comparison of ocular pathologies in vitamin A-deficient mice and RPE65 gene knockout mice.

PURPOSE: RPE65 gene knockout (Rpe65⁻/⁻) mice showed abolished isomerohydrolase activity in the visual cycle and were considered a model for vitamin A deficiency in the retina. The purpose of this study was to compare the retinal phenotypes between vitamin A-deficient (VAD) mice and Rpe65⁻/⁻ mice under normal diet. METHODS: The VAD mice were fed with a vitamin A-deprived diet after birth. The age-matched control mice and Rpe65⁻/⁻ mice were maintained under normal diet. The structure of photoreceptor outer segment was compared using electron microscopy. Photoreceptor-specific gene expression was determined using real-time RT-PCR. The isomerohydrolase and lecithin-retinol acyltransferase (LRAT) activities were measured using an in vitro enzymatic activity assay. Endogenous retinoid profiles were analyzed by HPLC in mouse eyecup homogenates. RESULTS: Compared to wild-type mice under normal diet, scanning and transmission electron microscopy showed that the outer segments of photoreceptors were disorganized in VAD mice and were not disorganized in Rpe65⁻/⁻ mice, although they were shortened in the latter. VAD mice showed more prominent downregulation of middle wavelength cone opsin, whereas Rpe65⁻/⁻ mice displayed more suppressed expression of short wavelength cone opsin. In vitro enzymatic activity assay and Western blot analysis showed that vitamin A deprivation downregulated LRAT expression and activity in the eyecup, but Rpe65⁻/⁻ mice showed unchanged LRAT expression and activity. The depressed LRAT activity in VAD mice was partially rescued by the intraperitoneal injection of retinoic acid. CONCLUSIONS: VAD and Rpe65⁻/⁻ mice are different in cone photoreceptor degeneration, photoreceptor-specific gene regulation, isomerohydrolase activity, endogenous retinoid profile, and LRAT activity.

Activation of the Wnt pathway plays a pathogenic role in diabetic retinopathy in humans and animal models.

Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of beta-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of beta-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target.

Systemic administration of HMG-CoA reductase inhibitor protects the blood-retinal barrier and ameliorates retinal inflammation in type 2 diabetes.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are frequently used lipid-lowering drugs in type 2 diabetes. Recent emerging evidence suggests that statins protect cardiovascular function via lipid-independent mechanisms. However, the potential role of statins in diabetic retinopathy in type 2 diabetes is largely unclear. In the present study we have investigated the effect of lovastatin on blood-retinal barrier and inflammatory status in the retina of db/db mice and in cultured retinal cells. Male C57BL/KsJ db/db mice were randomly chosen to receive gastric gavage of lovastatin (10mg/kg/day) or vehicle control for 6 weeks. Retinal vascular permeability, the tight junction and inflammation were determined. The results showed that db/db mice at the age of 19 weeks exhibited significantly increased retinal vascular leakage and decreased tight junction protein level in the retina. Moreover, the expression of pro-inflammatory factors, e.g. ICAM-1 and TNF-alpha, was drastically up-regulated in diabetic retina. Lovastatin treatment normalized all of these changes. In cultured bovine retinal capillary endothelial cells (RCECs) and human ARPE-19 cells, lovastatin attenuated the decrease of tight junction protein (occludin) and adherens junction protein (VE-cadherin) expression-induced by TNF-alpha, a major pro-inflammatory cytokine in diabetic retinopathy. Lovastatin also attenuated TNF-alpha expression in RCEC. Towards the mechanism, we showed that lovastatin ameliorated ICAM-1 expression-induced by hypoxia and TNF-alpha in both RCECs and ARPE-19 cells, in part through inhibition of NF-kappaB activation. Taken together, these findings indicate that lovastatin protects blood-retinal barrier in diabetic retinopathy, which is likely via its anti-inflammatory effects.

Anti-inflammatory effects of pigment epithelium-derived factor in diabetic nephropathy.

Previously, we have reported that pigment epithelium-derived factor (PEDF) ameliorates albuminuria and inhibits matrix protein deposition in the kidney of streptozotocin (STZ)-induced diabetic rats, suggesting a renoprotective effect of PEDF in early stages of diabetic nephropathy. As inflammation is a major contributor to the development and progression of diabetic nephropathy, we examined in the present study whether PEDF inhibits renal inflammation in diabetic kidney. Diabetic rats received an intravenous injection of an adenovirus expressing PEDF (Ad-PEDF) or the same titer of a control virus. Three wk after the injection, diabetic rats treated with the control virus showed significantly elevated renal levels of proinflammatory factors such as ICAM-1, MCP-1, TNF-alpha, and VEGF compared with age-matched nondiabetic controls. Ad-PEDF effectively suppressed the overexpression of these proinflammatory factors in diabetic kidneys. In cultured primary human renal mesangial cells (HMC), the high-glucose medium-induced upregulation of VEGF and MCP-1 was largely blocked by PEDF. Furthermore, PEDF inhibited high glucose-induced activation of NF-kappaB, a key transcription factor mediating inflammatory responses, and hypoxia-inducible factor-1, a major activator of VEGF expression in HMC. These results suggest that the renoprotective effect of PEDF against diabetic nephropathy may be partially through its anti-inflammatory activity, likely by blocking the NF-kappaB and HIF-1 pathways.

Rat strain-dependent susceptibility to ischemia-induced retinopathy associated with retinal vascular endothelial growth factor regulation.

Vascular endothelial growth factor (VEGF) is a potent inflammation, vascular permeability, and angiogenic factor. Variations of the VEGF gene are implicated in the pathogenesis of diabetic retinopathy. Previous studies have shown that Brown Norway (BN) rats have higher retinal VEGF levels and more severe retinal vascular leakage than Sprague-Dawley (SD) rats in response to ischemia and diabetes. To investigate the molecular mechanism of vascular leakage in this animal model, F2 progeny were generated by crossbreeding BN and SD rats. Neonatal rats were exposed to hyperoxia to induce oxygen-induced retinopathy (OIR) models. The F2 rats in response to ischemia have shown a linear distribution of retinal VEGF levels, which is significantly and positively correlated to retinal vascular leakage. We identified a single nucleotide polymorphism (SNP) at upstream stimulating factor-binding site in the VEGF promoter region between BN and SD rats. No differences were found in retinal vascular permeability or VEGF levels between F2 rats with BN, SD, and BN/SD alleles of VEGF SNP. The increased retinal VEGF levels are correlated to ischemia-induced retinal vascular leakage in the OIR rat model. The VEGF mRNA and promoter are not responsible for increased retinal VEGF level and vascular permeability. The up-regulation of VEGF expression activated by a yet to be identified upstream factor or mediator affecting VEGF stability may be associated with a high susceptibility to retinal vascular leakage in BN rats.

Salutary effect of pigment epithelium-derived factor in diabetic nephropathy: evidence for antifibrogenic activities.

Diabetic nephropathy is a major complication of diabetes and a leading cause of end-stage renal diseases in the U.S. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor that has been extensively studied in diabetic retinopathy. Recently, we reported that PEDF is expressed at high levels in normal kidneys and that PEDF levels are decreased in kidneys of streptozotocin (STZ)-induced diabetic rats. In the present study, we injected STZ-diabetic rats with an adenovirus expressing PEDF (Ad-PEDF) to evaluate its effects in diabetes. The results showed that increased expression of PEDF in the kidney in response to Ad-PEDF delivery significantly alleviated microalbuminuria in early stages of diabetes. Administration of Ad-PEDF was found to prevent the overexpression of two major fibrogenic factors, transforming growth factor-beta (TGF-beta)1 and connective tissue growth factor (CTGF), and to significantly reduce the production of an extracellular matrix (ECM) protein in the diabetic kidney. Moreover, PEDF upregulated metalloproteinase-2 expression in diabetic kidney, which is responsible for ECM degradation. In cultured human mesangial cells, PEDF significantly inhibited the overexpression of TGF-beta1 and fibronectin induced by angiotensin II. PEDF also blocked the fibronectin production induced by TGF-beta1 through inhibition of Smad3 activation. These findings suggest that PEDF functions as an endogenous anti-TGF-beta and antifibrogenic factor in the kidney. A therapeutic potential of PEDF in diabetic nephropathy is supported by its downregulation in diabetes; its prevention of the overexpression of TGF-beta, CTGF, and ECM proteins in diabetic kidney; and its amelioration of proteinuria in diabetic rats following Ad-PEDF injection.

Therapeutic potential of angiostatin in diabetic nephropathy.

Angiostatin is a proteolytic fragment of plasminogen and a potent angiogenic inhibitor. Previous studies have shown that angiostatin inhibits retinal neovascularization and reduces retinal vascular permeability in diabetic retinopathy. Here, it is reported for the first time that angiostatin is also implicated in diabetic nephropathy (DN). Angiostatin levels are dramatically decreased in the kidney of streptozotocin-induced diabetic rats. Consistently, diabetic kidneys also showed decreased expression and proteolytic activities of matrix metalloproteinase-2, an enzyme that releases angiostatin from plasminogen. Adenovirus-mediated delivery of angiostatin significantly alleviated albuminuria and attenuated the glomerular hypertrophy in diabetic rats. Moreover, angiostatin treatment downregulated the expression of vascular endothelial growth factor and TGF-beta1, two major pathogenic factors of DN, in diabetic kidneys. In cultured human mesangial cells, angiostatin blocked the overexpression of vascular endothelial growth factor and TGF-beta1 that were induced by high glucose while increasing the levels of pigment epithelium-derived factor, an endogenous inhibitor of DN. Moreover, angiostatin effectively inhibited the high-glucose-and TGF-beta1-induced overproduction of proinflammatory factors and extracellular matrix proteins via blockade of the Smad signaling pathway. These findings suggest that the decrease of angiostatin levels in diabetic kidney may contribute to the pathologic changes such as inflammation and fibrosis in DN. Therefore, angiostatin has therapeutic potential in DN as a result of its anti-inflammatory and antifibrosis activities.

Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor.

Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Reduced PEDF levels are associated with diabetic retinopathy. However, the mechanism for the protective effects of PEDF against diabetic retinopathy (DR) is presently unclear. As inflammation plays a role in DR, the present study determined the effect of PEDF on inflammation. Western blot analysis and ELISA demonstrated that retinal and plasma PEDF levels were drastically decreased in rats with endotoxin-induced uveitis (EIU), which suggests that PEDF is a negative acute-phase protein. Intravitreal injection of PEDF significantly reduced vascular hyper-permeability in rat models of diabetes and oxygen-induced retinopathy, correlating with the decreased levels of retinal inflammatory factors, including VEGF, VEGF receptor-2, MCP-1, TNF-alpha, and ICAM-1. In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF-alpha and ICAM-1 expression under hypoxia. Moreover, down-regulation of PEDF expression by siRNA resulted in significantly increases of VEGF and TNF-alpha secretion in retinal Müller cells. These findings suggest that PEDF is a novel endogenous anti-inflammatory factor in the eye. The decrease of ocular PEDF levels may contribute to inflammation and vascular leakage in DR.

Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy.

Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Previous studies have shown that decreased ocular levels of PEDF are associated with diabetic retinopathy. However, the implication of PEDF expression in diabetic nephropathy has not been revealed. In the present study, we demonstrated for the first time that the expression of PEDF was decreased at both the mRNA and protein levels in the kidney of diabetic rats, whereas transforming growth factor-beta (TGF-beta) and fibronectin levels were increased in the same diabetic kidneys. As shown by immunohistochemistry, the decrease of PEDF expression occurs primarily in the glomeruli. In vitro studies showed that high concentrations of glucose significantly decreased PEDF secretion in primary human glomerular mesangial cells (HMCs), suggesting that hyperglycemia is a direct cause of the PEDF decrease in the kidney. Toward the function of PEDF, we showed that PEDF blocked the high-glucose-induced overexpression of TGF-beta, a major pathogenic factor in diabetic nephropathy, and fibronectin in primary HMCs, suggesting that PEDF may function as an endogenous inhibitor of TGF-beta expression and fibronectin production in glomeruli. Therefore, decreased expression of PEDF in diabetic kidneys may contribute to extracellular matrix overproduction and the development of diabetic nephropathy.

Caring for civilians during peace keeping missions: priorities and decisions.

Humanitarian assistance is increasingly being offered by the military in operations other than war. Balancing issues of resources, priorities, and security is important but complicated. Sometimes errors are made that are costly, either in terms of public relations, morale, or lives. Unfortunately, not enough education of physicians and other medical personnel is done prior to the mission, as to how to weigh these aspects. No absolute guidelines can be recommended, as each situation is different, but some examples and guidelines are presented.