Sarah X. Zhang

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‐α, and ICAM‐1. In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF‐α and ICAM‐1 expression under hypoxia. Moreover, down‐regulation of PEDF expression by siRNA resulted in significantly increases of VEGF and TNF‐α 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.

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.

Ocular neovascularization: Implication of endogenous angiogenic inhibitors and potential therapy.

Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and as a consequence, there is no satisfactory therapy for ocular NV. In the last 10 years, a number of studies provided increasing evidence demonstrating that the imbalance between angiogenic stimulating factors and angiogenic inhibitors is a major contributor to the angiogenesis induced by various insults, such as hypoxia or ischemia, inflammation and tumor. The angiogenic inhibitors alone or in combination with other existing therapies are, therefore, believed to be promising in the treatment of ocular NV in the near future. This article reviews recent progress in studies on the mechanisms and treatment of ocular NV, focusing on the implication and therapeutic potential of endogenous angiogenic inhibitors in ocular NV.

Inhibition of Reactive Oxygen Species by Lovastatin Downregulates Vascular Endothelial Growth Factor Expression and Ameliorates Blood-Retinal Barrier Breakdown in db/db Mice: Role of NADPH Oxidase 4

OBJECTIVE Oxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy. RESEARCH DESIGN AND METHODS Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin. RESULTS Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice. CONCLUSIONS Activation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.

ER Stress and Apoptosis: A New Mechanism for Retinal Cell Death

The endoplasmic reticulum (ER) is the primary subcellular organelle where proteins are synthesized and folded. When the homeostasis of the ER is disturbed, unfolded or misfolded proteins accumulate in the ER lumen, resulting in ER stress. In response to ER stress, cells activate a set of tightly controlled regulatory programs, known as the unfolded protein response (UPR), to restore the normal function of the ER. However, if ER stress is sustained and the adaptive UPR fails to eliminate unfolded/misfolded proteins, apoptosis will occur to remove the stressed cells. In recent years, a large body of studies has shown that ER stress-induced apoptosis is implicated in numerous human diseases, such as diabetes and neurogenerative diseases. Moreover, emerging evidence supports a role of ER stress in retinal apoptosis and cell death in blinding disorders such as age-related macular degeneration and diabetic retinopathy. In the present review, we summarize recent progress on ER stress and apoptosis in retinal diseases, focusing on various proapoptotic and antiapoptotic pathways that are activated by the UPR, and discuss how these pathways contribute to ER stress-induced apoptosis in retinal cells.

Inhibition of reactive oxygen species by lovastatin down-regulates VEGF expression and ameliorates blood-retinal barrier breakdown in db/db mice: role of NADPH oxidase 4

OBJECTIVE Oxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy. RESEARCH DESIGN AND METHODS Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin. RESULTS Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice. CONCLUSIONS Activation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.

Activation of Endoplasmic Reticulum Stress by Hyperglycemia Is Essential for Müller Cell–Derived Inflammatory Cytokine Production in Diabetes

Inflammation plays an important role in diabetes-induced retinal vascular leakage. The purpose of this study is to examine the role of endoplasmic reticulum (ER) stress and the signaling pathway of ER stress–induced activating transcription factor 4 (ATF4) in the regulation of Müller cell–derived inflammatory mediators in diabetic retinopathy. In diabetic animals, elevated ER stress markers, ATF4, and vascular endothelial growth factor (VEGF) expression were partially localized to Müller cells in the retina. In cultured Müller cells, high glucose induced a time-dependent increase of ER stress, ATF4 expression, and inflammatory factor production. Inducing ER stress or overexpressing ATF4 resulted in elevated intracellular adhesion molecule 1 and VEGF proteins in Müller cells. In contrast, alleviation of ER stress or blockade of ATF4 activity attenuated inflammatory gene expression induced by high glucose or hypoxia. Furthermore, we found that ATF4 regulated the c-Jun NH2-terminal kinase pathway resulting in VEGF upregulation. ATF4 was also required for ER stress–induced and hypoxia-inducible factor-1α activation. Finally, we showed that administration of chemical chaperone 4-phenylbutyrate or genetic inhibition of ATF4 successfully attenuated retinal VEGF expression and reduced vascular leakage in mice with STZ-induced diabetes. Taken together, our data indicate that ER stress and ATF4 play a critical role in retinal inflammatory signaling and Müller cell–derived inflammatory cytokine production in diabetes.

Preconditioning with Endoplasmic Reticulum Stress Mitigates Retinal Endothelial Inflammation via Activation of X-box Binding Protein 1

Endoplasmic reticulum (ER) stress is widely implicated in various pathological conditions such as diabetes. Previously, we reported that enhanced ER stress contributes to inflammation and vascular damage in diabetic and ischemia-induced retinopathy. However, the exact role of the signaling pathways activated by ER stress in vascular inflammation remains poorly understood. In the present study, we investigated the role of X-box binding protein 1 (XBP1) in retinal adhesion molecule expression, leukostasis, and vascular leakage. Exposure of human retinal endothelial cells to low dose ER stress inducers resulted in a robust activation of XBP1 but did not affect inflammatory gene expression. However, ER stress preconditioning almost completely abolished TNF-α-elicited NF-κB activation and adhesion molecule ICAM-1 and VCAM-1 expression. Pharmaceutical inhibition of XBP1 activation or knockdown of XBP1 by siRNA markedly attenuated the effects of preconditioning on inflammation. Moreover, loss of XBP1 led to an increase in ICAM-1 and VCAM-1 expression. Conversely, overexpression of spliced XBP1 attenuated TNF-α-induced phosphorylation of IKK, IκBα, and NF-κB p65, accompanied by decreased NF-κB activity and reduced adhesion molecule expression. Finally, in vivo studies show that activation of XBP1 by ER stress preconditioning prevents TNF-α-induced ICAM-1 and VCAM-1 expression, leukostasis, and vascular leakage in mouse retinas. These results collectively indicate a protective effect of ER stress preconditioning against retinal endothelial inflammation, which is likely through activation of XBP1-mediated unfolded protein response (UPR) and inhibition of NF-κB activation.

Genetic Difference in Susceptibility to the Blood- Retina Barrier Breakdown in Diabetes and Oxygen- Induced Retinopathy

The breakdown of the blood-retina barrier (BRB) is a common feature of diabetic retinopathy. The purpose of the present study is to determine whether there are genetic differences in susceptibility to the breakdown of the BRB in diabetic retinopathy using two rat models. In streptozotocin (STZ)-induced diabetes, Brown Norway (BN) rats developed sustained vascular hyperpermeability in the retina during the entire experimental period (16 weeks of diabetes), while diabetic Sprague Dawley (SD) rats only showed retinal hyperpermeability from 3 to 10 days after the onset of diabetes. The strain difference in permeability was not correlated with the blood glucose levels in these two strains. In oxygen-induced retinopathy (OIR), BN rats developed retinal vascular hyperpermeability from postnatal day 12 (P12) to P22 with a peak at P16, which was 8.7-fold higher than that in the age-matched normal controls. In OIR-SD rats, however, hyperpermeability was observed from P14 to P18, with a peak only 2.2-fold higher than that in the controls. The strain difference in vascular hyperpermeability was correlated with the different overexpression of vascular endothelial growth factor (VEGF) in the retina of these two models. This finding suggests that genetic backgrounds contribute to the susceptibility to diabetic retinopathy.

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-β (TGF-β)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-β1 and fibronectin induced by angiotensin II. PEDF also blocked the fibronectin production induced by TGF-β1 through inhibition of Smad3 activation. These findings suggest that PEDF functions as an endogenous anti–TGF-β 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-β, CTGF, and ECM proteins in diabetic kidney; and its amelioration of proteinuria in diabetic rats following Ad-PEDF injection.