Elia J Duh

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (49)251.28 Total impact

  • Diabetes 03/2015; 64(3):1067. · 7.90 Impact Factor
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    ABSTRACT: Retinal ischemia plays a critical role in multiple vision-threatening diseases and leads to death of retinal neurons, particularly ganglion cells. Oxidative stress plays an important role in this ganglion cell loss. Nrf2 (NF-E2-related factor 2) is a major regulator of the antioxidant response, and its role in the retina is increasingly appreciated. We investigated the potential retinal neuroprotective function of Nrf2 after ischemia-reperfusion (I-R) injury. In an experimental model of retinal I/R, Nrf2 knockout mice exhibited much greater loss of neuronal cells in the ganglion cell layer than wild-type mice. Primary retinal ganglion cells (RGCs) isolated from Nrf2 knockout mice exhibited decreased cell viability compared to wild-type RGCs, demonstrating the cell-intrinsic protective role of Nrf2. The retinal neuronal cell line 661W exhibited reduced cell viability following siRNA-mediated knockdown of Nrf2 under conditions of oxidative stress, and this was associated with exacerbation of increase in reactive oxygen species (ROS). The synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide), a potent Nrf2 activator, inhibited ROS increase in cultured 661W under oxidative stress conditions and increased neuronal cell survival after I/R injury in wild-type, but not Nrf2, knockout mice. Our findings indicate that Nrf2 exhibits a retinal neuroprotective function in I-R and suggest that pharmacologic activation of Nrf2 could be a therapeutic strategy. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 02/2015; · 4.24 Impact Factor
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    ABSTRACT: Purpose: Chronic inflammation is a critical process in pterygium development and progression, including promotion of angiogenesis. Vascular endothelial cells (ECs) actively participate in and regulate inflammation. Pterygium research has uncovered multiple inflammatory cytokines that are upregulated, but there has been minimal focus on EC activation. The Receptor for Advanced Glycation Endproducts (RAGE), a major proinflammatory molecule expressed in the vascular endothelium and other cell types, is a major instigator of endothelial cell activation. In this study, we explored the hypothesis that RAGE is upregulated in ECs in pterygium. To this end, we examined RAGE expression and immunolocalization in human pterygium and normal conjunctival tissue, with a particular interest in assessing endothelial RAGE. Methods: Pterygium specimens were obtained from 25 patients during surgery at the King Khaled Eye Specialist Hospital (KKESH). In the same patients, conjunctiva were obtained from the autograft during surgery. Tissue specimens were formalin-fixed and paraffin-embedded. Tissue sections were analyzed with immunohistochemistry with anti-RAGE antibody. Expression and localization of RAGE were evaluated in pterygium and corresponding conjunctiva. Results: RAGE expression was detected in the vascular endothelium in all pterygium tissue specimens and most conjunctival specimens. Other cell types exhibited expression, notably epithelial cells, fibroblasts, and possibly macrophages. Strikingly, endothelial RAGE expression was increased in 19 of 25 pterygium tissue specimens, compared to the corresponding control conjunctiva. Conclusions: Our data reveal that RAGE expression is upregulated in vascular endothelial cells in pterygium. RAGE upregulation is an important mechanism by which endothelial cells amplify the overall inflammatory response, and suppression of RAGE has been shown to prevent the progression of some systemic disease processes in experimental models. This suggests that pharmacologic targeting of RAGE, which is already being attempted in clinical trials for some diseases, could be useful in treating pterygium.
    Molecular vision 12/2014; http://www.molvis.org/molvis/v20/1740(20):1740-1748. · 2.25 Impact Factor
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    ABSTRACT: Endothelial cells play a major role in the initiation and perpetuation of the inflammatory process in health and disease, including their pivotal role in leukocyte recruitment. The role of pro-inflammatory transcription factors in this process has been well-described, including NF-κB. However, much less is known regarding transcription factors that play an anti-inflammatory role in endothelial cells. Myocyte enhancer factor 2 C (MEF2C) is a transcription factor known to regulate angiogenesis in endothelial cells. Here, we report that MEF2C plays a critical function as an inhibitor of endothelial cell inflammation. Tumor necrosis factor (TNF)-α inhibited MEF2C expression in endothelial cells. Knockdown of MEF2C in endothelial cells resulted in the upregulation of pro-inflammatory molecules and stimulated leukocyte adhesion to endothelial cells. MEF2C knockdown also resulted in NF-κB activation in endothelial cells. Conversely, MEF2C overexpression by adenovirus significantly repressed TNF-α induction of pro-inflammatory molecules, activation of NF-κB, and leukocyte adhesion to endothelial cells. This inhibition of leukocyte adhesion by MEF2C was partially mediated by induction of KLF2. In mice, lipopolysaccharide (LPS)-induced leukocyte adhesion to the retinal vasculature was significantly increased by endothelial cell-specific ablation of MEF2C. Taken together, these results demonstrate that MEF2C is a novel negative regulator of inflammation in endothelial cells and may represent a therapeutic target for vascular inflammation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Journal of Cellular Physiology 12/2014; · 3.87 Impact Factor
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    ABSTRACT: A new diabetic mouse strain, the Akita.PlGF knockout ((-/-)), was generated to study the role of placental growth factor (PlGF) in the pathogenesis of diabetic retinopathy (DR). PlGF deletion did not affect blood glucose but reduced the body weight of Akita.PlGF(-/-) mice. Diabetes-induced retinal cell death, capillary degeneration, pericyte loss, and blood-retinal barrier breakdown were prevented in these mice. Protein expression of PlGF was upregulated by diabetes, particularly in vascular cells. Diabetes-induced degradation of ZO-1 and VE-cadherin was reversed due to PlGF deficiency; their expression was correlated with that of sonic hedgehog and angiopoietin-1. PlGF deletion in Akita mice resulted in an increased Akt phosphorylation. Diabetes-activated HIF1α-VEGF pathway, including expression of HIF1α , VEGF, VEGFR1-3 and the extent of phospho (p)-VEGFR1, p-VEGFR2 , and p-eNOS, was inhibited in the retinas of diabetic PlGF(-/-) mice. However, expression of ICAM-1, VCAM-1, CD11b, and CD18 was not inhibited by PlGF deletion, nor was retinal leukostasis. These results suggest that PlGF is critical for the development of DR and its genetic deletion protects the retina from diabetic damage. The protective mechanisms are associated with the Akt activation and HIF1α-VEGF pathway inhibition, but independent of retinal leukostasis in the retinas of diabetic PlGF(-/-) mice.
    Diabetes 09/2014; · 7.90 Impact Factor
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    ABSTRACT: Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR. Retinal expression of NRF2 was investigated in human donor and mouse eyes by immunohistochemistry. The effect of NRF2 modulation on oxidative stress was studied in the human Müller cell line MIO-M1. Non-diabetic and streptozotocin-induced diabetic wild-type and Nrf2 knockout mice were evaluated for multiple DR endpoints. NRF2 was expressed prominently in Müller glial cells and astrocytes in both human and mouse retinas. In cultured MIO-M1 cells, NRF2 inhibition significantly decreased antioxidant gene expression and exacerbated tert-butyl hydroperoxide- and hydrogen peroxide-induced oxidative stress. NRF2 activation strongly increased NRF2 target gene expression and suppressed oxidant-induced reactive oxygen species. Diabetic mice exhibited retinal NRF2 activation, indicated by nuclear translocation. Superoxide levels were significantly increased by diabetes in Nrf2 knockout mice as compared with wild-type mice. Diabetic Nrf2 knockout mice exhibited a reduction in retinal glutathione and an increase in TNF-α protein compared with wild-type mice. Nrf2 knockout mice exhibited early onset of blood-retina barrier dysfunction and exacerbation of neuronal dysfunction in diabetes. These results indicate that NRF2 is an important protective factor regulating the progression of DR and suggest enhancement of the NRF2 pathway as a potential therapeutic strategy.
    Diabetologia 11/2013; · 6.88 Impact Factor
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    ABSTRACT: To determine the percentage of ranibizumab-treated patients with retinal vein occlusion (RVO) who had resolution of edema for at least 6 months after the last injection, along with factors and outcomes that correlate with resolution. Post hoc analysis of open-label clinical trial. Twenty patients with branch RVO (BRVO) and 20 with central RVO (CRVO) received ranibizumab monthly for 3 months and as needed for recurrent/persistent macular edema, no more frequently than every 2 months. Patients still requiring injections after month 40 received scatter and grid laser photocoagulation to try to reduce the need for injections. Main outcome measures included the percentage of patients who had resolution of edema, change in best-corrected visual acuity (BCVA) from baseline, and change in area of retinal nonperfusion in central subfields. Nine patients with BRVO (45%) had edema resolution from injections alone after a mean of 20.2 months, 4 resolved after addition of laser, 4 were unresolved through 72 months, and 3 exited prior to resolution. Five patients with CRVO (25%) resolved from injections alone after a mean of 14.0 months, 8 remained unresolved through 72 months despite addition of laser, and 7 exited prior to resolution. For BRVO or CRVO, there was a negative correlation between posterior retinal nonperfusion area and BCVA at months 18, 24, and 36 (P < .05). In patients with RVO, infrequent ranibizumab injections to control edema may not be sufficient to prevent progression of retinal nonperfusion, which may contribute to loss of visual gains.
    American Journal of Ophthalmology 10/2013; 156(4):693-705.e11. · 4.02 Impact Factor
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    ABSTRACT: Angiogenesis, in which new blood vessels form via endothelial cell (EC) sprouting from existing vessels, is a critical event in embryonic development and multiple disease processes. Many insights have been made into key EC receptors and ligands/growth factors that govern sprouting angiogenesis, but intracellular molecular mechanisms of this process are not well understood. NF-E2-related factor 2 (Nrf2) is a transcription factor well-known for regulating the stress response in multiple pathologic settings, but its role in development is less appreciated. Here, we show that Nrf2 is increased and activated during vascular development. Global deletion of Nrf2 resulted in reduction of vascular density as well as EC sprouting. This was also observed with specific deletion of Nrf2 in ECs, but not with deletion of Nrf2 in the surrounding nonvascular tissue. Nrf2 deletion resulted in increased delta-like ligand 4 (Dll4) expression and Notch activity in ECs. Blockade of Dll4 or Notch signaling restored the vascular phenotype in Nrf2 KOs. Constitutive activation of endothelial Nrf2 enhanced EC sprouting and vascularization by suppression of Dll4/Notch signaling in vivo and in vitro. Nrf2 activation in ECs suppressed Dll4 expression under normoxia and hypoxia and inhibited Dll4-induced Notch signaling. Activation of Nrf2 blocked VEGF induction of VEGFR2-PI3K/Akt and downregulated HIF-2α in ECs, which may serve as important mechanisms for Nrf2 inhibition of Dll4 and Notch signaling. Our data reveal a function for Nrf2 in promoting the angiogenic sprouting phenotype in vascular ECs.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
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    ABSTRACT: Vision loss from ischemic retinopathies commonly results from the accumulation of fluid in the inner retina [macular edema (ME)]. Although the precise events that lead to the development of ME remain under debate, growing evidence supports a role for an ischemia-induced hyperpermeability state regulated, in part, by VEGF. Monthly treatment with anti-VEGF therapies is effective for the treatment of ME but results in a major improvement in vision in a minority of patients, underscoring the need to identify additional therapeutic targets. Using the oxygen-induced retinopathy mouse model for ischemic retinopathy, we provide evidence showing that hypoxic Müller cells promote vascular permeability by stabilizing hypoxia-inducible factor-1α (HIF-1α) and secreting angiogenic cytokines. Blocking HIF-1α translation with digoxin inhibits the promotion of endothelial cell permeability in vitro and retinal edema in vivo. Interestingly, Müller cells require HIF-but not VEGF-to promote vascular permeability, suggesting that other HIF-dependent factors may contribute to the development of ME. Using gene expression analysis, we identify angiopoietin-like 4 (ANGPTL4) as a cytokine up-regulated by HIF-1 in hypoxic Müller cells in vitro and the ischemic inner retina in vivo. ANGPTL4 is critical and sufficient to promote vessel permeability by hypoxic Müller cells. Immunohistochemical analysis of retinal tissue from patients with diabetic eye disease shows that HIF-1α and ANGPTL4 localize to ischemic Müller cells. Our results suggest that ANGPTL4 may play an important role in promoting vessel permeability in ischemic retinopathies and could be an important target for the treatment of ME.
    Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor
  • Elia J Duh
    Investigative ophthalmology & visual science 06/2013; 54(6). · 3.43 Impact Factor
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    ABSTRACT: Intravitreal injection of biodegradable nanoparticles (NP) holds promise for gene therapy and drug delivery to the back of the eye. In some cases, including gene therapy, NP need to diffuse rapidly from the site of injection in order to reach targeted cell types in the back of the eye, whereas in other cases it may be preferred for the particles to remain at the injection site and slowly release drugs that may then diffuse to the site of action. We studied the movements of polystyrene (PS) nanoparticles of various sizes and surface chemistries in fresh bovine vitreous. PS NP as large as 510 nm rapidly penetrated the vitreous gel when coated with polyethylene glycol (PEG), whereas the movements of NP 1190 nm in diameter or larger were highly restricted regardless of surface chemistry owing to steric obstruction. PS NP coated with primary amine groups (-NH(2)) possessed positively charged surfaces at the pH of bovine vitreous (pH = 7.2), and were immobilized within the vitreous gel. In comparison, PS NP coated with -COOH (possessing negatively charged surfaces) in the size range of 100-200 nm and at particle concentrations below 0.0025% (w/v) readily diffused through the vitreous meshwork; at higher concentrations (~0.1% w/v), these nanoparticles aggregated within vitreous. Based on the mobility of different sized PS-PEG NP, we estimated the average mesh size of fresh bovine vitreous to be ~550 ± 50 nm. The bovine vitreous behaved as an impermeable elastic barrier to objects sized 1190 nm and larger, but as a highly permeable viscoelastic liquid to non-adhesive objects smaller than 510 nm in diameter. Guided by these studies, we next sought to examine the transport of drug- and DNA-loaded nanoparticles in bovine vitreous. Biodegradable NP with diameter of 227 nm, composed of a poly(lactic-co-glycolic acid) (PLGA)-based core coated with poly(vinyl alcohol) rapidly penetrated vitreous. Rod-shaped, highly-compacted CK(30)PEG(10k)/DNA with PEG coating (neutral surface charge; diameter ~60 nm) diffused rapidly within vitreous. These findings will help guide the development of nanoparticle-based therapeutics for the treatment of vision-threatening ocular diseases.
    Journal of Controlled Release 01/2013; · 7.63 Impact Factor
  • Shuang Wang, James K Park, Elia J Duh
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    ABSTRACT: Proliferative diabetic retinopathy (PDR), characterized by pathologic retinal angiogenesis, is a major cause of blindness in the USA and globally. Treatments targeting vascular endothelial growth factor (VEGF) have emerged as a beneficial part of the therapeutic armamentarium for this condition, highlighting the utility of identifying and targeting specific pathogenic molecules. There continues to be active research into the molecular players regulating retinal angiogenesis, including pro-angiogenic factors, anti-angiogenic factors, and integrins and matrix proteinases. New insights have been especially prominent regarding molecules which regulate specialized endothelial cells called tip cells, which play a lead role in endothelial sprouting. Together, these research efforts are uncovering new, important molecular regulators of retinal angiogenesis, which provide fertile areas for therapeutic exploration. This review discusses potential molecular targets, with an emphasis towards newer targets.
    Current Diabetes Reports 05/2012; 12(4):355-63. · 3.38 Impact Factor
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    ABSTRACT: Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)-specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.
    American Journal Of Pathology 04/2012; 180(6):2548-60. · 4.60 Impact Factor
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    ABSTRACT: Endothelial cell dysfunction is a critical component of ocular diseases such as age-related macular degeneration and diabetic retinopathy. An important limitation in endothelial cell research is the difficulty in achieving efficient transfection of these cells. A new polymer library was here synthesized and utilized to find polymeric nanoparticles that can transfect macrovascular (human umbilical vein, HUVECs) and microvascular (human retinal, HRECs) endothelial cells. Nanoparticles were synthesized that can achieve transfection efficiency of up to 85% for HRECs and 65% for HUVECs. These nanoparticle systems enable high levels of expression while avoiding problems associated with viral gene delivery. The polymeric nanoparticles also show cell-specific behavior, with a high correlation between microvascular and macrovascular transfection (R(2) = 0.81) but low correlation between retinal endothelial and retinal epithelial transfection (R(2) = 0.21). These polymeric nanoparticles can be used in vitro as experimental tools and potentially in vivo to target and treat vascular-specific diseases. FROM THE CLINICAL EDITOR: Polymeric nanoparticles were synthesized with the goal of transfecting endothelial cells, which are commonly considered difficult targets. The authors report excellent transfection efficiency of up to 85% for human retinal and 65% for human umbilical vein endothelial cells. These NPs can be used in vitro as experimental tools and potentially in vivo to target and treat vascular-specific diseases.
    Nanomedicine: nanotechnology, biology, and medicine 02/2012; 8(7):1200-7. · 6.93 Impact Factor
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    ABSTRACT: Retinal angiogenesis is a major cause of blindness in ischemic retinopathies including diabetic retinopathy and retinopathy of prematurity. Integrin αvβ3 is a promising therapeutic target for ocular angiogenesis, modulating the pro-angiogenic actions of multiple growth factors. In this study, we sought to determine the effects of the integrin αvβ3 antagonist tetra-iodothyroacetic acid (tetrac) on the angiogenic actions of VEGF and erythropoietin (EPO) in cultured human retinal endothelial cells. In addition, we investigated the effect of tetrac and a nanoparticulate formulation of tetrac on retinal angiogenesis in vivo, in the mouse oxygen-induced retinopathy (OIR) model. Tetrac inhibitory activity was evaluated in human retinal endothelial cells treated with VEGF and/or EPO. Endothelial cell proliferation, migration, and tube formation were assessed, in addition to phosphorylation of ERK1/2. For the studies of the oxygen-induced retinopathy model, C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12, and then returned to room air. Tetrac and tetrac-nanoparticle (tetrac-NP) were administered at P12 and P15 by either intraperitoneal or intravitreal injection. Retinal neovascularization was quantitated at P18. Tetrac significantly inhibited pro-angiogenic effects of VEGF and/or EPO on retinal endothelial cells, indicating that the angiogenic effects of both growth factors are dependent on integrin αvβ3. Retinal neovascularization in the OIR model was significantly inhibited by both tetrac and tetrac-NP. These results indicate that the integrin αvβ3 antagonist, tetrac, is an effective inhibitor of retinal angiogenesis. The ability of tetrac to inhibit the pro-angiogenic effect of both VEGF and EPO on retinal endothelial cells suggests that tetrac (and antagonism of integrin αvβ3) is a viable therapeutic strategy for proliferative diabetic retinopathy.
    Experimental Eye Research 11/2011; 94(1):41-8. · 3.02 Impact Factor
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    ABSTRACT: Retinal ischemia-reperfusion (I/R) involves an extensive increase in reactive oxygen species as well as proinflammatory changes that result in significant histopathologic damage, including neuronal and vascular degeneration. Nrf2 has a well-known cytoprotective role in many tissues, but its protective function in the retina is unclear. We investigated the possible role of Nrf2 as a protective mechanism in retinal ischemia-reperfusion injury using Nrf2(-/-) mice. I/R resulted in an increase in retinal levels of superoxide and proinflammatory mediators, as well as leukocyte infiltration of the retina and vitreous, in Nrf2(+/+) mice. These effects were greatly accentuated in Nrf2(-/-) mice. With regard to histopathologic damage, Nrf2(-/-) mice exhibited loss of cells in the ganglion cell layer and markedly accentuated retinal capillary degeneration, as compared to wild-type. Treatment with the Nrf2 activator CDDO-Me increased antioxidant gene expression and normalized I/R-induced superoxide in the retina in wild-type but not Nrf2(-/-) mice. CDDO-Me treatment abrogated retinal capillary degeneration induced by I/R in wild-type but not Nrf2(-/-) mice. These studies indicate that Nrf2 is an important cytoprotective mechanism in the retina in response to ischemia-reperfusion injury and suggest that pharmacologic induction of Nrf2 could be a new therapeutic strategy for retinal ischemia-reperfusion and other retinal diseases.
    Free Radical Biology and Medicine 07/2011; 51(1):216-24. · 5.27 Impact Factor
  • Elia J Duh
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    ABSTRACT: In this issue of Blood, Joyal and colleagues make the insightful finding that Semaphorin3A (Sema3A) is secreted by hypoxic neurons in ischemic/avascular retina,thereby inhibiting vascular regeneration of the retina and enhancing pathologic preretinal neovascularization.
    Blood 06/2011; 117(22):5785-6. · 9.78 Impact Factor
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    ABSTRACT: Pigment epithelium-derived factor (PEDF) is an adipocyte-secreted factor involved in the development of insulin resistance in obesity. Previous studies have identified PEDF as a regulator of triacylglycerol metabolism in the liver that may act through adipose triglyceride lipase (ATGL). We used ATGL(-/-) mice to determine the role of PEDF in regulating lipid and glucose metabolism. Recombinant PEDF was administered to ATGL(-/-) and wild-type mice, and whole-body energy metabolism was studied by indirect calorimetry. Adipose tissue lipolysis and skeletal muscle fatty acid metabolism was determined in isolated tissue preparations. Muscle lipids were assessed by electrospray ionization-tandem mass spectrometry. Whole-body insulin sensitivity and skeletal muscle glucose uptake were assessed. PEDF impaired the capacity to adjust substrate selection, resulting in a delayed diurnal decline in the respiratory exchange ratio, and suppressed daily fatty acid oxidation. PEDF enhanced adipocyte lipolysis and triacylglycerol lipase activity in skeletal muscle. Muscle fatty acid uptake and storage were unaffected, whereas fatty acid oxidation was impaired. These changes in lipid metabolism were abrogated in ATGL(-/-) mice and were not attributable to hypothalamic actions. ATGL(-/-) mice were also refractory to PEDF-mediated insulin resistance, but this was not related to changes in lipid species in skeletal muscle. The results are the first direct demonstration that 1) PEDF influences systemic fatty acid metabolism by promoting lipolysis in an ATGL-dependent manner and reducing fatty acid oxidation and 2) ATGL is required for the negative effects of PEDF on insulin action.
    Diabetes 04/2011; 60(5):1458-66. · 7.90 Impact Factor
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    ABSTRACT: Blood-retinal barrier [BRB] breakdown, characteristic of diabetic retinopathy (DR), is believed to depend on inflammation and apoptosis. Retinal inflammation is almost completely suppressed in the absence of TNFα, which is also associated with apoptosis. This study was conducted to determine the role of TNFα in these diabetic complications. Diabetes was induced with streptozotocin in Tnfa knockout (KO) mice, to provide a chemical model of diabetes, and Tnfa (KO) mice were crossed with Ins2(Akita) mice to generate a genetic model, with both models being devoid of TNFα. The BRB was assessed at 1, 1.5, 3, and 6 months. Leukostasis was assessed using FITC-conjugated ConA to label leukocytes. Apoptosis was assessed with TUNEL and activated caspase-3 staining. PECAM1 identified endothelial cells, and SMA identified pericytes. At 1 month of diabetes, the absence of TNFα had no effect on DR-associated BRB breakdown, even though it prevented retinal leukostasis, demonstrating that neither TNFα nor inflammation is essential for early BRB breakdown in DR in either model of diabetes. At 3 months of diabetes, BRB breakdown was significantly suppressed and at 6 months, it was completely prevented in the absence of TNFα in both models, showing that TNFα is essential for progressive BRB breakdown. DR-mediated apoptosis in the retina, which appears to involve endothelial cells, pericytes, and neurons, was inhibited in the absence of TNFα in both models. Although neither TNFα nor inflammation is necessary for early BRB breakdown in DR, TNFα is critical for later complications and would be a good therapeutic target for the prevention of the progressive BRB breakdown, retinal leukostasis, and apoptosis associated with DR.
    Investigative ophthalmology & visual science 03/2011; 52(3):1336-44. · 3.43 Impact Factor
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    ABSTRACT: Pigment epithelium-derived factor (PEDF) is a potent inhibitor of vascular endothelial growth factor (VEGF)-induced endothelial permeability. The goal of this study was to understand the mechanism by which PEDF blocks VEGF-induced increases in vascular permeability. The paracellular permeability of bovine retinal endothelial (BRE) cells was measured by assaying transendothelial cell electrical resistance and tracer flux. Western blot analysis was used to show phosphorylation of VEGFR2, MAP kinases, and glycogen synthase kinase 3 (GSK3)-beta. Confocal imaging and Western blot analysis were used to determine subcellular distribution of beta-catenin. Real-time RT-PCR and Western blot analysis were used to quantify urokinase plasminogen activator receptor (uPAR) expression. PEDF blocked VEGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 MAP kinase, the p38 substrate MAP kinase-activated protein kinase-2 (MAPKAPK-2), and GSK3-beta, but it had no effect on the phosphorylation of VEGFR2. In addition, the VEGF-induced transcriptional activation of beta-catenin and uPAR expression were blocked by PEDF and by inhibitors of p38 and MEK. Finally, the VEGF-induced increase in permeability was blocked by both PEDF and the same kinase inhibitors. The data suggest that p38 MAP kinase and ERK act upstream of GSK/beta-catenin in VEGF-induced activation of the uPA/uPAR system and that PEDF-mediated inhibition of the VEGF-induced increase in vascular permeability involves blockade of this pathway. These findings are important for developing precise and potent therapies for treatment of diseases characterized by vascular barrier dysfunction.
    Investigative ophthalmology & visual science 06/2010; 51(6):3273-80. · 3.43 Impact Factor

Publication Stats

2k Citations
251.28 Total Impact Points


  • 2001–2015
    • Johns Hopkins University
      • • Wilmer Eye Institute
      • • Department of Chemical and Biomolecular Engineering
      Baltimore, Maryland, United States
  • 1999–2013
    • Johns Hopkins Medicine
      • Wilmer Eye Institute
      Baltimore, MD, United States
  • 2012
    • China-Japan Friendship Hospital
      Peping, Beijing, China
  • 2005
    • Regeneron
      Terryville, New York, United States