Suppression of Retinal Peroxisome ProliferatorActivated Receptor in Experimental Diabetes and Oxygen-Induced Retinopathy: Role of NADPH Oxidase

Amany Tawk,1 Tammy Sanders,1 Khalid Kahook,1 Sara Akeel,1 Ahmed Elmarakby,2,3 and Mohamed Al-Shabrawey1,4
PURPOSE. Recently, the authors have shown that NADPH oxidase is positively correlated with increased leukocyte adhesion and vascular leakage in diabetes and neovascularization in oxygen-induced retinopathy (OIR). Peroxisome proliferatoractivated receptor gamma (PPAR) agonists have been shown to prevent vascular inammation and leakage in an experimental model of diabetes. The goal of this study was to investigate whether there is a link between NADPH oxidase and PPAR that leads to vascular dysfunction in diabetic retina or OIR. METHODS. Diabetes was induced with streptozotocin in wildtype mice or NOX2 knockout mice. One group of wild-type mice was treated with apocynin. Bovine retinal endothelial cells (BRECs) were treated with normal glucose (5 mM) or high glucose (25 mM) in the presence or absence of superoxide dismutase (SOD) or NADPH oxidase inhibitors (apocynin or diphenyleneiodonium [DPI]). Western blotting and immunouorescence were used to evaluate PPAR expression. Activation of nuclear factor (NF)B was measured using the transcription factor assay kit and Western blot analysis of phosphoNFB. PPAR expression was also tested in OIR and lipopolysaccharide-induced retinal inammation. RESULTS. Retinal expression of PPAR was suppressed in experimental models of diabetes, OIR, and retinal inammation. This was associated with the activation of NFB in the diabetic retina. These effects were prevented by apocynin or deletion of NOX2. PPAR expression was also suppressed in endothelial cells treated with high glucose, and this was prevented by apocynin, DPI, and SOD. CONCLUSIONS. Suppression of PPAR is involved in the pathogenesis of diabetic retinopathy and OIR. NADPH oxidase could be an upstream mediator of these changes. (Invest Ophthalmol Vis Sci. 2009;50:878 884) DOI:10.1167/iovs.08-2005 perglycemia is a major risk factor that has been linked to the development of vascular dysfunction in diabetic retinopathy. Increased production of reactive oxygen species (ROS)2,3 and inammatory markers 4 6 have all been shown to be associated with high glucose treatment of endothelial cells. The retinas large oxygen uptake and glucose oxidation make it more susceptible than any other tissue to oxidative stress.6,7 Studies have shown that oxidative stress is implicated in the development of diabetic neuropathy,8 nephropathy,9 and retinopathy.10,11 The major sources of ROS are NADPH oxidase(s), cytochrome P450, and nitric oxide synthase. In particular, studies have linked NADPH oxidase to vascular complications of diabetes such as atherosclerosis,12 hypertension,13,14 nephropathy,15 and retinopathy.16,17 NADPH oxidase consists of two membranous subunits, NOX2 and p22phox; three cytosolic subunits, p40phox, p47phox, and p67phox; and the small GTP-binding protein rac-1.1,18 Recent research has shown that the inhibition of NADPH oxidase prevents retinal neovascularization in oxygen-induced retinopathy (OIR)19 and vascular leakage in the diabetic retina.20,21 The mechanism by which NADPH oxidase mediates vascular damage in diabetic or OIR is still under investigation, but evidence indicates that this may occur through VEGF expression19,22,23 or increased vascular inammation.16,20,21,24 In particular, leukocyte-endothelial interaction (leukostasis) is thought to be an early and key event in the pathogenesis of diabetic retinopathy25,26 and OIR.27,28 Peroxisome proliferator-activated receptor gamma (PPAR) is a member of a ligand-activated nuclear receptor superfamily and plays a critical role in a variety of biological processes, including adipogenesis, glucose metabolism, and angiogenesis.29 PPAR may also represent a target for cardiovascular risk reduction. Synthetic PPAR agonists such as pioglitazone and rosiglitazone increase insulin sensitivity, modify lipid proles, decrease blood pressure, and reduce biomarkers of inammation.30 32 Previous work has shown that the PPAR signaling pathway inhibits diabetes-induced vascular injury in retina33 and kidney34 through a mechanism involving the inhibition of leukocyte adhesion. The anti-inammatory effect of PPAR has been shown to be mediated through the inhibition of the transcription factor nuclear factor (NF), which plays a crucial role in inammation.3537 The goals of the present study were to characterize the changes in retinal expression of PPAR in diabetic and OIRs and to determine whether NADPH oxidase plays a specic role in these changes.

iabetic retinopathy is the most common cause of blindness in working adults. It is characterized by early retinal microvascular dysfunctions such as abnormal vascular ow, hyperpermeability, and the nonperfusion of capillaries.1 Hy-

From the 1Department of Oral Biology and Anatomy, School of Dentistry, the 2Vascular Biology Center, and the Departments of 3Pharmacology and Toxicology and 4Ophthalmology, Medical College of Georgia, Augusta, Georgia. Supported by the Scientist Development Grant AHA00104 from the American Heart Association. Submitted for publication March 11, 2008; revised August 18 and September 9, 2008; accepted December 9, 2008. Disclosure: A. Tawk, None; T. Sanders, None; K. Kahook, None; S. Akeel, None; A. Elmarakby, None; M. Al-Shabrawey, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked advertisement in accordance with 18 U.S.C. 1734 solely to indicate this fact. Corresponding author: Mohamed Al-Shabrawey, Oral Biology and Anatomy, School of Dentistry, Medical College of Georgia, Augusta, GA 30912; malshabrawey@mcg.edu.

METHODS
Animals
All experimental procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experiments were performed on female C57Bl/6J mice 6 to 8 weeks old and age-matched NOX2-decient mice backcrossed on a C57Bl/6
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background (Jackson Laboratories, Bar Harbor, ME), each weighing 21 to 25 g. Six to eight mice were used for each experimental group (control wild-type [WT], control NOX2 knockout, diabetic WT, diabetic WT treated with apocynin, and diabetic NOX2 knockout). NOX2 knockout mice were genotyped before the experiment.

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Western Blot Analysis

For analysis of PPAR and phospho (p)-NFB, one retina from each mouse in different groups and treated endothelial cells were homogenized in a modied RIPA buffer (20 mM Tris-HCl [pH 7.4], 2.5 mM ethylenediaminetetraacetic acid, 50 mM NaF, 10 mM Na4P2O7, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1 mM phenylmethylsulfonyl uoride). Homogenates (50 g protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using ready precast gel (Bio-Rad, Hercules, CA), transferred to nitrocellulose membrane, and reacted with anti-PPAR (1:200) and antip-NFB (p65) 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence (Amersham Pharmacia, San Francisco, CA). Membranes were stripped and reprobed for -actin or NFB to demonstrate equal loading, and results were analyzed with the use of densitometry.

Cell Culture
Primary cultures of bovine retinal endothelial cells (BRECs) passages 7 to 9 were used in these experiments as described.38 Parallel experiments were also conducted on primary cultured human umbilical vein endothelial cells (HUVECs) obtained from Clonetics (Walkersville, MD) and maintained in complete endothelial cell growth medium (EGM)-2 supplemented with single-use aliquots (EGM-2 SingleQuots; (Clonetics), which contained human broblast growth factor-basic (hFGF-B), VEGF, human recombinant epidermal growth factor (hEGF), human recombinant insulin-like growth factor (R3IGF)-1, ascorbic acid, hydrocortisone, heparin, 2% fetal bovine serum, gentamicin, and amphotericin.

PPAR Immunolocalization
Retinal frozen sections (12-m thick) from diabetic and age-matched control mice were prepared for PPAR immunolocalization. Retinal sections were xed with 4% paraformaldehyde for 5 minutes, followed by washing with PBS and blocking with 3% normal goat serum for 30 minutes. Sections were incubated with the endothelial cell-specic marker biotinylated Griffonia simplicifolia isolectin B4 (GSI; Vector Laboratories, Burlingame, CA) and anti-PPAR polyclonal antibody (Santa Cruz Biotechnology) 1:50 overnight at 4C, followed by avidinconjugated Texas red (Vector Laboratories) and Oregon green-labeled antirabbit antibody (Molecular Probes, Eugene, OR) to identify the expression and localization of PPAR in retinal sections using confocal microscopy (LSM 510; Carl Zeiss, Thornwood, NY). Specicity of the reaction was conrmed by omitting the primary antibody and isolectin B4. For densitometry analysis, we collected two representative images from each retinal section (ve sections per mouse) from six different mice in each group. Collected images were analyzed by computerassisted morphometry for uorescence intensity.

Normal and High-Glucose Treatment of Cell Culture

BRECs or HUVECs were grown until they were 80% to 90% conuent and were switched to serum-free medium overnight. The next day the cells were treated with 5 mM D-glucose (NG) or 25 mM D-glucose (HG) in the presence or absence of NADPH oxidase inhibitor, apocynin (100 M; Sigma, St. Louis, MO), and 5 M diphenyleneiodonium (DPI; Sigma), which is also a general inhibitor of avoprotein-containing enzyme, a group that includes NADPH oxidase and several other enzymes such as nitric oxide synthase (NOS)39,40 or 100 U cell-permeable superoxide dismutase polyethylene glycol (Sigma). Additional groups of cells were incubated in 5 mM D-glucose supplemented with 20 mM mannitol as an osmolarity control. Three days later, cells were harvested and processed for analysis of PPAR expression. This experiment was replicated with at least two different batches of endothelial cells.

Mouse Model of Diabetic Retinopathy

Wild-type (WT) and NOX2 knockout mice were made diabetic by multiple intraperitoneal injections of streptozotocin (STZ; 55 mg/kg; Sigma) dissolved in 0.1 M fresh citrate buffer (pH 4.5). The mean blood glucose level was 437 53 in WT and 441 61 in knockout mice. One group of diabetic WT mice received apocynin (10 mg/kg) in drinking water. After 5 weeks, diabetic and age-matched normal and knockout mice were processed for Western blot analysis and immunolocalization. One retina from each animal was immediately frozen in liquid nitrogen and stored at 80C until further Western blot analysis, and the other eyeball was embedded in OCT for sectioning.

Measurement of Retinal NFB Activity

Frozen retina was homogenized in complete lysis buffer for the preparation of whole cell extract using a nuclear extract kit (Active Motif, Carlsbad, CA). Homogenate was centrifuged at 6160g for 10 minutes, and the supernatant was portioned into aliquots and stored at 80C. Protein concentration was determined, and the 20 g whole-cell extract was used for the determination of NFB activity using the NFB p65 transcription factor assay kit (TransAM; Active Motif) as described.41 Each of the standards and samples was run in duplicate, and the amount of activated NFB was normalized per microgram of retinal protein.

Mouse Model of Acute Retinal Vascular Inammation

Additional groups of WT and knockout mice were studied after injection with lipopolysaccharide (LPS) from Salmonella typhimurium (0.1 mg/kg; Sigma) as a model of acute retinal vascular inammation. Retinas were collected and processed for analysis of PPAR expression by Western blot 24 hours later.

Statistical Analysis
Group differences were evaluated using ANOVA followed by Tukey post hoc test. Results were considered signicant when P 0.05. For in vivo studies, age-matched control was compared with diabetic WT mice treated with or without apocynin and with diabetic or nondiabetic mice lacking NOX2 (n 6 8). For in vitro studies, four dishes were prepared for each treatment group, and each experiment was replicated with at least two different batches of endothelial cells. Data are represented as mean SE from at least six animals in each group and three experiments from the in vitro study.

Mouse Model of Oxygen-Induced Retinopathy

Experimental retinal neovascularization has been developed by incubating a group of mice at postnatal day (P) 7 in high oxygen (75%) for 5 days, followed by 5 days in room air (normoxia). One group of mice was treated with apocynin (intraperitoneal, 10 mg/kg/d) from P12 to P16. Mice were then killed on P17, and PPAR expression was tested in retina using immunouorescence and Western blotting. Additional groups of oxygen-treated mice were tested at P14 and compared with age-matched controls.

RESULTS
Effect of Apocynin or Deletion of NOX2 on Retinal Expression of PPAR in Diabetic Mice
Recently, we have shown that the inhibition of NADPH oxidase by apocynin or the deletion of NOX2 leads to a signicant

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IOVS, February 2009, Vol. 50, No. 2 reduction in ROS formation in the diabetic retina.21 To determine whether there is a relationship between NADPH oxidase activation and PPAR expression in diabetic retina, we tested the effect of apocynin treatment or deletion of NOX2 on the retinal level of PPAR in diabetic mice using Western blot analysis. Results of this study demonstrated a noticeable decrease in PPAR levels in retinas of diabetic mice compared with control WT or NOX-knockout (0.49 0.051 vs. 1.49 0.048 and 1.33 0.03, respectively). However, PPAR expression was restored by the deletion of NOX2 (1.64 0.3) or apocynin treatment (1.55 0.19; Fig. 1A). These observations were conrmed by the immunouorescence technique using a specic endothelial cell marker (GSI) and anti-PPAR. PPAR immunoreactivity was stronger in the retinas of control WT and knockout mice than in those of diabetic WT mice, particularly in retinal vessels and surrounding retinal cells shaped similarly to Mu ller glial cells. However, the deletion of NOX2 or apocynin treatment restored the normal level of PPAR expression and its distribution in the diabetic retina (Figs. 1B, 1C). No immunouorescence reaction was observed in retinal sections treated only by the secondary antibody, indicating the specicity of the reaction.

Effect of HG on PPAR Expression in Cultured Endothelial Cells

Because our immunouorescence demonstrated that PPAR is localized in the endothelial cells, we tested the effect of HG on PPAR in cultured endothelial cells. Our experiment showed a signicant decrease in the level of PPAR in BRECS by HG compared with the NG or mannitol (1420.0 177 vs. 2466.0 67 and 2742 153, respectively). This effect was prevented by SOD (2806 267) and NADPH oxidase inhibitors (apocynin, 2314 57; DPI, 2446 54; Fig. 2). Similar results were noticed in HUVECs incubated under the same conditions (data not shown).

Effect of Apocynin or Deletion of NOX2 on Diabetes-Induced NFB Activation

Previous studies have demonstrated the link between NADPH oxidase and vascular inammation in diabetic retinopathy.20,21 In the present study, we tested whether this link occurs through the NFB-dependent signaling pathway. Our experiments demonstrated a signicant activation of retinal NFB, as shown by the increases in the level of p-NFB in diabetic retina

FIGURE 1. PPAR expression in diabetic retina. (A) Western blot analysis showed the suppression of PPAR expression in diabetic wild-type mice (D) compared with the control WT (C) and NOX2 knockout (NOX/) mice. Deletion of NOX2 (D-NOX/) or apocynin treatment (Dapo) restored retinal levels of PPAR in the diabetic mice. *P 0.05 vs. C and NOX/; #P 0.05 vs. D (n 6). (B) Immunouorescence of retinal sections using endothelial cell marker (red) and antiPPAR (green) shows more PPAR immunoreactivity in retinas of the normal WT (C) and NOX/ mice than in the WT diabetic (D) mice. Diabetic mice lacking NOX2 (D-NOX/) or treated with apocynin (Dapo) showed marked restoration of PPAR. Note that PPAR is expressed in different layers of retina, particularly in retinal vessels (arrows) and other cells that look morphologically similar to the Mu ller glial cells (arrowhead). (C) Densitometry analysis of the reaction intensity shows that the decrease in PPAR immunoreactivity by diabetes (D) was signicant compared with the control WT (C) or NOX knockout mice (NOX/). This decrease was signicantly restored by the deletion of NOX2 (D-NOX/) or by apocynin treatment (Dapo). *P 0.05 vs. C and NOX/; #P 0.05 vs. D (n 5).

FIGURE 2. PPAR expression in cultured BRECs. Western blot analysis showed the suppression of PPAR expression in high glucose-treated BRECs (HG) compared with the normal glucose (NG) and mannitol (M)-treated cells. Superoxide dismutase (HGSOD), apocynin (HGapo), and DPI (HGDPI) prevented the effect of HG on PPAR expression in cultured BRECs. *P 0.05 vs. NG and M; #P 0.05 vs. HG (n 3).

IOVS, February 2009, Vol. 50, No. 2 compared with the control (p-NFB, 4.5 1.3 vs. 0.19). This effect was prevented by apocynin (p-NFB, 1.6 0.4) or by the deletion of NOX2 (p-NFB, 0.98 0.3; Fig. 3A). This result was conrmed by the NFB p65 transcription factor assay kit (TransAM). The assay showed a signicant increase in the activity of NFB in diabetic retina compared with control WT and NOX knockout mice. This increase was blocked in mice treated with apocynin or lacking NOX2 (Fig. 3B).

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PPAR Expression in a Mouse Model of OIR

We also tested the changes in PPAR expression in relation to retinal neovascularization in a mouse model of OIR. Western blot analysis demonstrated a signicant suppression of PPAR expression in OIR at P17 (1.7 0.26 vs. 2.1 0.26) that was restored by apocynin treatment (2.7 0.44; Fig. 4A). The decrease in PPAR expression started early during the course of OIR at P14 and was more obvious than at P17 if compared with the age-matched control (2.2 0.3 vs. 15.3 7; Fig. 4B). In addition, immunolocalization using double labeling of retinal sections with endothelial cell marker (GSI) and anti-PPAR also showed the restoration of PPAR in the retina of OIR by apocynin (Fig. 4C).

FIGURE 3. Assay of NFB activity in retina. (A) Western blot analysis of p-NFB shows a signicant increase in the retinal level of p-NFB by diabetes (D) compared with control (C). Deletion of NOX2 (DNOX/) or apocynin treatment (Dapo) prevented the effects of diabetes on the retinal p-NFB. (B) Measurement of NFB activity by TransAM NFB p65 transcription factor assay kit demonstrated a signicant increase in the amount of active NFB in the retinas of diabetic wild-type mice compared with control WT (C) and NOX2 knockout (NOX/) mice. Deletion of NOX2 (D-NOX/) or apocynin treatment (Dapo) prevented the effect of diabetes on NFB activity. *P 0.05 vs. C and NOX/; #P 0.05 vs. D (n 6).

FIGURE 4. Analysis of PPAR expression in OIR. Western blot analysis of PPAR in retinas of P17 (A) and P14 (B) mice. PPAR expression increased in OIR compared with age-matched control (C). Note the early onset of PPAR expression decrease in OIR by P14. Administration of apocynin (OIRapo) restored the normal expression of PPAR in OIR. *P 0.05 vs. C; #P 0.05 vs. OIR (n 6). (C) Immunouorescence of retinal sections shows that PPAR (green) is expressed in retinal vessels (arrow) and surrounding retinal cells (arrowhead) of normal mice (C). There is a marked decrease in the PPAR immunoreactivity in OIR compared with the control (C). Restoration of PPAR in retinal vessels and related cells was noticed in the apocynin treated mice (OIRapo).

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IOVS, February 2009, Vol. 50, No. 2 tes for the past several years. Treatment with TZDs has been shown to have a protective role on the vasculature of patients with diabetes. These cardiovascular protective effects are distinct from and additive to any benecial vascular consequence of glucose lowering.42 PPAR is found in endothelial cells and in vascular smooth muscle cells, where its activation exerts anti-inammatory and antiproliferative effects, suggesting that they may be of benet in ameliorating chronic vascular inammation.43 However, little is known about its role in diabetic retinopathy. The present study demonstrated that PPAR is expressed in the vessels and glial cells of normal retina and is abrogated in the experimental models of diabetes, ischemic retinopathy, and retinal inammation, which are known to exhibit signicant vascular injury. This nding is consistent with what has been reported in diabetes. For example, PPAR has been shown to be decreased in the subcutaneous tissue of obese subjects with type 2 diabetes44 and in peritoneal macrophages from rats with alloxan-induced diabetes.45 In addition, PPAR expression was decreased in aorta and heart tissues of long-term glucose-fed rats and was restored by combination therapy of antioxidants and a-lipoic acid.46 Streptozotocin-induced diabetes has also been reported to suppress adipose tissue PPAR expression by 75% in normal mice with partial restoration during insulin treatment.47 However, this is the rst report to show the suppression of PPAR in diabetic retina. Furthermore, treating endothelial cells with HG caused a signicant decrease in PPAR expression. Suppression of PPAR in diabetes was associated with the activation of NFB. Because vascular inammation is crucial in the pathogenesis of vascular dysfunction, such as hyperpermeability26 and neovascularization27 associated with diabetic or ischemic retinopathy, we suggest that PPAR may exert its protective effect by way of an anti-inammatory pathway. This suggestion is consistent with the recent report by Muranaka et al.,33 who showed the inhibition of ICAM-1 expression, leukocyte adhesion, and retinal vascular leakage in experimental diabetes by the PPAR agonist rosiglitazone and the increase in the same parameters by deletion of the gene encoding PPAR. Moreover, rosiglitazone has been shown to inhibit retinal neovascularization in OIR by a mechanism downstream from VEGF.48 This probably occurs through targeting ICAM-1 because VEGFinduced angiogenesis is blocked in ICAM-1 decient mice.49,50 In addition, Miyahara et al.51 suggest that ICAM-1 is involved in VEGF-induced leukocyte-endothelial cell interactions and subsequent blood-retinal barrier (BRB) breakdown in the diabetic retina. Oxidative stress plays a crucial role in the pathogenesis of vascular dysfunction in diabetes. The sources and mechanism of reactive oxygen species effects on diabetic vasculature continue to be dened. Because NADPH oxidase-derived ROS are a major factor in triggering vascular dysfunctions in diabetes,20,52 OIR,19 and LPS-induced endotoxemia,53 we tested whether PPAR is involved in this process. Our experiments showed that the inhibition of NADPH oxidase using apocynin or the deletion of NOX2 restores the suppressed retinal PPAR in diabetic, OIR, and LPS-injected mice, indicating that NADPH oxidase has a negative regulatory effect on PPAR. The ndings of our in vitro experiments were consistent with the previously mentioned in vivo study ndings. These experiments showed that HG suppresses PPAR expression in cultured BRECs and HUVECs but that it is prevented by SOD and NADPH oxidase inhibitors (apocynin and DPI), indicating that superoxide generation by NADPH plays an important role in mediating the effect of HG on PPAR expression in endothelial cells. Restoration of the retinal level of PPAR by NADPH oxidase inhibition was associated with the abrogation of inammatory signaling, as shown by the decreases in NFB activation. Of

PPAR Expression in Acute Retinal Inammation

We have previously reported that injection of LPS into mice is associated with increased retinal expression of ICAM-1 and leukostasis and that the deletion of NOX2 prevents these effects.21 Here, we tested whether this was associated with any changes in the expression of PPAR. Our experiments demonstrated a signicant decrease in the retinal expression of PPAR in LPS-injected mice compared with control (2001 127 vs. 3121 205) that was prevented in mice lacking NOX2 (3139 208; Fig. 5).

DISCUSSION
This study examined changes in the expression of PPAR in diabetic and ischemic retina and the role NADPH oxidase plays, if any, in mediating these changes. There are two main ndings of this work. The rst is that PPAR is expressed in normal retina, particularly in blood vessels and Mu ller cells, and that it is suppressed in experimental models of diabetes, OIR, and acute retinal inammation and in endothelial cells treated with HG. The second is that the inhibition of NADPH oxidase by apocynin or the deletion of NOX2 restores normal levels of retinal PPAR, associated with decreased levels of activated NFB in diabetic retina. To the best of our knowledge, this is the rst report characterizing the expression of PPAR in retina under normal and pathologic conditions associated with vascular dysfunction. Furthermore, it is the rst report to show the role of NADPH oxidase in mediating changes in the levels of PPAR in diabetic retina and OIR. In our previous study on a mouse model of STZ-induced diabetes, we showed increases in retinal intracellular adhesion molecule (ICAM)-1 expression, leukocyte adhesion, and vascular permeability. These effects of diabetes have been prevented by apocynin or deletion of NOX2.21 We also showed that apocynin blocks retinal neovascularization in OIR.19 Here, we tested the changes in PPAR expression during diabetic retinopathy and OIR and whether NADPH oxidase plays any role in mediating these changes. Activation of PPAR using specic agonists such as thiazolidinediones (TZDs) has been used for the treatment of diabe-

FIGURE 5. Western blot analysis of PPAR in LPS-injected mice. There is a signicant decrease in the retinal expression of PPAR in the LPS-injected wild-type mice (LPS-WT) compared with the control (C). PPAR level was restored in mice lacking NOX2 (LPS-NOX/) (n 7). *P 0.05 vs. C; #P 0.05 vs. LPS-WT.

IOVS, February 2009, Vol. 50, No. 2 note, the effect of apocynin on retinal PPAR and NFB was similar to that of NOX2 deletion, which indicates its specicity as an NADPH oxidase inhibitor. Our previous ndings in diabetic and LPS-injected mice demonstrated the inhibition of ICAM-1 expression and the abrogation of leukocyte adhesion by apocynin or the deletion of NOX2, and this was associated with the preservation of BRB in diabetic mice.21 Furthermore, apocynin treatment blocked retinal neovascularization in OIR.19 Interestingly, the decrease in PPAR expression follows the same pattern of the increase in retinal expression of NOX2 in OIR. Although the decrease in PPAR expression was more obvious by P14, the increase in NOX2 expression was also more prominent by P14 than at P17.19 These data suggest an inverse relationship between the levels of NOX2 and PPAR that could be involved in the pathogenesis of retinal neovascularization. Correlating our previous ndings with the current data led us to suggest that the restoration of PPAR could be an effective therapeutic strategy in preventing retinal vascular inammation, a crucial event in the pathogenesis of diabetes or OIR. The inhibitory effect of NADPH oxidase on PPAR gives a novel insight for how NADPH oxidase mediates vascular inammation in diabetic retinopathy and in other vascular complications of diabetes such as atherosclerosis. Hwang et al.54 have reported that PPAR ligands reduce superoxide anion generation in vascular endothelial cells by inhibiting NADPH oxidase. Hence, PPAR restoration by NADPH oxidase inhibitors might lead to further inhibition of ROS generation by NADPH oxidase. More experiments are needed to elucidate how NADPH oxidase modulates PPAR expression and activity in diabetic retinopathy. Because NFB has been suggested to be the major redoxsensitive transcriptional regulator of endothelial adhesion molecules such as ICAM-1, and vascular cell adhesion molecule-1, we tested the effect of NADPH oxidase inhibition on its activation. Activation of NFB is associated with the phosphorylation and degradation of the inhibitor B (IB) and with the nuclear translocation of NFB subunit p65.55 In resting cells, NFB is inactive because IB proteins retain it in the cytoplasm and prevent DNA binding.56Our data show an activation of NFB in retinas of diabetic mice that was abrogated in mice lacking NOX2 or treated with apocynin. These ndings clearly indicate that vascular inammation associated with diabetic retinopathy is mediated through NADPH oxidase-dependent activation of NFB, which leads to increased ICAM-1 expression and leukocyte-endothelial interaction. These data, together with the decreased PPAR level in diabetic retina, support the link between PPAR and retinal vascular inammation in diabetic retinopathy. In summary, our ndings indicate that suppression of PPAR is downstream from NADPH oxidase activation in diabetic and ischemic retinopathies. Suppression of PPAR leads to activation of the NFB signaling pathway, including the upregulation of ICAM-1, increased leukocyte-endothelial interaction, and retinal vascular dysfunction. Targeting this signaling pathway could be benecial in preventing retinal vascular damage induced by hyperglycemia.

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