Journal of Diabetes and Its Complications 29 (2015) 275–281
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Expression of intraocular peroxisome proliferator-activated receptor gamma in patients with proliferative diabetic retinopathy Takashi Katome a, b, Kazuhiko Namekata b, Yoshinori Mitamura a,⁎, Kentaro Semba a, b, Mariko Egawa a, Takeshi Naito a, Chikako Harada b, Takayuki Harada a, b a b
Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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Article history: Received 30 September 2014 Received in revised form 18 October 2014 Accepted 21 October 2014 Available online 29 October 2014 Keywords: Angiogenesis Diabetic retinopathy Exosomes Peroxisome proliferator-activated receptor gamma Vascular endothelial growth factor
a b s t r a c t Aims: To determine whether peroxisome proliferator-activated receptor gamma (PPARγ), which is recognized as a component of the exosomes circulating in plasma, is expressed intraocularly in patients with proliferative diabetic retinopathy (PDR). Methods: The concentrations of PPARγ and vascular endothelial growth factor (VEGF) in the aqueous humor and vitreous of 50 eyes with PDR and 38 control eyes were determined by ELISA. The levels of the mRNA and protein of PPARγ were determined in proliferative membranes from 12 PDR and 5 control eyes by quantitative RT-PCR and immunohistochemical analyses. Results: PPARγ was detected in the culture media of human umbilical vein endothelial cells indicating that PPARγ can be released into the extracellular fluid. The PPARγ concentrations in the aqueous humor and vitreous fluid were significantly higher in PDR patients than in controls (P b 0.0005). There was a significant positive correlation between the PPARγ and VEGF concentrations (P b 0.0005). The level of PPARγ increased as the clinical stage advanced. The expressions of the mRNA and protein of PPARγ were higher in the membranes of PDR than those of controls. Anti-VEGF therapy significantly reduced the VEGF concentration (P b 0.0001) but not the PPARγ concentration. Conclusions: PPARγ may play an important role in the pathogenesis of PDR. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The presence of intraocular proliferative membranes has been associated with various clinical conditions including proliferatiave diabetic retinopathy (PDR), proliferative vitreoretinopathy, vitreous hemorrhage, congenital disorders, and idiopathic or spontaneous developments of membranes. Histopathological studies have shown that proliferative membranes are composed of various cell types such as glial cells, fibroblasts, and endothelial cells (Smiddy et al., 1989). Many cytokines are believed to be involved in the pathogenesis of PDR. Among them, vascular endothelial growth factor (VEGF) is the best candidate for causing the development of PDR (Mitamura, Harada, & Harada, 2005). The up-regulation of VEGF leads to the development of PDR because it promotes angiogenesis by enhancing endothelial cell proliferation, migration, and tube formation (Abu ElAsrar, Missotten, & Geboes, 2007). In fact, VEGF has been detected in
Conflict of Interest: There are no conflicts of interest. ⁎ Corresponding author at: Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. Tel.: +81 88 633 7163; fax: +81 88 631 4848. E-mail address: [email protected] (Y. Mitamura). http://dx.doi.org/10.1016/j.jdiacomp.2014.10.010 1056-8727/© 2015 Elsevier Inc. All rights reserved.
the vitreous fluid and proliferative membrane specimens obtained from eyes with PDR (Aiello et al., 1994). However, the precise molecular mechanisms involved in the formation of the membranes in eyes with PDR have not been fully determined. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor that is a critical component of a variety of biological processes, including adipogenesis, glucose metabolism, and inflammation (Rosen & Spiegelman, 2001). The results of recent experiments suggest that PPARγ activators also directly affect vascular cell function by modulating the secretion (Marx, Bourcier, Sukhova, Libby, & Plutzky, 1999) and proliferative responses of vascular cells (Murata et al., 2000; Murata, Hata, Ishibashi, et al., 2001; Panigrahy et al., 2002; Xin, Yang, Kowalski, & Gerritsen, 1999). The PPARγ ligand, rosiglitazone, inhibits endothelial cell proliferation in vitro and decreases the VEGF production by tumor cells (Panigrahy et al., 2002). Murata et al. (2000) reported on the antiangiogenic effects of PPARγ agonists on the ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and in eyes with experimental laser photocoagulation-induced CNV in vivo. Exosomes are nanovesicles that are released from cells for cell-free intercellular communications. Looze et al. (2009) identified PPARγ as a component of exosomes that circulated in human plasma, and they
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identified PPARγ as a potential new pathway for the paracrine transfer of nuclear receptors. This was possible because exosomes play a role as intercellular messengers. In an earlier study, we were able to detect the expression of promyelocytic leukemia protein, a nuclear protein, in the culture media of human umbilical vein endothelial cells (HUVECs) and in the aqueous humor and vitreous fluid of human eyes (Katome et al., 2012). These results stimulated us to examine the PPARγ levels in the aqueous humor and vitreous fluid of PDR patients which has not been made. Thus, the purpose of this study was to determine whether PPARγ is present in eyes with PDR and whether its level of expression is associated with the expression of VEGF. 2. Subjects and methods Undiluted aqueous humor and vitreous fluid were collected from 50 patients with PDR (one patient with Type 1 and 49 patients with Type 2 diabetes mellitus). There were 23 women and 27 men whose mean age was 51.7-years with a range from 25- to 75-years. The control patients included 38 patients with idiopathic epiretinal membranes (ERMs) (17 women and 21 men). The idiopathic ERM consists of a non-angiogenic fibroglial membrane that is not associated with diabetes mellitus, and is relatively common disease. The idiopathic ERM involving macular regions can cause a reduction in vision, metamorphopsia, micropsia, or occasionally monocular diplopia. The mean age of these control patients was 64.9-years with a range from 48- to 81-years. All of the patients including the controls underwent pars plana vitrectomy at the Tokushima University Hospital. Undiluted aqueous humor and vitreous fluid were collected at the beginning of the surgery. Patients with a history of vitreoretinal surgery or intraocular ischemia caused by diseases other than diabetic retinopathy were excluded from the study. The duration of the diabetes varied from 9to 30-years (mean, 16.3 years) in the 50 PDR subjects. Sixteen patients were being treated with insulin and 49 with oral hypoglycemic drugs. One patient was being treated by diet therapy alone. The mean concentration of HbA1c was 7.8% (61 mmol/mol) with a range of 6.2% to 10.3% (43–89 mmol/mol). Thirty-eight PDR subjects had hypertension. We also collected proliferative membranes from 12 PDR patients with Type 2 diabetes mellitus, and idiopathic ERMs from 5 control patients without diabetes. The proliferative membranes from the PDR patients were mainly composed of neovascular vessels, while the idiopathic ERMs consisted of non-angiogenic fibroglial cells (Yoshida-Hata et al., 2010). The samples of aqueous humor, vitreous fluid, and proliferative membranes were immediately frozen at −80 °C until assayed. An informed consent was obtained from each patient, and the Institutional Review Board of Tokushima University Hospital approved the study protocol. The procedures used conformed to the tenets of the Declaration of Helsinki as revised in 2000. HUVECs (CRL-1730; ATCC, Manassas, VA, USA) were transfected with plasmid encoding PPARγ tagged with myc. The exosomes were purified with the ExoQuick-TC™ Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) according to the manufacturer’s instructions. Briefly, after 24 h of transfection, 10 ml of the culture medium was collected and mixed with ExoQuick-TC™. The mixture was incubated for 16 h at 4 °C. The exosomes were centrifuged at 1500 × g for 30 min, and the pellets were resuspended in RIPA buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). The suspensions were subjected to SDS-PAGE followed by immunoblot analysis using anti-myc antibody. The concentrations of the PPARγ and VEGF proteins in the aqueous humor and vitreous fluid samples were determined by commercially available ELISA kits for human PPARγ (Cosmo Bio Co., Ltd., Tokyo, Japan) and for human VEGF (GE Healthcare, Little Chalfont, UK). The total protein concentrations in the aqueous humor and vitreous fluid
samples were also measured using Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). These assays were performed according to the manufacturer’s protocol. The assay detection limit was 0.08 nmol for PPARγ and 8.0 pg/ml for VEGF. For the statistical calculations, samples with undetectable concentrations were entered as the detection limit. An elevation of PPARγ or VEGF concentrations in the aqueous humor and vitreous fluid of patients with PDR can be due to an increase of total vitreal proteins observed in PDR patients due to the disruption of the blood-retinal barrier (Burgos et al., 2000). Thus, the PPARγ and VEGF concentrations were evaluated as ratios of PPARγ and VEGF to the total protein concentrations (Burgos et al., 2000; Tashimo et al., 2004). To examine the relationship between PPARγ concentrations and the clinical stage of PDR, the degree of fibrous proliferation in PDR patients was graded as follows: no apparent fibrous proliferation = 0; fibrous proliferation around the disc only = 1; fibrous proliferation around the vascular arcade = 2; and fibrous proliferation throughout the posterior pole = 3 (Mitamura et al., 2001). The degree of fibrous proliferation was determined according to the medical records and operative notes by two of the authors (ME and TN) who were masked to the PPARγ and VEGF concentrations. Bevacizumab, an anti-VEGF antibody, has been approved for the treatment for PDR. We compared the PPARγ and VEGF concentrations in PDR patients treated with and without intravitreous bevacizumab (IVB). The IVB was performed 3 or 4 days before the surgery when the samples were collected. The level of mRNA expression of PPARγ in the proliferative membranes from 12 PDR patients and in the idiopathic ERMs from 5 control patients was determined by quantitative real-time RT-PCR analysis (ABI 7500; Applied Biosystems, Foster City, CA, USA) (Harada et al., 2002; Yoshida-Hata et al., 2010). The initial amount of total RNA was normalized in every assay to β-actin, a housekeeping gene, as an internal standard. Primers specific for human PPARγ (sense: 5′CCTCATGAAGAGCCTTCCAA-3′; antisense: 5′-ACCCTTGCATCCTTCACAAG3′) and human β-actin (sense: 5′-AGAGCTACGAGCTGCCTGAC-3′; antisense: 5′-AGCACTGTGTTGGCGTACAG-3′) were used. Immunohistochemical analysis was performed as described (Harada et al., 2002; Mitamura et al., 2003). The antibodies used were: PPARγ (1:500; Cell Signaling Technology, Danvers, MA, USA) and von Willebrand factor (vWF) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1.0 μg/ml). The sections were counterstained with Hoechst to make the nuclei visible. Student’s t tests were used to determine the significance of the differences in the protein concentrations or mRNA expression between the two groups. Data were analyzed using one-factor ANOVA and Fisher’s PSLD to compare PPARγ concentrations in four PDR subgroups. The correlations between PPARγ and VEGF concentrations were determined by Pearson’s correlation tests. All analyses were done with the SPSS version 20.0 (SPSS Japan, Tokyo, Japan). A P value b 0.05 was considered statistically significant. 3. Results We first determined whether PPARγ in the exosomes in the cell culture media could be measured. HUVECs were transfected with plasmids encoding PPARγ, and the level of PPARγ in the exosome fraction from the HUVEC culture media was measured by immunoblot analysis. The positive findings of PPARγ indicated that PPARγ is released from these cells into the extracellular fluid (Fig. 1). These results suggest that PPARγ can be secreted into the aqueous humor and vitreous fluid. The total protein concentrations in the aqueous humor and vitreous fluid were significantly higher in PDR patients (4.95 ± 2.25 mg/ml and 5.82 ± 2.65 mg/ml, respectively; mean ± SD), than in control patients (2.97 ± 1.23 mg/ml; P b 0.0001 and 4.16 ± 1.66 mg/ml; P = 0.0012, respectively). The ratio of the PPARγ concentration to total protein
T. Katome et al. / Journal of Diabetes and Its Complications 29 (2015) 275–281
Fig. 1. Immunoblot analysis of myc-tagged PPARγ in exosomes from human umbilical vascular endothelial cells (HUVECs). PPARγ is detected in the exosome fraction.
concentration in the aqueous humor was 0.072 ± 0.078 nmol/mg in the control patients and 0.375 ± 0.466 nmol/mg in the PDR patients. The ratios of the PPARγ protein to the total protein in the vitreous fluid were 0.077 ± 0.090 nmol/mg in the control patients and 0.581 ± 0.683 nmol/mg in the PDR patients. When the PPARγ concentration was corrected for the total proteins, the PPARγ concentration was significantly higher in PDR patients than in control patients in both the aqueous humor (P = 0.0002) and vitreous fluid (P b 0.0001, Fig. 2a, b). To determine whether there was a significant association between the expression levels of PPARγ and VEGF, we also examined the concentration of the VEGF protein. The ratios of VEGF to total proteins in the aqueous humor (49.2 ± 9.33 pg/mg) and in the vitreous fluid (84.6 ± 16.1 pg/mg) of PDR patients were significantly higher than those of control patients (7.32 ± 3.09 pg/mg, P = 0.0007; and 3.06 ± 1.00 pg/mg, P = 0.0004, respectively). We found a significant positive correlation between the concentrations of PPARγ and VEGF in
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the aqueous humor (r = 0.395, P = 0.0001; Fig. 2c) and in the vitreous fluid (r = 0.729, P b 0.0001; Fig. 2d). In the PDR patients, the fibrous proliferation ranged from grade 0 to grade 3. After correcting for the total protein concentration, the PPARγ concentration in the aqueous humor was 0.080 ± 0.081 nmol/mg in eyes with grade 0 (11 eyes), 0.340 ± 0.369 nmol/mg in eyes with grade 1 (20 eyes), 0.432 ± 0.388 nmol/mg in eyes with grade 2 (15 eyes), and 1.184 ± 0.965 nmol/mg in eyes with grade 3 (4 eyes; Fig. 3a). Significant differences were found among these four groups in the aqueous PPARγ concentrations (P = 0.0004). When the aqueous PPARγ concentrations in eyes of the four PDR subgroups were compared independently with each group, the ratio of PPARγ to total protein was significantly higher in grade 3 than in grades 0, 1, and 2 eyes (P b 0.0001, P = 0.0004, P = 0.0019, respectively). In addition, the PPARγ concentrations were significantly higher in grade 2 than in grade 0 (P = 0.0336). The ratio of the PPARγ protein to the total protein in the vitreous fluid was 0.071 ± 0.075 nmol/mg in the eyes with grade 0 (11 eyes), 0.407 ± 0.421 nmol/mg in eyes with grade 1 (20 eyes), 0.967 ± 0.784 nmol/mg in eyes with grade 2 (15 eyes), and 1.323 ± 0.821 nmol/mg in eyes with grade 3 (4 eyes; Fig. 3b). Significant differences were found among these four groups in the vitreous PPARγ concentrations (P = 0.0001). When the vitreous PPARγ concentrations in the eyes of the four PDR subgroups were compared independently with each of other, the ratio of PPARγ protein to the total protein was significantly higher in grade 3 than in grades 0 and 1 eyes (P = 0.0003, P = 0.0040, respectively). In addition, the PPARγ concentration was significantly higher in eyes with grade 2 than in eyes with grades 0 and 1 (P = 0.0002, P = 0.0048, respectively). To investigate the effect of IVB on the vitreous PPARγ and VEGF concentrations, we compared the PPARγ and VEGF concentrations in PDR patients treated with IVB to those without IVB. The ratio of PPARγ
Fig. 2. PPARγ protein concentrations in aqueous humor and vitreous fluid. a and b: PPARγ protein concentrations in aqueous humor (a) and vitreous fluid (b) of control and PDR patients. The PPARγ concentrations in the aqueous humor and vitreous fluid are significantly higher in PDR than in controls. *P b 0.0005. The horizontal lines represent the mean. c and d: Correlation between the concentrations of PPARγ and VEGF proteins in aqueous humor (c) and vitreous fluid (d) of PDR patients. The PPARγ concentrations are significantly correlated with VEGF concentrations in both the aqueous humor and vitreous fluid (r = 0.395, P = 0.0001; r = 0.729, P b 0.0001; respectively).
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Fig. 3. PPARγ concentrations in aqueous humor (a) and vitreous fluid (b) samples from PDR patients with grade 0 (n = 11), grade 1 (n = 20), grade 2 (n = 15), and grade 3 (n = 4) fibrous proliferation. Significant differences are present among these four groups in the aqueous and vitreous PPARγ concentrations (P = 0.0004, P = 0.0001, respectively). The horizontal lines indicate the mean concentration in each group.
protein to the total protein in the vitreous fluid was significantly higher in PDR patients treated with IVB than those not treated with IVB (1.321 ± 0.746 nmol/mg, 0.247 ± 0.180 nmol/mg, respectively; P b 0.0001; Fig. 4a). In contrast, the vitreous VEGF concentrations were significantly lower in PDR patients treated with IVB than those without IVB (1.431 ± 0.755 nmol/mg, 84.608 ± 93.615 nmol/mg, respectively; P = 0.0009; Fig. 4b). The expression level of the mRNA of PPARγ was significantly higher in the proliferative membrane samples from PDR patients (104.820 ± 80.893) than in the idiopathic ERM samples from control patients (17.343 ± 12.520; P = 0.0321; Fig. 5a). Immunohistochemical analyses were performed on the membrane samples derived from control patients and PDR patients treated with or without IVB. Intense staining of the PPARγ protein was observed in membrane samples derived from PDR patients treated with IVB (Fig. 5b). The cells were double-labeled with anti-PPARγ and antivWF antibodies over a large area of the PDR membranes, suggesting that PPARγ is mainly expressed in the endothelial cells of the PDR membranes.
4. Discussion To the best of our knowledge, this is the first study that has demonstrated the expression of PPARγ in intraocular samples from eyes with PDR. In addition, our results showed that the expression of the mRNA of PPARγ was significantly higher in the proliferative membranes of eyes with PDR than in the ERM of the controls. Immunohistochemical analyses showed that the PPARγ protein was mainly expressed in endothelial cells of the PDR membranes. Our results showed that the PPARγ concentrations in the aqueous humor and vitreous fluid of PDR patients were significantly higher than those in the controls, and the concentrations were correlated with the VEGF concentrations. There was a tendency toward higher PPARγ concentrations in PDR patients with more severe fibrous proliferation. These results suggest that PPARγ may play an important role in the pathogenesis of PDR. The PPARγ concentration in the vitreous fluid of PDR patients treated with IVB was significantly higher than that in those without IVB. However, this may be because IVB was used mainly in severe PDR
Fig. 4. PPARγ (a) and VEGF (b) levels in the vitreous samples from PDR patients treated with or without intravitreous bevacizumab (IVB) injection. The PPARγ concentrations in the PDR patients treated with IVB are significantly higher than those without IVB. In contrast, vitreous VEGF concentrations in PDR patients treated with IVB are significantly lower than those without IVB. *P b 0.001. The horizontal lines indicate the mean concentration in each group.
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Fig. 5. Expression of PPARγ mRNA and protein in epiretinal membrane (ERM) samples from control patients and proliferative membrane samples from PDR patients. a: Expression level of PPARγ mRNA. Plots represent means ± SD. *P = 0.0321. b: Double-labeling immunohistochemistry of PPARγ and von Willebrand factor (vWF) in membrane samples derived from control patients and PDR patients treated with or without IVB. Nuclear staining was done in the same section. The cells over a large area of the PDR membranes are double-labeled with antiPPARγ and anti-vWF antibodies. Intense staining of PPARγ protein is observed in membrane samples derived from PDR patients treated with IVB. Bar; 100 μm.
cases. In contrast, the vitreous VEGF concentration in PDR patients treated with IVB was significantly lower than that in patients without IVB treatment. Immunohistochemical analyses showed intense staining for the PPARγ protein in the endothelial cells of PDR membranes treated with IVB. It has been reported that PPARγ is expressed in vascular endothelial cells, and that the PPARγ ligands can inhibit their proliferation induced by growth factors or their apoptosis in vitro (Bishop-Bailey & Hla, 1999; Marx et al., 1999; Xin et al., 1999). Taken together, these results suggest that PPARγ ligands may be useful in treating angiogenic diseases by inhibiting angiogenesis. Although we cannot exclude the possibility that IVB increased the expression of PPARγ, our results suggest that therapy targeting PPARγ may have a synergistic or additional effect to anti-VEGF therapy. PPARγ is a nuclear receptor but we were able to detect its expression in the exosome fraction found in the media of HUVECs transfected with plasmid encoding PPARγ. This suggests that it can be released from endothelial cells into the extracellular fluid. Recently, Looze et al. (2009) identified PPARγ as a component of exosomes that circulates in human plasma. Consistent with these results, we could detect the PPARγ protein in both the aqueous humor and vitreous fluid. PPARs are ligand-activated transcription factors of the nuclear hormone receptor superfamily related to retinoid, steroid, and thyroid hormone receptors (Uchiyama et al., 2013). The PPARs family is represented by three members: PPARα, PPARβ/δ, and PPARγ. PPARγ is a key transcription factor involved in adipocyte differentiation, and lipid and glucose homeostasis. It is an important therapeutic target for type 2 diabetes mellitus and the metabolic syndrome (Széles, Töröcsik, & Nagy, 2007). PPARγ may interfere with glucose signaling by increasing the insulin sensitivity in muscles and adipose tissues (Escher & Wahli, 2000). PPARγ acts by forming a heterodimer with the retinoid X receptor which binds to direct repeats of the hormonal response elements (Murata et al., 2000). Upon activation, the ligandinducible transcription factors stimulate gene expression by binding to the promoter of the target genes (Escher & Wahli, 2000). PPARγ is capable of both positive and negative regulation of gene expression in response to ligand binding (Li, Pascual, & Glass, 2000). PPARγ is expressed heterogeneously in the mammalian eye (Pershadsingh & Moore, 2008). PPARγ is most prominent in the retinal pigmented epithelium (RPE), photoreceptor outer segments, choriocapillaris, choroidal endothelial cells, corneal epithelium, and endothelium, and to a lesser extent, in the intraocular muscles, retinal
photoreceptor inner segments, outer plexiform layer, and the iris (Pershadsingh & Moore, 2008). The effect of the PPARγ ligands on retinal neovascularization was examined by Murata et al. (2001). The expression of PPARγ was confirmed in the retinal endothelial cells by Western immunoblotting, and the PPARγ ligands suppressed the VEGF-induced migration, proliferation, and tube formation by the retinal endothelial cells in vitro. In their in vivo study on the oxygen-induced ischemia model of retinal neovascularization which can be considered as an animal model of PDR, an intravenous injection of PPARγ ligands inhibited the development of the retinal neovascularization. The authors concluded that PPARγ ligands should be evaluated for their potential to inhibit the progression of PDR. Telmisartan and irbesartan were recently shown to belong to a unique subset of angiotensin receptor blockers (ARBs) and were capable of activating PPARγ (Schupp, Janke, Clasen, Unger, & Kintscher, 2004). Telmisartan was shown to down-regulate angiotensin II type 1 receptor through activation of PPARγ (Imayama et al., 2006). Telmisartan was also shown to have therapeutic benefits in rodent models of PDR (Nagai et al., 2007). Pershadsingh and Moore (2008) reported that telmisartan and irbesartan may improve the efficacy of treating PDR by activating PPARγ. On the other hand, there have been several studies showing that activation of PPARγ induces angiogenesis through the up-regulation of VEGF. Biscetti et al. (2013) demonstrated that cilostazol is able to induce angiogenesis in vivo in streptozotocin-induced diabetic mice by the up-regulation of pro-angiogenic genes and proteins. This phenomenon is dependent on PPARγ and may be suppressed by the systemic inhibition of PPARγ. Terrasi et al. (2013) reported that PPARγ agonists stimulated angiogenesis through the up-regulation of VEGF and leptin, and other yet unidentified proangiogenic factors. Biscetti et al. (2008, 2009) investigated angiogenic potentials of selective synthetic PPARα and PPARγ agonists in vitro and in vivo. In their study, activation of PPARα and PPARγ led to endothelial tube formation in an endothelial/interstitial cell co-culture assay, and this effect was associated with increased production of VEGF. In addition, PPARα and PPARγ agonists also induced neovascularization in the murine corneal angiogenic model. These results are consistent with our results that the PPARγ concentrations were significantly correlated with the VEGF concentrations in the aqueous humor and vitreous fluid of PDR patients.
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There is evidence that the expression of PPARγ is increased in patients with cancers, e.g., breast, prostate, pancreas, and colon, but it is decreased in others, e.g., esophagus and uterus cancers (Terrasi et al., 2013). The role of PPARγ in cancer progression remains controversial. While some studies demonstrated anti-proliferative role of PPARγ ligands in cellular and animal models of human cancers (Grommes, Landreth, & Heneka, 2004), other investigations showed that activation of PPARγ can induce cell growth and tumor proliferation (Talbert, Allred, Zaytseva, & Kilgore, 2008; Terrasi et al., 2013). One explanation for these discrepancies is that PPARγ is a multivalent receptor whose ligand binding domain can accommodate different PPARγ ligands (Pershadsingh & Moore, 2008). The PPARγ ligands, such as the full agonist, partial agonist, and selective PPARγ modulators, are capable of inducing distinct receptor combinations leading to selective gene expressions (Pershadsingh & Moore, 2008). For example, the thiazolidinediones, e.g., rosiglitazones, are full PPARγ agonist, while the ARBs, e.g., telmisartan or irbesartan, function as partial PPARγ agonists. Each ligand–receptor complex assumes a somewhat different three-dimensional conformation leading to unique and differential interactions with cofactors, histones, and other transcription factors. Consequently, each PPARγ ligand–receptor complex leads to a different and overlapping pattern of gene expression. Thus, each ligand will activate or repress multiple genes leading to a differential overlapped expression of different sets of genes. With respect to the two conflicting roles of PPARγ in PDR progression, Sassa et al. (2004) reported bifunctional properties of PPARγ in the regulation of the VEGF receptor gene. The VEGF receptor plays a critical role in mediating a variety of vasculogenic and angiogenic processes including PDR. PPARγ enhanced the expression of the gene of the VEGF receptor at the transcriptional level in the absence of its ligands in retinal capillary endothelial cells. The PPARγ ligands, however, suppressed the gene expression of VEGF receptor by decreasing the interaction between PPARγ and transcriptional factors Sp1/Sp3 (Sassa et al., 2004). They concluded that PPARγ had bifunctional properties in the regulation of gene expression of the VEGF receptor in retinal capillary endothelial cells. The PPARγ concentrations in the aqueous humor and vitreous fluid of PDR patients were significantly higher than those in the controls. In addition, there was a tendency toward higher PPARγ concentrations in PDR patients with increasing severity of fibrous proliferation. However, how this over-expression of PPARγ is involved in the pathogenesis of PDR remains unclear. Moreover, we cannot determine whether the increased PPARγ concentrations are the cause or the effect of PDR in this study. Further studies to investigate the precise molecular machanism by which PPARγ contributes to the pathogenesis of PDR are needed. In conclusion, intraocular expression of PPARγ was significantly higher in PDR patients than in controls subjects. Anti-VEGF therapy significantly reduced the VEGF concentration but not the PPARγ concentration. These findings suggest that PPARγ may play an important role in the pathogenesis of PDR by the independent mechanisms from those associated with VEGF. Acknowledgements There are no conflicts of interest. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (T. Katome, K. Namekata, Y. Mitamura, C. Harada, T. Harada), and the Funding Program for Next Generation World-Leading Researchers (NEXT Program) (T. Harada). References Abu El-Asrar, A. M., Missotten, L., & Geboes, K. (2007). Expression of hypoxia-inducible factor-1alpha and the protein products of its target genes in diabetic fibrovascular epiretinal membranes. British Journal of Ophthalmology, 91, 822–826.
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Expression of intraocular peroxisome proliferator-activated receptor gamma in patients with proliferative diabetic retinopathy Takashi Katome a, b, Kazuhiko Namekata b, Yoshinori Mitamura a,⁎, Kentaro Semba a, b, Mariko Egawa a, Takeshi Naito a, Chikako Harada b, Takayuki Harada a, b a b
Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 30 September 2014 Received in revised form 18 October 2014 Accepted 21 October 2014 Available online 29 October 2014 Keywords: Angiogenesis Diabetic retinopathy Exosomes Peroxisome proliferator-activated receptor gamma Vascular endothelial growth factor
a b s t r a c t Aims: To determine whether peroxisome proliferator-activated receptor gamma (PPARγ), which is recognized as a component of the exosomes circulating in plasma, is expressed intraocularly in patients with proliferative diabetic retinopathy (PDR). Methods: The concentrations of PPARγ and vascular endothelial growth factor (VEGF) in the aqueous humor and vitreous of 50 eyes with PDR and 38 control eyes were determined by ELISA. The levels of the mRNA and protein of PPARγ were determined in proliferative membranes from 12 PDR and 5 control eyes by quantitative RT-PCR and immunohistochemical analyses. Results: PPARγ was detected in the culture media of human umbilical vein endothelial cells indicating that PPARγ can be released into the extracellular fluid. The PPARγ concentrations in the aqueous humor and vitreous fluid were significantly higher in PDR patients than in controls (P b 0.0005). There was a significant positive correlation between the PPARγ and VEGF concentrations (P b 0.0005). The level of PPARγ increased as the clinical stage advanced. The expressions of the mRNA and protein of PPARγ were higher in the membranes of PDR than those of controls. Anti-VEGF therapy significantly reduced the VEGF concentration (P b 0.0001) but not the PPARγ concentration. Conclusions: PPARγ may play an important role in the pathogenesis of PDR. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The presence of intraocular proliferative membranes has been associated with various clinical conditions including proliferatiave diabetic retinopathy (PDR), proliferative vitreoretinopathy, vitreous hemorrhage, congenital disorders, and idiopathic or spontaneous developments of membranes. Histopathological studies have shown that proliferative membranes are composed of various cell types such as glial cells, fibroblasts, and endothelial cells (Smiddy et al., 1989). Many cytokines are believed to be involved in the pathogenesis of PDR. Among them, vascular endothelial growth factor (VEGF) is the best candidate for causing the development of PDR (Mitamura, Harada, & Harada, 2005). The up-regulation of VEGF leads to the development of PDR because it promotes angiogenesis by enhancing endothelial cell proliferation, migration, and tube formation (Abu ElAsrar, Missotten, & Geboes, 2007). In fact, VEGF has been detected in
Conflict of Interest: There are no conflicts of interest. ⁎ Corresponding author at: Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. Tel.: +81 88 633 7163; fax: +81 88 631 4848. E-mail address: [email protected] (Y. Mitamura). http://dx.doi.org/10.1016/j.jdiacomp.2014.10.010 1056-8727/© 2015 Elsevier Inc. All rights reserved.
the vitreous fluid and proliferative membrane specimens obtained from eyes with PDR (Aiello et al., 1994). However, the precise molecular mechanisms involved in the formation of the membranes in eyes with PDR have not been fully determined. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor that is a critical component of a variety of biological processes, including adipogenesis, glucose metabolism, and inflammation (Rosen & Spiegelman, 2001). The results of recent experiments suggest that PPARγ activators also directly affect vascular cell function by modulating the secretion (Marx, Bourcier, Sukhova, Libby, & Plutzky, 1999) and proliferative responses of vascular cells (Murata et al., 2000; Murata, Hata, Ishibashi, et al., 2001; Panigrahy et al., 2002; Xin, Yang, Kowalski, & Gerritsen, 1999). The PPARγ ligand, rosiglitazone, inhibits endothelial cell proliferation in vitro and decreases the VEGF production by tumor cells (Panigrahy et al., 2002). Murata et al. (2000) reported on the antiangiogenic effects of PPARγ agonists on the ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and in eyes with experimental laser photocoagulation-induced CNV in vivo. Exosomes are nanovesicles that are released from cells for cell-free intercellular communications. Looze et al. (2009) identified PPARγ as a component of exosomes that circulated in human plasma, and they
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identified PPARγ as a potential new pathway for the paracrine transfer of nuclear receptors. This was possible because exosomes play a role as intercellular messengers. In an earlier study, we were able to detect the expression of promyelocytic leukemia protein, a nuclear protein, in the culture media of human umbilical vein endothelial cells (HUVECs) and in the aqueous humor and vitreous fluid of human eyes (Katome et al., 2012). These results stimulated us to examine the PPARγ levels in the aqueous humor and vitreous fluid of PDR patients which has not been made. Thus, the purpose of this study was to determine whether PPARγ is present in eyes with PDR and whether its level of expression is associated with the expression of VEGF. 2. Subjects and methods Undiluted aqueous humor and vitreous fluid were collected from 50 patients with PDR (one patient with Type 1 and 49 patients with Type 2 diabetes mellitus). There were 23 women and 27 men whose mean age was 51.7-years with a range from 25- to 75-years. The control patients included 38 patients with idiopathic epiretinal membranes (ERMs) (17 women and 21 men). The idiopathic ERM consists of a non-angiogenic fibroglial membrane that is not associated with diabetes mellitus, and is relatively common disease. The idiopathic ERM involving macular regions can cause a reduction in vision, metamorphopsia, micropsia, or occasionally monocular diplopia. The mean age of these control patients was 64.9-years with a range from 48- to 81-years. All of the patients including the controls underwent pars plana vitrectomy at the Tokushima University Hospital. Undiluted aqueous humor and vitreous fluid were collected at the beginning of the surgery. Patients with a history of vitreoretinal surgery or intraocular ischemia caused by diseases other than diabetic retinopathy were excluded from the study. The duration of the diabetes varied from 9to 30-years (mean, 16.3 years) in the 50 PDR subjects. Sixteen patients were being treated with insulin and 49 with oral hypoglycemic drugs. One patient was being treated by diet therapy alone. The mean concentration of HbA1c was 7.8% (61 mmol/mol) with a range of 6.2% to 10.3% (43–89 mmol/mol). Thirty-eight PDR subjects had hypertension. We also collected proliferative membranes from 12 PDR patients with Type 2 diabetes mellitus, and idiopathic ERMs from 5 control patients without diabetes. The proliferative membranes from the PDR patients were mainly composed of neovascular vessels, while the idiopathic ERMs consisted of non-angiogenic fibroglial cells (Yoshida-Hata et al., 2010). The samples of aqueous humor, vitreous fluid, and proliferative membranes were immediately frozen at −80 °C until assayed. An informed consent was obtained from each patient, and the Institutional Review Board of Tokushima University Hospital approved the study protocol. The procedures used conformed to the tenets of the Declaration of Helsinki as revised in 2000. HUVECs (CRL-1730; ATCC, Manassas, VA, USA) were transfected with plasmid encoding PPARγ tagged with myc. The exosomes were purified with the ExoQuick-TC™ Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) according to the manufacturer’s instructions. Briefly, after 24 h of transfection, 10 ml of the culture medium was collected and mixed with ExoQuick-TC™. The mixture was incubated for 16 h at 4 °C. The exosomes were centrifuged at 1500 × g for 30 min, and the pellets were resuspended in RIPA buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). The suspensions were subjected to SDS-PAGE followed by immunoblot analysis using anti-myc antibody. The concentrations of the PPARγ and VEGF proteins in the aqueous humor and vitreous fluid samples were determined by commercially available ELISA kits for human PPARγ (Cosmo Bio Co., Ltd., Tokyo, Japan) and for human VEGF (GE Healthcare, Little Chalfont, UK). The total protein concentrations in the aqueous humor and vitreous fluid
samples were also measured using Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). These assays were performed according to the manufacturer’s protocol. The assay detection limit was 0.08 nmol for PPARγ and 8.0 pg/ml for VEGF. For the statistical calculations, samples with undetectable concentrations were entered as the detection limit. An elevation of PPARγ or VEGF concentrations in the aqueous humor and vitreous fluid of patients with PDR can be due to an increase of total vitreal proteins observed in PDR patients due to the disruption of the blood-retinal barrier (Burgos et al., 2000). Thus, the PPARγ and VEGF concentrations were evaluated as ratios of PPARγ and VEGF to the total protein concentrations (Burgos et al., 2000; Tashimo et al., 2004). To examine the relationship between PPARγ concentrations and the clinical stage of PDR, the degree of fibrous proliferation in PDR patients was graded as follows: no apparent fibrous proliferation = 0; fibrous proliferation around the disc only = 1; fibrous proliferation around the vascular arcade = 2; and fibrous proliferation throughout the posterior pole = 3 (Mitamura et al., 2001). The degree of fibrous proliferation was determined according to the medical records and operative notes by two of the authors (ME and TN) who were masked to the PPARγ and VEGF concentrations. Bevacizumab, an anti-VEGF antibody, has been approved for the treatment for PDR. We compared the PPARγ and VEGF concentrations in PDR patients treated with and without intravitreous bevacizumab (IVB). The IVB was performed 3 or 4 days before the surgery when the samples were collected. The level of mRNA expression of PPARγ in the proliferative membranes from 12 PDR patients and in the idiopathic ERMs from 5 control patients was determined by quantitative real-time RT-PCR analysis (ABI 7500; Applied Biosystems, Foster City, CA, USA) (Harada et al., 2002; Yoshida-Hata et al., 2010). The initial amount of total RNA was normalized in every assay to β-actin, a housekeeping gene, as an internal standard. Primers specific for human PPARγ (sense: 5′CCTCATGAAGAGCCTTCCAA-3′; antisense: 5′-ACCCTTGCATCCTTCACAAG3′) and human β-actin (sense: 5′-AGAGCTACGAGCTGCCTGAC-3′; antisense: 5′-AGCACTGTGTTGGCGTACAG-3′) were used. Immunohistochemical analysis was performed as described (Harada et al., 2002; Mitamura et al., 2003). The antibodies used were: PPARγ (1:500; Cell Signaling Technology, Danvers, MA, USA) and von Willebrand factor (vWF) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1.0 μg/ml). The sections were counterstained with Hoechst to make the nuclei visible. Student’s t tests were used to determine the significance of the differences in the protein concentrations or mRNA expression between the two groups. Data were analyzed using one-factor ANOVA and Fisher’s PSLD to compare PPARγ concentrations in four PDR subgroups. The correlations between PPARγ and VEGF concentrations were determined by Pearson’s correlation tests. All analyses were done with the SPSS version 20.0 (SPSS Japan, Tokyo, Japan). A P value b 0.05 was considered statistically significant. 3. Results We first determined whether PPARγ in the exosomes in the cell culture media could be measured. HUVECs were transfected with plasmids encoding PPARγ, and the level of PPARγ in the exosome fraction from the HUVEC culture media was measured by immunoblot analysis. The positive findings of PPARγ indicated that PPARγ is released from these cells into the extracellular fluid (Fig. 1). These results suggest that PPARγ can be secreted into the aqueous humor and vitreous fluid. The total protein concentrations in the aqueous humor and vitreous fluid were significantly higher in PDR patients (4.95 ± 2.25 mg/ml and 5.82 ± 2.65 mg/ml, respectively; mean ± SD), than in control patients (2.97 ± 1.23 mg/ml; P b 0.0001 and 4.16 ± 1.66 mg/ml; P = 0.0012, respectively). The ratio of the PPARγ concentration to total protein
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Fig. 1. Immunoblot analysis of myc-tagged PPARγ in exosomes from human umbilical vascular endothelial cells (HUVECs). PPARγ is detected in the exosome fraction.
concentration in the aqueous humor was 0.072 ± 0.078 nmol/mg in the control patients and 0.375 ± 0.466 nmol/mg in the PDR patients. The ratios of the PPARγ protein to the total protein in the vitreous fluid were 0.077 ± 0.090 nmol/mg in the control patients and 0.581 ± 0.683 nmol/mg in the PDR patients. When the PPARγ concentration was corrected for the total proteins, the PPARγ concentration was significantly higher in PDR patients than in control patients in both the aqueous humor (P = 0.0002) and vitreous fluid (P b 0.0001, Fig. 2a, b). To determine whether there was a significant association between the expression levels of PPARγ and VEGF, we also examined the concentration of the VEGF protein. The ratios of VEGF to total proteins in the aqueous humor (49.2 ± 9.33 pg/mg) and in the vitreous fluid (84.6 ± 16.1 pg/mg) of PDR patients were significantly higher than those of control patients (7.32 ± 3.09 pg/mg, P = 0.0007; and 3.06 ± 1.00 pg/mg, P = 0.0004, respectively). We found a significant positive correlation between the concentrations of PPARγ and VEGF in
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the aqueous humor (r = 0.395, P = 0.0001; Fig. 2c) and in the vitreous fluid (r = 0.729, P b 0.0001; Fig. 2d). In the PDR patients, the fibrous proliferation ranged from grade 0 to grade 3. After correcting for the total protein concentration, the PPARγ concentration in the aqueous humor was 0.080 ± 0.081 nmol/mg in eyes with grade 0 (11 eyes), 0.340 ± 0.369 nmol/mg in eyes with grade 1 (20 eyes), 0.432 ± 0.388 nmol/mg in eyes with grade 2 (15 eyes), and 1.184 ± 0.965 nmol/mg in eyes with grade 3 (4 eyes; Fig. 3a). Significant differences were found among these four groups in the aqueous PPARγ concentrations (P = 0.0004). When the aqueous PPARγ concentrations in eyes of the four PDR subgroups were compared independently with each group, the ratio of PPARγ to total protein was significantly higher in grade 3 than in grades 0, 1, and 2 eyes (P b 0.0001, P = 0.0004, P = 0.0019, respectively). In addition, the PPARγ concentrations were significantly higher in grade 2 than in grade 0 (P = 0.0336). The ratio of the PPARγ protein to the total protein in the vitreous fluid was 0.071 ± 0.075 nmol/mg in the eyes with grade 0 (11 eyes), 0.407 ± 0.421 nmol/mg in eyes with grade 1 (20 eyes), 0.967 ± 0.784 nmol/mg in eyes with grade 2 (15 eyes), and 1.323 ± 0.821 nmol/mg in eyes with grade 3 (4 eyes; Fig. 3b). Significant differences were found among these four groups in the vitreous PPARγ concentrations (P = 0.0001). When the vitreous PPARγ concentrations in the eyes of the four PDR subgroups were compared independently with each of other, the ratio of PPARγ protein to the total protein was significantly higher in grade 3 than in grades 0 and 1 eyes (P = 0.0003, P = 0.0040, respectively). In addition, the PPARγ concentration was significantly higher in eyes with grade 2 than in eyes with grades 0 and 1 (P = 0.0002, P = 0.0048, respectively). To investigate the effect of IVB on the vitreous PPARγ and VEGF concentrations, we compared the PPARγ and VEGF concentrations in PDR patients treated with IVB to those without IVB. The ratio of PPARγ
Fig. 2. PPARγ protein concentrations in aqueous humor and vitreous fluid. a and b: PPARγ protein concentrations in aqueous humor (a) and vitreous fluid (b) of control and PDR patients. The PPARγ concentrations in the aqueous humor and vitreous fluid are significantly higher in PDR than in controls. *P b 0.0005. The horizontal lines represent the mean. c and d: Correlation between the concentrations of PPARγ and VEGF proteins in aqueous humor (c) and vitreous fluid (d) of PDR patients. The PPARγ concentrations are significantly correlated with VEGF concentrations in both the aqueous humor and vitreous fluid (r = 0.395, P = 0.0001; r = 0.729, P b 0.0001; respectively).
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Fig. 3. PPARγ concentrations in aqueous humor (a) and vitreous fluid (b) samples from PDR patients with grade 0 (n = 11), grade 1 (n = 20), grade 2 (n = 15), and grade 3 (n = 4) fibrous proliferation. Significant differences are present among these four groups in the aqueous and vitreous PPARγ concentrations (P = 0.0004, P = 0.0001, respectively). The horizontal lines indicate the mean concentration in each group.
protein to the total protein in the vitreous fluid was significantly higher in PDR patients treated with IVB than those not treated with IVB (1.321 ± 0.746 nmol/mg, 0.247 ± 0.180 nmol/mg, respectively; P b 0.0001; Fig. 4a). In contrast, the vitreous VEGF concentrations were significantly lower in PDR patients treated with IVB than those without IVB (1.431 ± 0.755 nmol/mg, 84.608 ± 93.615 nmol/mg, respectively; P = 0.0009; Fig. 4b). The expression level of the mRNA of PPARγ was significantly higher in the proliferative membrane samples from PDR patients (104.820 ± 80.893) than in the idiopathic ERM samples from control patients (17.343 ± 12.520; P = 0.0321; Fig. 5a). Immunohistochemical analyses were performed on the membrane samples derived from control patients and PDR patients treated with or without IVB. Intense staining of the PPARγ protein was observed in membrane samples derived from PDR patients treated with IVB (Fig. 5b). The cells were double-labeled with anti-PPARγ and antivWF antibodies over a large area of the PDR membranes, suggesting that PPARγ is mainly expressed in the endothelial cells of the PDR membranes.
4. Discussion To the best of our knowledge, this is the first study that has demonstrated the expression of PPARγ in intraocular samples from eyes with PDR. In addition, our results showed that the expression of the mRNA of PPARγ was significantly higher in the proliferative membranes of eyes with PDR than in the ERM of the controls. Immunohistochemical analyses showed that the PPARγ protein was mainly expressed in endothelial cells of the PDR membranes. Our results showed that the PPARγ concentrations in the aqueous humor and vitreous fluid of PDR patients were significantly higher than those in the controls, and the concentrations were correlated with the VEGF concentrations. There was a tendency toward higher PPARγ concentrations in PDR patients with more severe fibrous proliferation. These results suggest that PPARγ may play an important role in the pathogenesis of PDR. The PPARγ concentration in the vitreous fluid of PDR patients treated with IVB was significantly higher than that in those without IVB. However, this may be because IVB was used mainly in severe PDR
Fig. 4. PPARγ (a) and VEGF (b) levels in the vitreous samples from PDR patients treated with or without intravitreous bevacizumab (IVB) injection. The PPARγ concentrations in the PDR patients treated with IVB are significantly higher than those without IVB. In contrast, vitreous VEGF concentrations in PDR patients treated with IVB are significantly lower than those without IVB. *P b 0.001. The horizontal lines indicate the mean concentration in each group.
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Fig. 5. Expression of PPARγ mRNA and protein in epiretinal membrane (ERM) samples from control patients and proliferative membrane samples from PDR patients. a: Expression level of PPARγ mRNA. Plots represent means ± SD. *P = 0.0321. b: Double-labeling immunohistochemistry of PPARγ and von Willebrand factor (vWF) in membrane samples derived from control patients and PDR patients treated with or without IVB. Nuclear staining was done in the same section. The cells over a large area of the PDR membranes are double-labeled with antiPPARγ and anti-vWF antibodies. Intense staining of PPARγ protein is observed in membrane samples derived from PDR patients treated with IVB. Bar; 100 μm.
cases. In contrast, the vitreous VEGF concentration in PDR patients treated with IVB was significantly lower than that in patients without IVB treatment. Immunohistochemical analyses showed intense staining for the PPARγ protein in the endothelial cells of PDR membranes treated with IVB. It has been reported that PPARγ is expressed in vascular endothelial cells, and that the PPARγ ligands can inhibit their proliferation induced by growth factors or their apoptosis in vitro (Bishop-Bailey & Hla, 1999; Marx et al., 1999; Xin et al., 1999). Taken together, these results suggest that PPARγ ligands may be useful in treating angiogenic diseases by inhibiting angiogenesis. Although we cannot exclude the possibility that IVB increased the expression of PPARγ, our results suggest that therapy targeting PPARγ may have a synergistic or additional effect to anti-VEGF therapy. PPARγ is a nuclear receptor but we were able to detect its expression in the exosome fraction found in the media of HUVECs transfected with plasmid encoding PPARγ. This suggests that it can be released from endothelial cells into the extracellular fluid. Recently, Looze et al. (2009) identified PPARγ as a component of exosomes that circulates in human plasma. Consistent with these results, we could detect the PPARγ protein in both the aqueous humor and vitreous fluid. PPARs are ligand-activated transcription factors of the nuclear hormone receptor superfamily related to retinoid, steroid, and thyroid hormone receptors (Uchiyama et al., 2013). The PPARs family is represented by three members: PPARα, PPARβ/δ, and PPARγ. PPARγ is a key transcription factor involved in adipocyte differentiation, and lipid and glucose homeostasis. It is an important therapeutic target for type 2 diabetes mellitus and the metabolic syndrome (Széles, Töröcsik, & Nagy, 2007). PPARγ may interfere with glucose signaling by increasing the insulin sensitivity in muscles and adipose tissues (Escher & Wahli, 2000). PPARγ acts by forming a heterodimer with the retinoid X receptor which binds to direct repeats of the hormonal response elements (Murata et al., 2000). Upon activation, the ligandinducible transcription factors stimulate gene expression by binding to the promoter of the target genes (Escher & Wahli, 2000). PPARγ is capable of both positive and negative regulation of gene expression in response to ligand binding (Li, Pascual, & Glass, 2000). PPARγ is expressed heterogeneously in the mammalian eye (Pershadsingh & Moore, 2008). PPARγ is most prominent in the retinal pigmented epithelium (RPE), photoreceptor outer segments, choriocapillaris, choroidal endothelial cells, corneal epithelium, and endothelium, and to a lesser extent, in the intraocular muscles, retinal
photoreceptor inner segments, outer plexiform layer, and the iris (Pershadsingh & Moore, 2008). The effect of the PPARγ ligands on retinal neovascularization was examined by Murata et al. (2001). The expression of PPARγ was confirmed in the retinal endothelial cells by Western immunoblotting, and the PPARγ ligands suppressed the VEGF-induced migration, proliferation, and tube formation by the retinal endothelial cells in vitro. In their in vivo study on the oxygen-induced ischemia model of retinal neovascularization which can be considered as an animal model of PDR, an intravenous injection of PPARγ ligands inhibited the development of the retinal neovascularization. The authors concluded that PPARγ ligands should be evaluated for their potential to inhibit the progression of PDR. Telmisartan and irbesartan were recently shown to belong to a unique subset of angiotensin receptor blockers (ARBs) and were capable of activating PPARγ (Schupp, Janke, Clasen, Unger, & Kintscher, 2004). Telmisartan was shown to down-regulate angiotensin II type 1 receptor through activation of PPARγ (Imayama et al., 2006). Telmisartan was also shown to have therapeutic benefits in rodent models of PDR (Nagai et al., 2007). Pershadsingh and Moore (2008) reported that telmisartan and irbesartan may improve the efficacy of treating PDR by activating PPARγ. On the other hand, there have been several studies showing that activation of PPARγ induces angiogenesis through the up-regulation of VEGF. Biscetti et al. (2013) demonstrated that cilostazol is able to induce angiogenesis in vivo in streptozotocin-induced diabetic mice by the up-regulation of pro-angiogenic genes and proteins. This phenomenon is dependent on PPARγ and may be suppressed by the systemic inhibition of PPARγ. Terrasi et al. (2013) reported that PPARγ agonists stimulated angiogenesis through the up-regulation of VEGF and leptin, and other yet unidentified proangiogenic factors. Biscetti et al. (2008, 2009) investigated angiogenic potentials of selective synthetic PPARα and PPARγ agonists in vitro and in vivo. In their study, activation of PPARα and PPARγ led to endothelial tube formation in an endothelial/interstitial cell co-culture assay, and this effect was associated with increased production of VEGF. In addition, PPARα and PPARγ agonists also induced neovascularization in the murine corneal angiogenic model. These results are consistent with our results that the PPARγ concentrations were significantly correlated with the VEGF concentrations in the aqueous humor and vitreous fluid of PDR patients.
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There is evidence that the expression of PPARγ is increased in patients with cancers, e.g., breast, prostate, pancreas, and colon, but it is decreased in others, e.g., esophagus and uterus cancers (Terrasi et al., 2013). The role of PPARγ in cancer progression remains controversial. While some studies demonstrated anti-proliferative role of PPARγ ligands in cellular and animal models of human cancers (Grommes, Landreth, & Heneka, 2004), other investigations showed that activation of PPARγ can induce cell growth and tumor proliferation (Talbert, Allred, Zaytseva, & Kilgore, 2008; Terrasi et al., 2013). One explanation for these discrepancies is that PPARγ is a multivalent receptor whose ligand binding domain can accommodate different PPARγ ligands (Pershadsingh & Moore, 2008). The PPARγ ligands, such as the full agonist, partial agonist, and selective PPARγ modulators, are capable of inducing distinct receptor combinations leading to selective gene expressions (Pershadsingh & Moore, 2008). For example, the thiazolidinediones, e.g., rosiglitazones, are full PPARγ agonist, while the ARBs, e.g., telmisartan or irbesartan, function as partial PPARγ agonists. Each ligand–receptor complex assumes a somewhat different three-dimensional conformation leading to unique and differential interactions with cofactors, histones, and other transcription factors. Consequently, each PPARγ ligand–receptor complex leads to a different and overlapping pattern of gene expression. Thus, each ligand will activate or repress multiple genes leading to a differential overlapped expression of different sets of genes. With respect to the two conflicting roles of PPARγ in PDR progression, Sassa et al. (2004) reported bifunctional properties of PPARγ in the regulation of the VEGF receptor gene. The VEGF receptor plays a critical role in mediating a variety of vasculogenic and angiogenic processes including PDR. PPARγ enhanced the expression of the gene of the VEGF receptor at the transcriptional level in the absence of its ligands in retinal capillary endothelial cells. The PPARγ ligands, however, suppressed the gene expression of VEGF receptor by decreasing the interaction between PPARγ and transcriptional factors Sp1/Sp3 (Sassa et al., 2004). They concluded that PPARγ had bifunctional properties in the regulation of gene expression of the VEGF receptor in retinal capillary endothelial cells. The PPARγ concentrations in the aqueous humor and vitreous fluid of PDR patients were significantly higher than those in the controls. In addition, there was a tendency toward higher PPARγ concentrations in PDR patients with increasing severity of fibrous proliferation. However, how this over-expression of PPARγ is involved in the pathogenesis of PDR remains unclear. Moreover, we cannot determine whether the increased PPARγ concentrations are the cause or the effect of PDR in this study. Further studies to investigate the precise molecular machanism by which PPARγ contributes to the pathogenesis of PDR are needed. In conclusion, intraocular expression of PPARγ was significantly higher in PDR patients than in controls subjects. Anti-VEGF therapy significantly reduced the VEGF concentration but not the PPARγ concentration. These findings suggest that PPARγ may play an important role in the pathogenesis of PDR by the independent mechanisms from those associated with VEGF. Acknowledgements There are no conflicts of interest. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (T. Katome, K. Namekata, Y. Mitamura, C. Harada, T. Harada), and the Funding Program for Next Generation World-Leading Researchers (NEXT Program) (T. Harada). References Abu El-Asrar, A. M., Missotten, L., & Geboes, K. (2007). Expression of hypoxia-inducible factor-1alpha and the protein products of its target genes in diabetic fibrovascular epiretinal membranes. British Journal of Ophthalmology, 91, 822–826.
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