Imbalanced levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of patients with proliferative diabetic retinopathy

Journal of Diabetes and Its Complications 26 (2012) 435–441

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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

Imbalanced levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of patients with proliferative diabetic retinopathy☆ Nithyakalyani Mohan, Finny Monickaraj, Muthuswamy Balasubramanyam, Mohan Rema, Viswanathan Mohan ⁎ Madras Diabetes Research Foundation and Dr. Mohans' Diabetes Specialities Centre, Gopalapuram, Chennai-600 086, India

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Article history: Received 31 March 2012 Received in revised form 4 May 2012 Accepted 4 May 2012 Available online 12 June 2012 Keywords: Proliferative diabetic retinopathy Vascular endothelial growth factor Erythropoietin Pigment epithelial derived factor Vitreous

a b s t r a c t A role for vascular endothelial growth factor (VEGF) has been clearly implicated in the pathogenesis of proliferative diabetic retinopathy (PDR). However, other molecules and mechanisms may be operating independently, or in conjunction with VEGF in the pathogenesis of this disease. Therefore, we made an attempt to comparatively investigate the levels of angiogenic and angiostatic factors in vitreous, plasma and postmortem retinal tissue of subjects with Proliferative Diabetic Retinopathy (PDR) compared to control subjects. The vitreous and plasma concentrations of VEGF, EPO (Erythropoietin) and PEDF (Pigment Epithelium Derived Factor) were measured using Enzyme Linked Immunosorbent Assay (ELISA) and the postmortem retinal tissue was subjected to Western blot analysis. The mean vitreous and plasma levels of VEGF and EPO in patients with PDR were significantly (p b 0.001) higher than those in subjects without diabetes. Conversely, the vitreous and plasma levels of PEDF were significantly (p b 0.001) lower in the PDR patients compared to control subjects. Multivariate logistic-regression analyses indicated that EPO was more strongly associated with PDR than VEGF. The protein expression of the VEGF and EPO in the retinal tissue was significantly higher in PDR and diabetes without complication groups compared to controls. Compared to controls, the protein expression of PEDF was significantly lower in retinal tissues from diabetes patients without complications and in patients with PDR. The fact that the vitreous and plasma levels and the retinal tissue protein expression of EPO were strongly associated with PDR implies a definite role of ‘hypererythropoietinemia' in neovascularization processes. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Intraocular neovascularization develops in numerous ischemic retinal disorders, such as diabetic retinopathy, ischemic retinal vein occlusion, and retinopathy of prematurity (Patz, 1980). In neovascular retinopathies such as proliferative diabetic retinopathy (PDR), there is subtle active and extensive proliferation of new vessels. This proliferation often leads to vitreous hemorrhage, retinal detachment, and neovascular glaucoma, with subsequent visual loss. A surgical procedure called vitrectomy can alleviate some of the complications of PDR but to a limited extent. The underlying mechanism of retinal neovascularization is not well understood, although it has been demonstrated that growth factors play an important part in the neovascularization associated with PDR (Paques, Massin, & Gaudric, 1997). In normal ocular tissues, angiogenic homeostasis is controlled by the balance between

☆ Conflicts of interest: None. ⁎ Corresponding author. Tel.: +91 44 4396 8888; fax: +91 44 2835 0935. E-mail address: [email protected] (V. Mohan). 1056-8727/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2012.05.005

various angiogenic stimulators, such as vascular endothelial growth factor (VEGF) and angiogenic inhibitors, such as pigment epithelium-derived factor (PEDF) (Dawson et al., 1999; Simó, Carrasco, García-Ramírez, & Hernández, 2006). Studies have shown that VEGF causes changes of the tight junction proteins of retinal vascular endothelial cells, and the role of VEGF in increasing vascular permeability in diabetic eyes is well established. Although inhibition of VEGF reduces retinal neovascularization (Aiello et al., 1995; Antonetti, Lieth, Barber, & Gardner, 1999; Bainbridge et al., 2002), it does not completely inhibit ischemia-driven retinal neovascularization. Thus, the involvement of other angiogenic factors in this process seems likely. Erythropoietin (EPO) stimulates the formation of red cells by enhancing both their proliferation and differentiation (Jelkmann, 1992) and hypoxia is a major signal that regulates the production of erythropoietin in these tissues. Erythropoietin shows an angiogenic activity in vascular endothelial cells, stimulating proliferation, migration, and angiogenesis in vitro, probably by means of the erythropoietin receptor expressed in those cells (Anagnostou et al., 1994). There is some evidence that erythropoietin is a potent retinal angiogenic factor independent of VEGF and is capable of

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stimulating ischemia-induced retinal angiogenesis in proliferative diabetic retinopathy (Watanabe et al., 2005). Pigment epithelium-derived factor (PEDF), is a member of the serine protease inhibitor family (Steele, Chader, Johnson, & Tombran-Tink, 1993). Recently, PEDF has been shown to be a highly effective inhibitor of angiogenesis in animal and cell culture models (Dawson et al., 1999; Stellmach, Crawford, Zhou, & Bouck, 2001). Production of PEDF is decreased by hypoxia, and hyperglycemia may influence the expression of PEDF in the eye. The biological activities of VEGF and PEDF are similar in some cases, but they have counterbalancing proangiogenic and antiangiogenic activities in situations such as retinal ischemia, proliferative diabetic retinopathy, and neovascular age-related macular degeneration (AMD) (Gao, Li, Zhang, Gee, Crosson, & Ma, 2001; Holekamp, Bouck, & Volpert, 2002; Spranger et al., 2001). The balance between the angiogenic and antiangiogenic factors rather than angiogenic factors alone will be crucial in determining the progression of PDR. There are reports but of isolated studies of vitreous, plasma and retinal tissue levels of angiogenic and angiostatic factors in patients with PDR. We here report on the VEGF, EPO and PDEF levels both comparatively and comprehensively in vitreous and plasma samples along with the protein expression of these growth factors in retinal tissues from patients with PDR compared to nondiabetic controls. 2. Materials and methods 2.1. Collection of human vitreous and plasma samples We conducted this study in accordance with the Declaration of Helsinki and received institutional approval from the ethics committee of the Madras Diabetes Research Foundation and the Regional Institute of Government Ophthalmic Hospital Egmore, Chennai. Undiluted vitreous fluid samples (0.3 to 0.5 ml) were harvested from the mid vitreous at the start of vitrectomy. An informed consent was obtained from all patients after an explanation of the purpose and procedures of the study. Undiluted vitreous samples were collected from 56 type 2 diabetes subjects with PDR who underwent vitrectomy for vitreous hemorrhage, retinal detachment and from 49 control subjects (non diabetic) who underwent vitrectomy for other reasons like macular hole. While majority of the PDR patients were on statins and/or ACE inhibitors along with oral antidiabetic drugs, 49% of the patients were on insulin in addition to other drugs. Vitreous and plasma samples were transported in ice, and then it was centrifuged at 5000 g for 15 min. The supernatant was then transferred on to cryovials and snap frozen in liquid nitrogen until analyzed. Ocular exclusion criteria included previous ocular surgery, intravitreal therapy, history of ocular inflammation and ophthalmic disorders associated with macular edema. Systemic exclusion criteria included ischemic cerebrovascular disorders, ischemic cardiovascular disorder, hepatic dysfunction and any other major systemic inflammatory diseases. None of the diabetes patients had CRF and no one was on erythropoietin. 2.2. Collection of human cadaver retinal eyes Human cadaver eyes were procured from the corneal eye bank of the Regional Institute of Government Ophthalmic Hospital (RIGOH), Egmore, Chennai, after the cornea was removed for transplant purposes. An order agreeing to supply the retina of human eyes for research purposes, which was left over after the corneal button was removed, was approved at the RIGOH. Retinal tissues were collected from non-diabetic subjects, which served as controls and type 2 diabetic subjects with and without PDR.

2.3. Measurement of growth factors by ELISA Concentrations of VEGF, EPO and PEDF in vitreous and plasma samples were measured by an enzyme-linked immunosorbent assay for human VEGF, EPO and PEDF (R & D Systems Inc., Minneapolis, Minnesota, USA). 2.4. Protein expression studies by western blot The postmortem retinal tissue (n = 10 each) from controls, diabetes without complications and patients with PDR were subjected to Western blot analysis for quantifying the protein expression patterns of various growth factors and their receptors. Retinal tissues were washed with cold PBS thrice and scraped into 100 μl RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1 mM EGTA, 1% triton-X 100, 1% Sodium deoxy cholate, 1% SDS and 1 μg/ml leupeptin), sonicated and centrifuged at 12,000 g for 15 min. The supernatant was collected and the protein was quantified using Nano drop. Aliquots of 50 μg protein were added with gel loading buffer (5 ×) and it was separated in 10% SDS-PAGE and then western blot was performed. Blots were then probed with respective primary and ALP conjugated secondary antibodies of the protein of interest and was developed using the NBT-BCIP method. All the test proteins were normalized to the βactin levels. Densitometry analysis of the protein expression was done using Image J software. 2.5. Statistics All statistical analyses were performed using SPSS for Windows version 10.0 software (SPSS Inc., Chicago, Illinois). Numbers were expressed as mean ± standard error. Differences among the groups were analyzed by paired Student's t-test or one way analysis of variance (ANOVA), followed by Fisher's test for individual comparisons between groups. Pearson correlation coefficient analysis was done to inter-relate erythropoietin, VEGF and PEDF levels in the proliferative diabetic retinopathy group. Multivariate logistic-regression analysis was done to associate the risk factors with disease status. The differences between the parameters were considered statistically significant at p b 0.05. 3. Results Of the total 105 subjects who underwent vitrectomy, 56 were type 2 diabetic patients with Proliferative Diabetic Retinopathy and 49 subjects were controls who underwent vitrectomy for other causes like macular hole, retinal detachment etc. The mean age (mean ± SE) of the non-diabetic subjects was 40 ± 0.74 years and 58 ± 1.0 years for the PDR subjects (Table 1). The mean duration of the diabetes mellitus in the PDR patients was 14 ± 0.8 years. The glycated hemoglobin levels in the controls (5% ± 0.4%) were significantly lower than in the PDR patients (10 ± 0.2). The age and duration of diabetes correlate significantly (p b 0.001) with the HbA1c in diabetes patients with PDR. The mean vitreous VEGF levels in patients with PDR were significantly higher than those in subjects without diabetes (919.22 vs 69.75 pg/ml, p b 0.001). The mean EPO levels in patients with PDR were also significantly higher compared to subjects without diabetes

Table 1 Clinical characteristics of the study subjects. Group (Nos)

Age (years) (Mean ± SE)

Gender M

F

Control (49) PDR (56)

40 ± 0.7 58 ± 1.0

24 25

25 31

Duration (yrs) (Mean ± SE)

HbA1c (%) (Mean ± SE)

14 ± 0.8

5.0 ± 0.4 10 ± 0.2

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Fig. 1. Levels of Angiogenic and Angiostatic Factors in the Vitreous of the Study Subjects; control (n = 49) and PDR (n = 56): A) VEGF B) EPO and C) PEDF. *p b 0.001 compared to control subjects.

(146.9 vs 7.76 mIU/ml, p b 0.001). Conversely, the vitreous levels of PEDF were lower in the PDR patients compared to the controls (25.7 vs 5.06 ng/ml, p b 0.001) (Fig. 1). The mean plasma VEGF and EPO levels in patients with PDR were significantly higher than those in subjects without diabetes (515 vs 41.5 pg/ml, p b 0.001) (135.9 vs 6.0 mIU/ml, p b 0.001). In contrast, the plasma levels of PEDF were lower

in the PDR patients compared to the controls (11.7 vs 4 ng/ml, p b 0.001) (Fig. 2). In the PDR group, the vitreous levels of angiogenic factors (VEGF, EPO) had a positive correlation with the clinical parameters (age, duration of diabetes and HbA1c) whereas the PEDF showed a negative correlation with the clinical parameters (Table 2). On comparison

Fig. 2. Levels of Angiogenic and Angiostatic Factors in the Plasma of the Study Subjects; control (n = 49) and PDR (n = 56): A) VEGF B) EPO C) PEDF. *p b 0.001 compared to control subjects.

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Table 2 Correlation of Vitreous levels of angiogenic/angiostatic factors with the clinical parameters. Clinical parameters V-Vitreous

V_VEGF r

p

r

V_EPO p

r

p

Age (yrs) Duration of DM (Yrs) HbA1c (%)

0.698 0.715 0.786

b 0.001 b 0.001 b 0.001

0.716 0.780 0.840

b0.001 b0.001 b0.001

− 0.690 − 0.729 − 0.788

b 0.001 b 0.001 b 0.001

Table 4 Logistic Regression Analysis showing the association of the Angiogenic and Angiostatic factors with Proliferative Diabetic retinopathy.

V_PEDF

between the two angiogenic factors, the vitreous level of VEGF correlated positively with the vitreous levels of EPO, and the vitreous levels of these two angiogenic factors correlated negatively with the levels of PEDF. Similar association of these growth factors was also observed in plasma (Table 3). Multivariate logistic regression analysis using PDR as dependent variable showed that VEGF and EPO were significantly associated with PDR even after adjusting for age and duration of diabetes mellitus. Odds ratio analysis revealed that compared to VEGF, EPO levels were strongly associated with PDR (Table 4). The protein expression of the VEGF and EPO was significantly increased in the retinal tissue from patients with PDR and in diabetic subjects without complications compared to the controls (Fig. 3). Conversely, the protein expression of PEDF was significantly lower in retinal samples from patients with PDR and in diabetics without complications compared to control group.

4. Discussion This study showed that vitreous VEGF levels were significantly elevated in patients with PDR compared to nondiabetic control subjects. Studies done earlier also reported elevated vitreous levels of VEGF in patients with proliferative retinopathy (Aiello et al., 1994). It has been reported that VEGF is localized in a number of retinal cells (Lutty, McLeod, Merges, Diggs, & Plouét, 1996) and it is well established that VEGF is a powerful inducer of increased vascular permeability (Senger, Galli, Dvorak, Perruzzi, Harvey, & Dvorak, 1983). The intravitreal injection of VEGF increases vascular permeability in rats (Aiello et al., 1997) and in primates (Tolentino et al., 1996). The mechanism that underlies the increase of VEGF production is uncertain. It is known to be upregulated by ischemia, advanced glycation end products, and insulin-like growth factor (Punglia et al., 1997; Scheiki, Itin, Soffer, & Keshet, 1992) while other factors have also been implicated. Since the inhibition of VEGF is not associated to total regression of retinal neovascularization secondary to PDR (Avery et al., 2006; Watanabe et al., 2005), it is important to realize that other angiogenic factors might as well play a role in retinal neovascularization processes in diabetic patients. EPO has been found to have actions beyond stimulation of red cell precursors, including pro-angiogenic functions. In our study, the vitreous Erythropoietin and VEGF levels correlated significantly

Factors V-Vitreous P-Plasma

Odds ratio (CI)

Significance

V_VEGF V_EPO V_PEDF P_VEGF P_EPO P_PEDF

1.020 1.912 0.089 1.046 2.880 0.099

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

(1.008–1.032) (1.022–2.010) (0.028–0.613) (1.010–1.083) (1.009–2.932) (0.023–0.428)

with the HbA1c in the patients with proliferative diabetic retinopathy. We also observed a strong correlation between the vitreous and plasma levels of erythropoietin and VEGF in patients with proliferative diabetic retinopathy. Erythropoietin may have a biphasic effect on endothelial proliferation much as VEGF does (Ashley, Dubuque, Dvorak, Woodward, Williams, & Kling, 2002; Chow, Ogunshola, Fan, Li, Ment, & Madri, 2001; Hamma-Kourbali et al., 2001). Erythropoietin in the vitreous of patients with proliferative diabetic retinopathy is bioactive and stimulates proliferation of BRECs (Watanabe et al., 2005). The blockade of erythropoietin has been shown to inhibit the stimulation of cell growth in vitro as efficiently as did VEGF. Our study is consistent with these results and we have shown similar increase in EPO levels from PDR patients in both vitreous and plasma samples. In this study, we found that vitreous EPO concentrations were markedly higher in PDR patients than in control non-diabetic patients who underwent vitrectomy for macular hole or retinal detachment. These results are consistent with those of Inomata, Hirata, Takahashi, Kawaji, Fukushima, and Tanihara (2004) and Katsura et al. (2005) who also reported that vitreous EPO concentrations are higher in PDR than in other ocular diseases. Katsura et al. (2005) found no significant correlation between intravitreous and serum concentrations of EPO and weak correlation between intravitreous concentrations of EPO and VEGF in patients with PDR. However in our study, there was a strong correlation between the levels of VEGF and EPO levels and there was also a strong and significant correlation between intravitreous and plasma concentrations of EPO in patients with PDR. Although increased systemic and vitreous EPO levels imply a possible breakdown of the blood–retinal barrier, it appears that EPO must also be produced locally in the retina. In fact, Hernández et al. (2006) found EPO concentrations in the vitreous 30 times higher than in serum in patients with PDR. Several reports show the efficacy of treatment with erythropoietin for various diseases. Correcting anemia with erythropoietin therapy may slow the progression of renal failure (Kuriyama, Tomonari, Yoshida, Hashimoto, Kawaguchi, & Sakai, 1997) and may reduce the progression of tumors (Glaspy & Dunst, 2004). Case studies of patients with diabetic nephropathy indicate that treating anemia with erythropoietin improves diabetic retinopathy (Ehrenreich et al., 2002; Friedman, Brown, & Berman, 1995)] and this may

Table 3 Correlation of Vitreous and Plasma levels of Angiogenic and Angiostatic factors in the PDR subjects. Factors V-Vitreous P-Plasma

V_VEGF r

p

r

V_EPO p

r

V_PEDF p

r

P_VEGF p

r

P_EPO p

r

P_PEDF p

V_VEGF V_EPO V_PEDF P_VEGF P_EPO P_PEDF

0.624 − 0.689 0.630 0.499 − 0.701

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

0.624 − 0.751 0.792 0.545 − 0.821

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

− 0.689 − 0.751 − 0.693 − 0.573 0.748

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

0.630 0.792 − 0.693 0.544 − 0.752

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

0.449 0.545 − 0.573 0.544 −0.534

b0.001 b0.001 b0.001 b0.001 b0.001

− 0.701 − 0.821 0.748 − 0.752 − 0.534 -

b0.001 b0.001 b0.001 b0.001 b0.001 -

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Fig. 3. Protein Expression pattern in retinal tissues of the study subjects (n = 10 in each group). A) Representative protein blots of VEGF, EPO, PEDF and β-actin. B) Cumulative protein expression data of VEGF, C) cumulative protein expression data of EPO and D) cumulative protein expression data of PEDF. A-Control, B-Diabetes without PDR, C-Diabetes with PDR. *p b 0.05 compared to controls; ** p b 0.001 compared to controls and diabetes without PDR, #p b 0.001 compared to controls.

be a confounded observation, since anemia is a risk factor for diabetic retinopathy (Sinclair, DelVecchio, & Levin, 2003). Although there is evidence that EPO plays a role in the pathogenesis of PDR, the interaction appears to be complex and not uniform. Chen, Connor, Aderman, and Smith (2008) showed in a mouse model of hypoxia-induced retinopathy that the administration of exogenous EPO during the early phase prevents vessel dropout and protects against hypoxia-induced retinal neuron apoptosis, whereas late EPO treatment enhances pathological neovascularization. Previous studies also found protective effects of EPO against photochemical and ischaemia/reperfusion injury of retinal cells (Grimm et al., 2002; Junk et al., 2002). Recently, McVicar, Hamilton, Colhoun, Gardiner, Brines, and Cerami (2011) have demonstrated that intervention with an erythropoietin-derived peptide protected against neuroglial and vascular degeneration during diabetic retinopathy. Wang, Zhao, and Zhao (2011) have also proposed that EPO therapy can be beneficial at least in early diabetic retinopathy through its protective effects on retinal pericytes. Therefore, further studies are needed to determine the tissue-specific effects of EPO and its bioactive role in the various phases of the progression of diabetic retinopathy. We found that the vitreous and plasma concentration of PEDF in the non-diabetic subjects was higher compared to the PDR group. The vitreous humor has been shown to have antiangiogenic properties (Dawson et al., 1999) and removal of pigment epithelium-derived factor from the vitreous fluid abrogates this antiangiogenic activity. Our findings indicate that PEDF is most likely associated with the protective metabolism in patients with diabetes mellitus and may be impaired during prolonged hyperglycemia. PEDF has been shown in several cell model systems to counteract VEGF and EPO and function beneficially at multiple levels in the retinal vasculature (Wang et al., 2010).

In our study, both plasma and vitreous levels of VEGF and EPO were shown to be increased several folds in patients with PDR compared to control subjects. Similarly PEDF levels were decreased several folds in patients with PDR compared to control subjects. It is unlikely that the observed results are influenced by medication in patients with PDR. The drug regimen such as statins or ACE inhibitors could have only decreased the growth factor levels (VEGF, EPO). The only concern is with use of insulin. Therefore, we have analyzed the whole data on the growth factors (both plasma and vitreous) in PDR patients into two groups, viz., those on insulin and those who were not on insulin and found that none of the growth factor levels were significantly altered in the presence of insulin (data not shown). Thus the strikingly increased levels of VEGF and EPO and decreased levels of PEDF in patients with PDR compared to control subjects in our study strongly indicate the imbalance of angiogenic and angiostatic factors in PDR. The switch to an angiogenic phenotype of proliferating tissues requires both up-regulation of angiogenic stimulators and downregulation of angiogenesis inhibitors. This has been consistently proved in our study wherein there was an increased protein expression of VEGF and EPO and downregulated expression of PEDF in retinal tissues from patients with PDR and diabetic patients without complications compared to control group. Michaelson (1948) and Ashton (1957) put forward the initial hypothesis that retinal neovascularization is triggered by a hypoxia induced vasoformative factor. Thus augmented expression of hypoxia induced angiogenic factor in the diabetic patient may tip the balance between angiogenesis stimulators and angiogenesis inhibitors known to be present in the vitreous and retina (Bensaid, Malecaze, Bayard, & Tauber, 1989; Chen & Chen, 1980; Glazer, D'Amore, Michels, Patz, & Fenselau, 1980; Lutty, Chandler, Bennett, Fait, & Patz, 1986; McIntosh, Gaal, & Forrester, 1989). Consistent with our results, García-Ramírez,

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Hernández, and Simó (2008) have found a higher protein expression of EPO in the retinas from diabetic donors in comparison with the retinas from nondiabetic donors. The mechanisms that regulate EPO expression in the human retina remain to be elucidated. Apart from hypoxia, other factors including hyperglycemia and hyperglycemiadriven cellular mediators could regulate EPO expression. A reduction in EPO catabolism could also contribute to the higher EPO levels detected in the retina and vitreous fluid from diabetic donors. In this regard, the glycosylation of EPO reduces its affinity for EPOR (Darling, Kuchibhotla, & Glaesner, 2002). Because EPO is degraded only by EPOR-expressing cells and their receptor binding determines the rate of intracellular degradation, it is possible that a higher degree of EPO glycosylation may dictate lower clearance of EPO. Alternatively, the association between EPO and DR could be originated by genetic polymorphisms. In fact, Tong et al. (2008) and Abhary, Burdon, Casson, Goggin, Petrovsky, and Craig (2010) have recently demonstrated association between erythropoietin gene polymorphisms and diabetic retinopathy. Therefore, a genetically determined ability of increased EPO synthesis and EPO levels might also predispose diabetic patients to the development of PDR. 5. Conclusion To conclude, our work is unique in that it has comprehensively demonstrated the imbalance of angiogenic and angiostatic factors by simultaneous analysis of plasma, vitreous and postmortem retinal tissues from patients with PDR. Our data suggest an association of ‘hypererythropoietinemia’ in addition to VEGF in the pathogenesis of proliferative diabetic retinopathy. This implies that perhaps, in the therapy of DR, modulating other angiogenic factors in addition to VEGF may lead to better results. A significant decrease in PEDF levels and expression in PDR also suggests that boosting the endogenous PEDF by therapeutic measures is also important in the treatment of DR. Acknowledgments Authors thank the Department of Biotechnology (DBT) and the Department of Science & Technology (DST), New Delhi, India for the financial assistance. References Abhary, S., Burdon, K. P., Casson, R. J., Goggin, M., Petrovsky, N. P., & Craig, J. E. (2010). Association between erythropoietin gene polymorphisms and diabetic retinopathy. Archives of Ophthalmology, 128, 102–106. Aiello, L. P., Avery, R. L., Arrigg, P. G., Keyt, B. A., Jampel, H. D., Shah, S. T., et al. (1994). Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. New England Journal of Medicine, 331, 1480–1487. Aiello, L. P., Bursell, S. E., Clermont, A., Duh, E., Ishii, H., Takagi, C., et al. (1997). Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective B-isoform-selective inhibitor. Diabetes, 46, 1473–1480. Aiello, L. P., Pierce, E. A., Foley, E. D., Takagi, H., Chen, H., Riddle, L., et al. (1995). Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proceedings of the National Academy of Sciences of the United States of America, 92, 10457–104561. Anagnostou, A., Liu, Z., Steiner, M., Chin, K., Lee, E. S., Kessimian, N., et al. (1994). Erythropoietin receptor mRNA expression in human endothelial cells. Proceedings of the National Academy of Sciences of the United States of America, 91, 3974–3978. Antonetti, D. A., Lieth, E., Barber, A. J., & Gardner, T. W. (1999). Molecular mechanisms of vascular permeability in diabetic retinopathy. Seminars in Ophthalmology, 14, 240–248. Ashley, R. A., Dubuque, S. H., Dvorak, B., Woodward, S. S., Williams, S. K., & Kling, P. J. (2002). Erythropoietin stimulates vasculogenesis in neonatal rat mesenteric microvascular endothelial cells. Pediatric Research, 51, 472–478. Ashton, N. (1957). Retinal vascularization in health and disease. American Journal of Ophthalmology, 44, 7–17. Avery, R. L., Pearlman, J., Pieramici, D. J., Rabena, M. D., Castellarin, A. A., Nasir, M. A., et al. (2006). Intravitreal bevacizumab (Avastin) in the treatment of proliferative diabetic retinopathy. Ophthalmology, 113, 1695–1705.

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