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ORIGINAL ARTICLE
Year : 2007  |  Volume : 14  |  Issue : 1  |  Page : 3-6 Table of Contents     

Vitreous, aqueous, and serum levels of vascular endothelial growth factor and angiopoietin-2 in patients with proliferative diabetic retinopathy and diabetic macular edema


1 Department of Clinical and Chemical Pathology, Giza, Egypt
2 Department of Ophthalmology, Research Institute of Ophthalmology, Giza, Egypt

Date of Web Publication11-Nov-2009

Correspondence Address:
Hoiyda A Abdel Rasol
Research Institute of Ophthalmology, 3 Refaa Street, El Dokki, Giza 12611
Egypt
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Source of Support: None, Conflict of Interest: None


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   Abstract 

Purpose. Proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) may be a response to abnormal angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2. The purpose of this study is to compare vitreous, aqueous, and serum levels of VEGF and Ang-2 in diabetic patients who have PDR and DME with those of nondiabetic patients, and to identify correlations between the vitreous, aqueous, and serum levels of these angiogenic cytokines.
Patients and Methods. Aqueous humor, vitreous fluid, and serum samples were obtained during cataract and vitreous surgery from 16 eyes of 16 patients with PDR (10 eyes) and DME (6 eyes), as well as from 8 eyes of 8 nondiabetic patients with macular holes, and analyzed for levels of VEGF and Ang-2.
Results.
The mean vitreous level of VEGF was significantly higher in the samples from patients with PDR (182.1 ± 19.0 ng/mL) and from patients with DME (152.2 ± 46.3 ng/mL) when compared to that of the samples from the control group (63.0 ± 37.2 ng/mL) (P<.001; P<.01, respectively). The mean vitreous level of Ang-2 was significantly higher in patients with PDR (369.3 ± 352.2 pg/mL) and in patients with DME (495.3 ± 434.7 pg/mL) than in the control group (28.6 ± 20.9 pg/mL) (P < .001; P<.05, respectively). There was a significant correlation between vitreous and aqueous levels of VEGF (r) = 0.985; P<.001), but not between vitreous and aqueous levels of Ang-2 (r) = 0.087; P = not significant [NS]). There was no correlation between glycosylated hemoglobin (HbA 1c ) and intraocular levels of VEGF (vitreous and aqueous) or Ang-2.
Conclusion. VEGF and Ang-2 are significantly elevated in vitreous and aqueous humor in the eyes of patients with PDR and DME when compared to control eyes of patients without diabetes. Among diabetics, there is no correlation between HbA 1C and intraocular levels of VEGF or Ang-2. Correlations do exist between the vitreous, aqueous, and serum levels of VEGF, but not of Ang-2, in patients with PDR and DME. Measuring the aqueous humor level of VEGF may be useful in analyzing the pathogenesis of PDR and in predicting disease activity.

Keywords: angiopoietin, diabetes, macular edema, proliferative diabetic retinopathy, vascular endothelial growth factor


How to cite this article:
Abdel Rasol HA, Azab A. Vitreous, aqueous, and serum levels of vascular endothelial growth factor and angiopoietin-2 in patients with proliferative diabetic retinopathy and diabetic macular edema. Middle East Afr J Ophthalmol 2007;14:3-6

How to cite this URL:
Abdel Rasol HA, Azab A. Vitreous, aqueous, and serum levels of vascular endothelial growth factor and angiopoietin-2 in patients with proliferative diabetic retinopathy and diabetic macular edema. Middle East Afr J Ophthalmol [serial online] 2007 [cited 2019 Sep 19];14:3-6. Available from: http://www.meajo.org/text.asp?2007/14/1/3/57682

Proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) are common microvascular complications in patients with diabetes, which may have a debilitating impact on visual acuity and eventually lead to blindness. [1] Angiogenesis is the major feature of PDR. The factor that stimulates the growth of retinal vessels has not been fully identified; however, circumstantial evidence indicates that potent angiogenic cytokines, such as vascular endothelial growth factor (VEGF), may play a role. [2] Several types of retinal cells are known to produce VEGF. [3]

Indeed, VEGF has been shown to be responsible for the initiation of angiogenesis through the recruitment and proliferation of endothelial cells, a process that angiopoietin(Ang)-1 potentiates. [4] There are 4 known angiopoietins (angiopoietins 1-4). [5] Ang-1 exerts its biologic effect by binding to Tie-2, including phosphorylation of Tie-2. Ang-2 binds to Tie-2 with an affinity similar to that of Ang-1 but does not stimulate its phosphorylation. Ang-2 is antagonistic to Ang-1 and, thus, leads to endothelial destabilization, which in turn results in angiogenesis in the presence of VEGF. [6] Ang-2 has been reported to be markedly upregulated and more associated with pathologic angiogenesis. Its expression was found in human choroidal neovascular membrane [7] and in human epiretinal membranes of proliferative retinopathy. [8]

Several studies have shown that the expression of many cytokines is increased in diabetes and its level is increased in ocular fluids. [3] ,[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] It has been suggested that the activity or severity of diabetic retinopathy can be assessed by measuring VEGF and Ang-2 levels in aqueous or vitreous samples. [9] Because surgical harvesting of vitreous samples is associated with a small risk of vitreous hemorrhage, retinal detachment, and endophthalmitis, it is preferable to perform the less risky harvesting of aqueous samples. This recommendation can be made if a correlation can be demonstrated between vitreous and aqueous levels of VEGF and Ang-2. To determine whether correlations exist between the vitreous, aqueous, and serum levels of VEGF and Ang-2, we obtained samples from all 3 sources in patients with PDR and DME at the beginning of combined cataract and vitrectomy procedures in eyes with these disorders. Age-matched nondiabetic patients with macular holes were used as controls.


   Patients and Methods Top


Patients with PDR, DME, and macular holes who were scheduled for planned combined phacoemulsification and pars plana vitrectomy in conjunction with treatment of their vitreoretinal pathology were considered for inclusion in this prospective study. Patients with rubeosis iridis, with or without neovascular glaucoma, were excluded from the study. After providing a thorough explanation of the nature of the study and obtaining written informed consent, patients were enrolled in the study.

At the time of surgery, samples of undiluted aqueous humor were collected in sterile tubes at the beginning of phacoemulsification, and undiluted vitreous samples were collected in sterile tubes at the beginning of pars plana vitrectomy. Samples that were obtained in eyes in which zonular dialysis or other major complications occurred were excluded from statistical analysis. Samples were centrifuged at 3000 rpm for 5 minutes to separate the cell contents and then rapidly frozen at -70ΊC. An EDTA blood sample was taken for determination of glycosylated hemoglobin (HbA 1c ). Blood samples were allowed to clot overnight at 4ΊC and then centrifuged for 10 minutes at 3000 rpm. Serum was removed and stored at-20ΊC. Multiple freeze-thaw cycles were avoided.

The VEGF levels were measured using a competitive enzyme immunoassay (ACCUCYTE , CytImmune Sciences, Inc., College Park, MD, USA). The Ang-2 levels were determined using a sandwich enzyme immunoassay (Research and Diagnostic Systems, Inc., Minneapolis, MN, USA). The VEGF and Ang-2 measurements were performed according to the protocols supplied by the manufacturers.

Recent glucose control was assessed by using a cation exchange chromatography method to measure the HbA 1c . The procedure is a microchromatographic methodology for the quantification of glycosylated hemoglobin (nondiabetic reference, 5.5%-7.7%) (Glyco Hb Quik Column Kit;Helena Laboratories, Beaumont, TX, USA ).

A statistical software package (SPSS version 10.0) was used for tabulation and statistical analysis. Data were subjected to the Kolmogorov-Smirnov test to determine the distribution and method of analysis. Because of the skewed distribution, results were analyzed statistically using a nonparametric test (Mann-Whitney U test) and were expressed as mean ± SD when comparing groups. To examine the correlation, the Spearman rank correlation coefficients were calculated by linear regression analysis. A P value (two-tailed) less than .05 was considered statistically significant.


   Results Top


This study included 16 diabetic patients (10 with PDR and 6 with DME), as well as 8 age- and sex-matched nondiabetic patients with macular holes. Vitreous, aqueous, and serum samples of VEGF and Ang-2 were taken from these patients and included in the statistical analysis [Table 1].

The mean vitreous level of VEGF was significantly higher in the samples from patients with PDR (182.1 ± 19.0 ng/mL) and from patients with DME (152.2 ± 46.3 ng/mL) when compared to that of the control group (63.0 ± 37.2 ng/mL) (P<.001; P<.01, respectively). Serum VEGF levels in both patients with PDF and those with DME (137.4 ± 42.0 ng/mL; 153.5 ± 30.7 ng/mL, respectively) were also significantly higher compared to controls (49.5 ± 30.5 ng/mL) (P<.05; P<.01, respectively).

The mean vitreous level of Ang-2 was significantly higher in patients with PDR (369.3 ± 352.2 pg/mL) and in patients with DME (495.3 ± 434.7 pg/mL) than in the control group (28.6 ± 20.9 pg/mL) (P<.001; P<.05, respectively). Serum Ang-2 levels in both patients with PDR and those with DME (644.8 ± 434.5 pg/mL; 1179.5 ± 757.8 pg/mL, respectively) were also significantly higher compared to controls (152.8 ± 40.6 pg/mL) (P<.05; P<.01, respectively).

There was a significant correlation between the vitreous and aqueous levels of VEGF (r = 0.985; P<.001), but not between the vitreous and aqueous levels of Ang-2 (r = 0.087, P = not significant [NS]) [Table 2],[Table 3]. There was no correlation between HbA 1c and intraocular levels of VEGF (vitreous and aqueous) or Ang-2.


   Discussion Top


This study confirmed that levels of VEGF and Ang-2 are significantly elevated in vitreous and aqueous humor in the eyes of patients with PDR and of those with DME when compared to control eyes of patients without diabetes, thus further supporting the role of these molecules in the pathogenesis of diabetic retinopathy. Of the patients with diabetes, correlations were found to exist between the vitreous, aqueous, and serum levels of VEGF (but not of Ang-2) in patients with PDR and also in those with DME. There was no correlation between HbA 1C and the intraocular levels of VEGF or Ang-2, as reported in previous studies. [9] ,[19]

To rule out the influence of VEGF and Ang-2 originating in the serum, we compared the VEGF and Ang-2 levels in the serum to those in the vitreous fluid. The VEGF level was higher in the vitreous fluid than in the serum of diabetic eyes with PDR, but not with DME, whereas Ang-2 levels were higher in the serum of diabetic eyes with PDR and of those with DME. These results suggest that intravitreal VEGF does not originate in serum but rather is generated locally in eyes with PDR. Our results are supported by previous studies, which have indicated that intraocular synthesis is the main factor contributing to high levels of VEGF in eyes with PDR. [17] ,[18]

There are several important implications for the correlation between vitreous and aqueous levels of VEGF that were observed in this study. The occurrence of anterior segment neovascularization in association with retinal ischemia may be related to a vitreous-to-aqueous diffusion gradient, as initially suggested by Aiello et al. [3] Our study supports this hypothesis because levels of vitreous VEGF were always higher than those of aqueous VEGF. This gradient may be maintained by rapid clearance of VEGF from the anterior chamber or by more rapid degradation in aqueous than in vitreous humor. [19] For diagnostic purposes, aqueous samples can be collected and assayed for VEGF, without subjecting patients to the higher risks of vitreous aspiration, to evaluate angiogenicactivity in eyes with PDR.

 
   References Top

1.Ciulla TA, Amador AG, Zinman B. Diabetic retinopathy and diabetic macular edema: pathophysiology, screening, and novel therapies. Diabetes Care 2003;26:2653-2664.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]  
2.Funatsu H, Yamashita H, Nakanishi Y, Hori S. Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Br J Ophthalmol 2002;86:311-315.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994;331:1480-1487.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]  
4.Chung NA, Makin AJ, Lip GY. Measurement of the soluble angiopoietin receptor tie-2 in patients with coronary artery disease: development and application of an immunoassay. Eur J Clin Invest 2003;33:529-535.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]  
5.Koh GY, Kim I, Kwak HJ, et al. Biomedical significance of endothelial cell specific growth factor, angiopoietin. Exp Mol Med 2002;34:1-11.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 1999;284:1994-1998.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Otani A, Takagi H, Oh H, et al. Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1999;40:1912-1920.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
8.Watanabe D, Suzuma K, Suzuma I, et al. Vitreous levels of angiopoietin 2 and vascular endothelial growth factor in patients with proliferative diabetic retinopathy. Am J Ophthalmol 2005;139:476-481.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]  
9.Funatsu H, Yamashita H, Noma H, et al. Aqueous humor levels of cytokines are related to vitreous levels and progression of diabetic retinopathy in diabetic patients. Graefes Arch Clin Exp Ophthalmol 2005;243:3-8.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]  
10.Adamis AP, Miller JW, Bernal MT, et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994;118:445-450.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]  
11.Burgos R, Simo R, Audi L, et al. Vitreous levels of vascular endothelial growth factor are not influenced by its serum concentrations in diabetic retinopathy. Diabetologia 1997;40:1107-1109.  Back to cited text no. 11      
12.Noma H, Funatsu H, Yamashita H, et al. Regulation of angiogenesis in diabetic retinopathy: possible balance between vascular endothelial growth factor and endostatin. Arch Ophthalmol 2002;120:1075-1080.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]  
13.Ogata N, Nishikawa M, Nishimura T, et al. Unbalanced vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic retinopathy. Am J Ophthalmol 2002;134:348-353.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
14.Simo R, Lecube A, Segura RM, et al. Free insulin growth factor-I and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Am J Ophthalmol 2002;134:376-382.  Back to cited text no. 14      
15.Patel JI, Hykin PG, Gregor ZJ, et al. Angiopoietin concentrations in diabetic retinopathy. Br J Ophthalmol 2005;89:480-483.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]  
16.Mandriota SJ, Pepper MS. Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ Res 1998;83:852-859.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]  
17.Lip PL, Chatterjee S, Caine GJ, et al. Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy: effects of laser photocoagulation and angiotensin receptor blockade. Br J Ophthalmol 2004;88:1543-1546.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]  
18.Itakura H, Kishi S, Kotajima N, Murakami M. Persistent secretion of vascular endothelial growth factor into the vitreous cavity in proliferative diabetic retinopathy after vitrectomy. Ophthalmology 2004;111:1880-1884.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]  
19.Funatsu H, Yamashita H, Ikeda T, et al. Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology 2003;110:1690-1696.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]  



 
 
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  [Table 1], [Table 2], [Table 3]



 

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