Aqueous VEGF and IL-6 Levels in Diabetic Retinopathy

Diabetes has reached epidemic proportions. There are 150 million diabetics worldwide with one third in developing countries. India has approximately 30 million diabetics and is ahead of China and USA. The prevalence of diabetic retinopathy among diabetics in India is 25% [1].

Many cytokines are implicated in the pathogenesis of diabetic retinopathy. Vascular Endothelial Growth Factor (VEGF) is an endothelial cell mutagen and vasopermeability factor [2-4]. Interleukin 6 (IL-6) by inducing the expression of VEGF increases angiogenesis and vasopermeability [2,5]. The VEGF and IL-6 levels are elevated in both vitreous and aqueous humor of patients with diabetes especially in the presence of proliferative diabetic retinopathy [2,6]. The major stimulus for VEGF secretion is retinal hypoxia [4]. The local secretion of VEGF in intraocular fluids is reported to be higher in patients with diabetic retinopathy than their serum levels [6,7]. Its activity as vascular permeability factor matches perfectly with the findings of leaky vessels in diabetic retinopathy [4]. Aqueous VEGF levels have been reported to increase by two folds in patients with non-proliferative diabetic retinopathy [6]. IL-6 level in the vitreous has been correlated with VEGF levels in diabetic macular edema and in various stages of diabetic retinopathy [7-10]. The aqueous and plasma levels of IL-6 have not been correlated with VEGF and severity of diabetic retinopathy. Hence our study paves the way in finding the marker which may help us to predict the risk for developing diabetic retinopathy. Early detection of the risk for proliferative retinopathy, especially in patients undergoing cataract surgery (which may accelerate the progression of retinopathy), may help to plan on close follow-up of these patients and early treatment preventing blinding complications of diabetic retinopathy.

The present study was undertaken to measure aqueous VEGF and IL-6 levels in eyes of diabetics with and without retinopathy undergoing cataract surgery and to assess the predictive value of aqueous VEGF and IL-6 levels in diabetic retinopathy.

The study was conducted at a tertiary care hospital in south India. Institute ethics committee approval was taken. Patients aged 40 years or above undergoing cataract surgery were recruited in the study. Patients with other retinal diseases, those with pseudoexfoliation, presence of uveitis and those on immunosuppressant therapy were excluded from the study. Study subjects were divided into three groups- Group I included non-diabetic subjects, Group II were diabetics without retinopathy while group III included subjects with retinopathy.

Demographic data was noted. A detailed ophthalmic evaluation was performed including dilated fundus evaluation to stage diabetic retinopathy. The severity of diabetic retinopathy was graded based on Early Treatment of Diabetic Retinopathy Study (ETDRS) criteria.

In diabetic patients serum blood glucose levels, glycosylated Hemoglobin (HbA1c) and lipid profile were assessed. All patients underwent extracapsular cataract extraction. Intraoperatively 0.2 ml aqueous humor (which otherwise is lost during any intraocular surgery) was withdrawn in a tuberculin syringe using 26 gauze needle, before entering the anterior chamber. Extracapsular cataract extraction with posterior chamber intraocular lens implantation was done. The aqueous sample collected was immediately transferred and stored at -80°C until assay of VEGF and IL-6 levels was done.

Enzyme-linked Immunosorbent Assay (ELISA) of aqueous humor samples was performed to quantify the levels of VEGF with the Human VEGF ELISA Kit (Ray Biotech Inc., Norcross, GA, United States of America), which was designed to measure VEGF level with a sensitivity of less than 10 pg/ml. The intra-assay Co-efficient of Variation (CV) was <10% and inter-assay CV was <12%. Each sample was assayed in duplicate and mean of the measurements was taken to increase validity of the assay.

Enzyme-linked Immunosorbent Assay (ELISA) of aqueous humor samples was performed to quantify the levels of IL-6 with the Avibion Human IL-6 ELISA kit (Orgenium Laboratories, Helsinki, Finland), which was designed to measure IL-6 level with a sensitivity of less than 2 pg/ml. The intra-assay CV was ≤9.4% and inter-assay CV was ≤8.6%. Each sample was assayed in duplicate and mean of the measurements was taken to increase validity of the assay. The association of aqueous VEGF and IL6 levels to the stage of diabetic retinopathy was assessed using ANOVA test.

A total of 140 patients were recruited in our study. The group 1 (non-diabetic controls) included 60 patients, group 2 (diabetics without retinopathy) included 53 patients while 27 were recruited into the group 3 (diabetics with retinopathy). The mean age was 55 in groups 1 and 2, while it was 59 in group 3. There were 71 males and 59 female patients in the study. Comparison of the three groups is shown in table 1.

	Group 1 (Non diabetic control)	Group 2 (Diabetic patients without retinopathy)	Group 3 (Diabetics with retinopathy)	Total
Number of subjects	60	53	27	140
Mean age	55	55	59	55.8
Males	31	25	15	71
Females	29	28	12	69
Mean IL-6 levels average (pg/ml)	42.01	161.66	310.98	139.67
Mean VEGF levels (pg/ml)	130.69	131.69	397.6	182.54

Table 1: Comparison of three group patients.

The duration of diabetes mellitus in group 2 varied from one month to 27 years with an average of 6.07 years. In group 3, duration of diabetes varied from 15 days to twenty years with an average of 7 years. The levels of IL-6 had a negative correlation with the increase in duration of diabetes in both group 2 and 3, though it was not statistically significant (p=0.0805 for group 2 and p=0.0266 for group 3). In group 2, Spearman r=-0.2422 with 95% confidence interval of -0.4874 to 0.03827 while in group 3, Spearman r=-0.3168 with 95% confidence interval of -0.5551 to -0.03051. Aqueous VEGF levels showed a negative correlation on Spearman correlation analysis and was statistically significant in group 2 (p=0.0165) while in group 3, it was not statistically significant (p=0.1177). In group 2, Spearman r=-0.3596 with 95% confidence interval: -0.5991 to -0.06117. In group 3, Spearman r=-0.269 with 95% confidence interval of -0.6147 to 0.163.

IL-6 levels in non-diabetic controls varied from 22.2 to 69.05 pg/ml with an average of 41.88 pg/ml; in diabetics without retinopathy it varied from 117.6 to 190 pg/ml with an average of 161.65 pg/ml and in diabetics with retinopathy, values ranged between 278 to 355 pg/ml with an average of 310 pg/ml (p value <0.0001). VEGF levels in non-diabetic controls varied from 22.2 to 69.05 pg/ml with an average of 42 pg/ml; in diabetics without retinopathy, from 117.6 to 190 pg/ml with an average of 117.59 pg and in diabetics with retinopathy, values were between 278 to 355 pg/ml with an average of 310 pg/ml (p value <0.0001). ANOVA test revealed that the IL-6 and VEGF levels were higher in diabetic patients especially those with retinopathy. Kruskal-Wallis test was found to be extremely significant (p -value was <0.0001).

However, the levels showed a negative correlation with ETDRS grades of diabetic retinopathy and were statistically not significant. In group 3, there were fifteen patients with mild retinopathy, six with moderate and four with severe diabetic retinopathy while two patients had proliferative diabetic retinopathy.

Our study showed higher levels of aqueous IL-6 and VEGF especially in those with retinopathy. Prior studies showed similar increase in cases of diabetics with macular edema [2,5-7]. Vascular endothelial growth factors are implicated in the pathogenesis of diabetic retinopathy. The VEGF levels in the aqueous and vitreous are elevated in diabetics [7,11]. The rise in the levels of VEGF may correlate with the onset and progression of diabetic retinopathy. However, we did not find statistically significant change in the VEGF levels in patients with various degrees of diabetic retinopathy. A recent study showed that there is a decline in inflammatory and angiogenic markers namely IL-6 and VEGF with the onset of proliferative diabetic retinopathy changes [12]. The reduction in angiogenic factors with worsening of retinopathy may explain the role of VEGF in initiating the proliferative changes and systemic worsening [13]. However, the final end point is secondary to the sequel arising out of the fibrovascular proliferation. A larger study is required as the sample size in the subsets of diabetic retinopathy group was small to draw conclusions. The increase in VEGF levels may thus be an early indicator for predicting the patients developing diabetic retinopathy. However, the role of VEGF levels and progression of diabetic retinopathy is not well established.

IL-6 is a cytokine which is implicated in increased vascular permeability and thus considered to be important in pathogenesis of diabetic retinopathy. IL-6 has shown a paracrine effect on VEGF by increasing the vascular permeability. IL-6 levels were increased in diabetic patients especially in those with retinopathy. Blood retinal barrier breakdown is implicated in the pathogenesis of diabetic retinopathy and increased release of inflammatory cytokines is believed to be resulting in macular edema. The rise in VEGF and IL-6 levels may help us in early detection of diabetic retinopathy changes, thereby reducing the incidence of vision threatening complications of diabetic retinopathy.

There is an increase in the IL-6 and VEGF levels in diabetic patients as compared to non-diabetic controls. Further, the increase in IL-6 and VEGF levels are more in diabetics with retinopathy. This may help us to predict the risk for developing diabetic retinopathy. The levels may reduce with the worsening of retinopathy to proliferative stage.