High vitreous concentration of vascular endothelial growth factor in diabetic patients with proliferative retinopathy using statins

Abstract

Background. Vascular endothelial growth factor (VEGF) plays an important role in the development of diabetic retinopathy. Previous studies have suggested that angiotensin‐converting enzyme (ACE) inhibitor therapy may reduce vitreous VEGF concentration in diabetic retinopathy. Also HMG CoA reductase inhibitors (statins), known for their beneficial effects on vascular endothelium, might influence vitreous VEGF concentration in diabetic retinopathy.

Aim. Vitreous VEGF concentration of diabetic patients with proliferative retinopathy using statin therapy and/or ACE inhibitor therapy was studied.

Methods. Fifty diabetic patients with proliferative diabetic retinopathy, 21 diabetic control patients without proliferative retinopathy, and 43 non‐diabetic control subjects underwent vitrectomy. Vitreous samples were collected at the beginning of surgery, and VEGF concentration was assessed using a chemiluminescent VEGF immunoassay.

Results. Vitreous VEGF concentration was significantly higher in diabetic patients with proliferative retinopathy using statins than in those not using statins. The diabetic patients with proliferative retinopathy were divided into subgroups according to use of ACE inhibitor and/or statin medication. These groups did not differ significantly in concentration of vitreous VEGF.

Conclusions. Statin therapy is associated with high vitreous VEGF concentration in diabetic patients with proliferative retinopathy. In contrast to previous reports, ACE inhibitor use did not significantly influence vitreous VEGF concentration in these patients.

• Diabetes
• diabetic complication
• proliferative retinopathy
• retinopathy

Statins	=	HMG CoA reductase inhibitors
VEGF	=	vascular endothelial growth factor

Introduction

In diabetic retinopathy, angiogenesis and increased permeability of retinal microvascular vessels are mediated through the action of vascular endothelial growth factor (VEGF). Increased concentrations of VEGF in vitreous reflect retinal VEGF production and are associated with proliferative retinopathy as well as non‐proliferative retinopathy. Several medical therapies for diabetic retinopathy have been investigated, but apart from tight glycaemic control and blood pressure control no preventive medication is hitherto introduced 1. The effect of intravitreal VEGF antibody therapy in severe diabetic retinopathy is under intensive investigation.

The renin‐angiotensin system is activated in the eye and high concentrations of angiotensin II in diabetic vitreous are associated with high concentrations of vitreous VEGF 2. A previous study has suggested that angiotensin‐converting enzyme (ACE) inhibitor use is associated with decreased concentration of vitreous VEGF in proliferative diabetic retinopathy in a dose‐dependent manner 3. However, the effect of ACE inhibitors on the progression of diabetic retinopathy is under debate, since a protective effect on developing proliferative retinopathy was found in an EUCLID Study 4 but not in other studies 5.

In addition to their cholesterol‐lowering effect, statins have beneficial effects on several pathologic conditions, including diabetes. The anti‐angiogenic effect of statins has been elucidated 6, but the action seems to be biphasic; high doses of statins being anti‐angiogenic and low therapeutic doses have been found to increase endothelial cell proliferation 7. Statins also reduce expression of VEGF through down‐regulation of several transcription factors involved in VEGF expression 8.

Both ACE inhibitors and statins are beneficial and widely used in diabetic patients; therefore, it is essential to investigate their action on VEGF in diabetic retinopathy. We hypothesized that vitreous VEGF concentrations are lower in the diabetic patients with proliferative retinopathy using statin and/or ACE inhibitor medication.

Key messages

• Statin therapy is associated with increased vitreous vascular endothelial growth factor (VEGF) concentration in diabetic patients with proliferative retinopathy.

• The potentially protective effect of statins on diabetic retinopathy in previous studies may not have been mediated through a decrease in vitreous VEGF.

Materials and methods

Subjects

The study population consisted of 50 diabetic patients with proliferative retinopathy, 21 diabetic control patients without proliferative retinopathy, and 43 consecutive non‐diabetic control subjects who underwent vitreous surgery in Oulu University Hospital, Oulu, Finland. Three diabetic patients had vitrectomy for both eyes; therefore the total number of eyes was 53 in diabetic patients with proliferative retinopathy group. The study was performed in accordance with the declaration of Helsinki, and the study design was approved by the Ethical Committee of The Northern Ostrobothnia Hospital District. All study subjects gave informed consent before the study. None of the diabetic patients or non‐diabetic control subjects had end stage renal failure.

The clinical characteristics of the non‐diabetic control subjects and diabetic patients are shown in Table I. In non‐diabetic control subjects the indications for surgery were: retinal detachment (n = 17), vitreous opacities (n = 4), macular pucker (n = 8), macular hole (n = 5), prior vitreous haemorrhage (n = 7), and intra‐ocular lens luxation (n = 2). In diabetic patients without proliferative retinopathy, the indications for surgery were retinal detachment (n = 8), macular hole (n = 2), prior vitreous haemorrhage (n = 3), and diabetic maculopathy (n = 8). The patients were interviewed, and their medications, diabetic complications, and the age of diabetes onset were recorded. On the same day the ophthalmologist performed a routine preoperative eye examination including visual acuity, intra‐ocular pressure, and ocular fundus examination.

Table I. Clinical characteristics of diabetic patients and non‐diabetic control subjects. The data are expressed as means and standard deviation in parentheses, except for vitreous VEGF, which is presented as median and range.

	Non‐diabetic control subjects		Diabetic patients without proliferative retinopathy		Diabetic patients with proliferative retinopathy
	Mean (SD)	n	Mean (SD)	n	Mean (SD)	n
Age (years)	65.5 (14.4)	43	58.3 (15.6)	21	57.3 (13.8)	50
Gender (M/F)	18/25	43	8/13	21	25/25	50
Fasting plasma glucose (mmol/L)	5.2 (0.7)	43	8.7 (7.4)	21	9.5 (4.2)	49
Glycated haemoglobin (%)	5.56 (0.49)	43	8.4 (3.1)	21	8.8 (1.6)	49
Duration of diabetes (years)	‐	‐	7.7 (1.8)	21	19.1 (9.9)	50
Systolic blood pressure (mmHg)	148 (20)	43	154 (18)	21	155 (23)	48
Diastolic blood pressure (mmHg)	86 (11)	43	84 (10)	21	84 (12)	48
Plasma cholesterol (mmol/L)	5.15 (1.03)	43	4.67 (1.07)	21	4.90 (1.06)	50
LDL cholesterol (mmol/L)	3.33 (0.89)	43	2.78 (0.96)	21	2.94 (0.89)	50
HDL cholesterol (mmol/L)	1.41 (0.40)	43	1.33 (0.41)	21	1.24 (0.31)	50
Triglycerides (mmol/L)	1.38 (0.60)	43	1.83 (0.85)	21	1.99 (1.21)	49
Creatinine (μmol/L)	78 (21)	43	75 (25)	21	92 (49)	50
Plasma VEGF (pg/mL)	172 (101)	43	127 (85)	20	163 (91)	49
Vitreous VEGF (pg/mL):		42		21		53
Mean (SD)	48 (112)		424 (753)		1383 (1534)
Median (range)	9 (0–631)		119 (9–3351)		744 (117–7110)

LDL = low‐density lipoprotein; HDL = high‐density lipoprotein; VEGF = vascular endothelial growth factor

Sample collection

Vitreous collection was performed at the beginning of the classic three‐port pars plana vitrectomy before starting intravitreal infusion with balanced salt solution. Undiluted vitreous samples (200–500 µL) were aspirated into a tuberculin syringe attached into a vitreous cutter and immediately refrigerated after collection. Vitreous was aliquoted, transferred into sterile tubes, and frozen at −70 °C.

Venous blood samples were obtained after an overnight fast on the first postoperative day. Plasma was separated out by centrifuging at +4 °C and stored at −70 °C for VEGF measurements.

Determination of vitreous VEGF concentration

Plasma and vitreous VEGF concentrations were assessed using a QuantiGlo Chemiluminescent human VEGF immunoassay (cat. no QVE00B, R&D Systems, Minneapolis, MN, USA). The assay was performed according to the manufacturer's instructions. The detection limit of the assay is 6.4 pg/mL, and the assay is linear up to 20,000 pg/mL. According to the manufacturer, the intra‐assay coefficient of variation was 4.0% and the inter‐assay coefficient of variation 7.9%.

Statistical analyses

The statistical analyses were carried out using SPSS version 14.0 (Chicago, IL, USA). The statistical difference between the means was assessed by independent samples two‐tailed Student's t‐test or analysis of variance (ANOVA), except for vitreous VEGF in which logarithmic transformation was used for comparison between two groups and Kruskal‐Wallis test for comparison between three or more groups. Linear regression analysis was used to calculate the effect of diabetes variables such as diabetes duration or glycaemic control on vitreous VEGF concentration. The data are expressed as means and standard deviations (SD) or medians and range (for skewed distributions). P‐values<0.05 were considered significant.

Results

The clinical characteristics of the diabetic patients and non‐diabetic control subjects are shown in Tables I and II. In diabetic patients with proliferative retinopathy, there were no significant changes in fasting plasma glucose, glycated haemoglobin, diabetes duration, blood pressure, body mass index, age, or diabetes type between the users or non‐users of ACE inhibitors (ACE+ or ACE‐, respectively), or users or non‐users of statin medication (Statin+ or Statin‐, respectively) (t‐test) (data not shown). Vitreous VEGF concentration was significantly higher in the Statin+ group than the in Statin‐ group (medians 1361 pg/mL versus 666 pg/mL, P = 0.006, respectively) (Figure 1), but did not differ significantly between the groups ACE+ versus ACE‐ (medians 703 versus 744, respectively, P = 0.98) (Figure 1). Vitreous VEGF concentration was also higher in non‐diabetic control subjects using statins than in those not using statins (100 pg/mL versus 34 pg/mL, respectively, P = 0.001, data not shown).

Table II. Clinical characteristics of the diabetic subjects.

	Diabetic patients with proliferative retinopathy	Diabetic patients without proliferative retinopathy
	n (%)	n (%)
Diabetes type:
Type 1	26 (52%)	3 (14%)
Type 2	24 (48%)	18 (86%)
Medication:
Insulin	38 (76%)	9 (43%)
Oral diabetes medication	19 (38%)	9 (43%)
ACE inhibitor	29 (56%)	11 (52%)
Statin	15 (30%)	10 (48%)
Complications:
Nephropathy (mild)	19 (38%)	4 (19%)
Neuropathy	17 (34%)	3 (14%)
Laser:
Macular laser	8 (16%)	9 (43%)
Panretinal laser	42 (84%)	3 (14%)

ACE = angiotensin‐converting enzyme

Figure 1 Vitreous VEGF concentration in diabetic patients with proliferative retinopathy according to statin use or ACE inhibitor use. The data are expressed as follows: black line inside the box indicating median; box between 25 and 75 percentiles; whiskers, 5 and 95 percentiles. ACE = angiotensin‐converting enzyme; VEGF = vascular endothelial growth factor.

Statin use increased vitreous VEGF levels in diabetic patients with proliferative retinopathy (Table III). Diabetic patients without proliferative retinopathy were found to have a minor increase in vitreous VEGF concentration (Table III), and statin users to have higher vitreous VEGF, but it did not differ significantly due to the low number of subjects (550 pg/mL, n = 11, versus 286 pg/mL, n = 10, respectively, P = 0.341) (data not shown). However, the diabetes duration and glycaemic control were significantly better in diabetic patients without proliferative retinopathy than in diabetic patients with proliferative retinopathy.

Table III. Diabetes duration, glycaemic control, blood pressure and vitreous VEGF, in non‐diabetic control subjects, diabetic patients without proliferative retinopathy, or in diabetic patients with proliferative retinopathy according to statin medication. The data are expressed as means and standard deviation for others, and vitreous VEGF as median and range. The n for vitreous VEGF is shown in parentheses. Statistical testing was done between diabetic groups with ANOVA, except for vitreous VEGF where Kruskal‐Wallis was used.

	Non‐diabetic control subjects	Diabetic patients without proliferative retinopathy	Diabetic patients with proliferative retinopathy without statin use	Diabetic patients with proliferative retinopathy with statin use	P
n	43	21	35	15
Duration of diabetes (years)	‐	8.7 (7.4)	18.2 (9.5)	21.7 (9.3)	0.000
Fasting plasma glucose (mmol/L)	5.2 (0.7)	8.4 (3.1)	9.6 (4.6)	9.3 (3.1)	0.632
Glycated haemoglobin (%)	5.56 (0.49)	7.7 (1.8)	8.8 (1.7)	8.8 (1.5)	0.032
Systolic blood pressure (mmHg)	148 (20)	154 (18)	153 (22)	158 (26)	0.768
Diastolic blood pressure (mmHg)	86 (11)	84 (10)	84 (12)	83 (12)	0.867
Vitreous VEGF (pg/mL)	9 (0–631)	119 (9–3351)	666 (117–7110) (n = 37)	1361 (273–6620) (n = 16)	0.000

ANOVA = analysis of variance; VEGF = vascular endothelial growth factor

The diabetic patients with proliferative retinopathy were divided into four subgroups according to ACE inhibitor (A) or statin (S) medication: A‐/S‐, A+/S‐, A‐/S+, and A+/S+ (Table IV). The glycaemic control was slightly better, but diabetes duration longer in the A‐/S+ group, but they were not statistically significant in Kruskal‐Wallis test. The vitreous VEGF concentration was higher in groups using statins: median 2592 pg/mL in A‐/S+ group and 686 pg/mL in A‐/S‐ group (P = 0.035). Furthermore, vitreous VEGF median was 1361 pg/mL in A+/S+ group, while it was 589 pg/mL in A+/S‐ group (P = 0.045) (Table IV). The comparison between A‐/S‐ and A+/S‐ (P = 0.93) or A‐/S+ and A+/S+ groups (P = 0.309) did not reach statistical significance.

Table IV. Diabetes duration, glycaemic control, blood pressure and vitreous VEGF in diabetic patients with proliferative retinopathy in the four medication groups divided according to ACE inhibitor (A) or statin (S) medication: A‐/S‐, A+/S‐, A‐/S+ and A+/S+. The data are expressed as means and standard deviation for others, and vitreous VEGF as median and range. The n for vitreous VEGF is shown in parentheses.

	A‐/S‐ n = 16	A+/S‐ n = 19	A‐/S+ n = 5	A+/S+ n = 10
Duration of diabetes (years)	16.9 (9.3)	18.7 (10.1)	24.0 (5.9)	21.0 (10.2)
Fasting plasma glucose (mmol/L)	9.3 (4.4)	9.8 (4.9)	8.4 (1.9)	9.8 (3.6)
Glycated haemoglobin (%)	8.8 (1.6)	8.8 (1.7)	7.8 (1.2)	9.4 (1.3)
Systolic blood pressure (mmHg)	158 (16)	150 (26)	141 (31)	166 (19)
Diastolic blood pressure (mmHg)	85 (13)	84 (12)	73 (13)	84 (12)
Vitreous VEGF (pg/mL)	686 (117–4421) ^c (n = 16)	589 (250–7110) ^a (n = 21)	2592 (744–5081) ^b (n = 5)	1361 (273–6620) ^d (n = 11)

^aP<0.05 between groups A+/S‐ and A+/S+ (P = 0.045)

^bP<0.05 between groups A‐/S‐ and A‐/S+ (P = 0.035)

^cP = NS between groups A‐/S‐ and A+/S‐ (P = 0.930)

^dP = NS between groups A‐/S+ and A+/S+ (P = 0.309) VEGF = vascular endothelial growth factor; ACE = angiotensin‐converting enzyme; NS = not significant

The possible effect of diabetes duration or diabetes type, age, glycated haemoglobin, fasting plasma glucose, statin or ACE inhibitor use, body mass index, or blood pressure on vitreous VEGF was ruled out by performing linear regression analysis, and none of the above‐mentioned was found to have significant effect on vitreous VEGF.

Discussion

The current study shows that in diabetic patients with proliferative retinopathy using statins, the vitreous VEGF concentration was significantly higher than in those not using statin medication. This study is the first one assessing the effect of HMG CoA reductase inhibitors, statins, on vitreous VEGF concentration in diabetic patients with proliferative retinopathy, and it seems that statins have no beneficial effect in reducing VEGF production in the human diabetic eye. Previously, two clinical studies have suggested that a short‐time intervention with statins may retard the progression of diabetic retinopathy in hypercholesterolaemic patients 9, 10. However, the number of patients was low in both studies, and the study of Gordon et al. was not blinded. So far there is no clinical evidence that statins would be useful in primary or secondary prevention of diabetic retinopathy. In the light of the present results it is most likely that if a beneficial effect of statins on diabetic retinopathy would be found, it is not transmitted through a decrease in VEGF expression.

As the key regulatory element in angiogenesis, VEGF modulates endothelial cell migration and proliferation 11, 12. Though a theoretical rationale exists for statins in decreasing VEGF expression in vitro, it seems, however, that statins may even increase VEGF production in the human eye. In certain cells, statins augment VEGF expression 13. In a recent publication VEGF concentration in mouse retinal cell lysate was not affected by a clinically effective cholesterol‐lowering dose of pitavastatin 14. Statins may have biphasic effects as they seem to induce angiogenesis in vitro at low concentrations, equivalent to clinically used doses, and prevent angiogenesis at higher doses 6, 7. The enhancing effect of clinically equivalent doses of statins on angiogenesis is also present in vivo in patients with coronary heart disease 15. The desired statin‐induced angiogenesis in the heart would possibly be accompanied by detrimental effects in the diabetic eye. This question of potential benefit or harm of statin therapy for diabetic retinopathy patients should be solved urgently in a prospective clinical study.

Angiotensin II levels are high in the diabetic eye 2, and angiotensin II acts by stimulating VEGF expression and endothelial cell growth. One previous study has suggested that ACE inhibitor medication use is associated with lower concentration of vitreous VEGF in proliferative diabetic retinopathy in a dose‐dependent manner 3. However, in such a study the evaluation of glycaemic control is of utmost importance, since poor glycaemic control has been shown to increase VEGF expression in the diabetic retina causing blood‐retinal breakdown 1. The discrepancy between current results and the previous study 3 could probably be explained by a difference in glycaemic control between the groups in the study of Hogeboom van Buggenum et al. 3. They checked the glycaemic control only in a subset of patients, leaving the possibility of different glucose values in the total series. Therefore, in this study we emphasized the control of diabetes by measuring fasting plasma glucose and glycated haemoglobin in all our subjects, and the similarity in glycaemic control, duration of diabetes, or hypertension between the groups increases the reliability of our results, showing a lack of any significant effect of ACE inhibition on vitreous VEGF.

In conclusion, statin use is associated with high vitreous VEGF concentration in diabetic patients with proliferative diabetic retinopathy. However, since statins may enhance angiogenesis at clinically used doses, further studies evaluating the effect of statins and ACE inhibitors on the progression of diabetic retinopathy are warranted.

Acknowledgements

The authors wish to thank Drs Kaisu Järvinen, Ulla Lahtela, and Tapani Palosaari, who performed the vitrectomies. We are also very grateful to the staff of the Oulu University Hospital, Department of Ophthalmology for their excellent co‐operation. The skilful technical assistance of Saara Korhonen, Marja‐Leena Kytökangas, and Sari Pyrhönen is greatly appreciated. Dr Liinamaa was supported by a grant from the Eye Foundation, Helsinki, Finland and Professor Savolainen by Academy of Finland and Sigrid Juselius Foundation.