Polymorphisms of the vascular endothelial growth factor and susceptibility

to diabetic microvascular complications in patients

with type 1 diabetes mellitus

Bingmei Yang, Deborah F. Cross, Martin Ollerenshaw,Beverly A. Millward, Andrew G. Demaine*

Molecular Medicine Research Group, Plymouth Postgraduate Medical School, University of Plymouth, ITTC Building,

Tamar Science Park, Derriford Road, Plymouth, PL6 8BX, UK

Received 7 December 2001; received in revised form 27 March 2002; accepted 3 April 2002

Abstract

There is increasing evidence implicating genetic factors in the susceptibility to diabetic microvascular complications. Recent studies

suggest that increased expression of the cytokine vascular endothelial growth factor (VEGF) may play a role in the pathogenesis of diabetic

complications. A number of polymorphisms in the promoter region of the VEGF gene have been identified. The aim was to investigate

whether an 18 base pair (bp) deletion (D)/insertion (I) polymorphism at position � 2549 in the promoter region of the VEGF gene is

associated with the susceptibility to diabetic microvascular complications. Two hundred and thirty-two patients with type 1 diabetes mellitus

(T1DM) and 141 normal healthy controls were studied. The D/D genotype was significantly increased in those patients with nephropathy

(n = 102) compared to those with no complications after 20 years duration of diabetes (uncomplicated, n= 66) (40.2% vs. 22.7%,

respectively, c2 = 5.5, P < .05). The combination of polymorphisms of VEGF together with the aldose reductase (ALR2) gene showed that in

the nephropaths, 8 of the 83 subjects had the VEGF I allele together with the Z + 2 50ALR2 allele compared with 27 of the 62 uncomplicated

patients (c2 = 26.7, P< .00001). The functional role of the D/I polymorphism was examined by cloning the region into a luciferase reporter

assay system and transient transfection into HepG2 cells. The construct containing the 18 bp deletion had a 1.95-fold increase in

transcriptional activity compared with its counterpart that had the insert (P< .01). These results suggest that polymorphisms in the promoter

region of the VEGF gene together with the ALR2 may be associated with the pathogenesis of diabetic nephropathy. D 2003 Elsevier

Science Inc. All rights reserved.

Keywords: VEGF; Aldose reductase; Diabetic microvascular complications; Type 1 diabetes

1. Introduction

Diabetic microvascular complications are the major

causes of morbidity and early mortality in diabetes (Ander-

sen, Christiansen, Andersen, Kreiner, & Deckert, 1993). It is

well established that hyperglycaemia is a necessary risk

factor for development of diabetic complications (DCCT,

1993; Molitch, 1997; Molyneaux, Constantino, McGill, Zil-

kens, & Yue, 1998; Steffes, 1997). It is becoming clear that

genetic factors also play a major role in the susceptibility to

diabetic nephropathy, retinopathy and neuropathy (Chowd-

hury, Kumar, Barnett, & Bain, 1995; Doria, Warram, &

Krolewski, 1995; Parving, Tarnow, & Rossing, 1996). The

precise molecular and cellular cascades that provoke the

tissue damage following exposure to hyperglycaemia have

still to be elucidated. There is increasing evidence to suggest

that growth factors may play an important role in modifying

as well as accelerating the tissue damage caused by hyper-

glycaemia (Adler, Pahl, & Seldin, 2000; Stevens, Feldman,

& Greene, 1995).

Vascular endothelial growth factor (VEGF) is a cytokine

that has been proposed to play a key role in the pathogene-

sis of diabetic microvascular complications (Aiello et al.,

1994; Grone, 1995; Williams, 1997). The expression of the

gene is regulated by changes in oxygen tension and redox

1056-8727/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved.

PII: S1056 -8727 (02 )00181 -2

* Corresponding author. Tel.: +44-1752-764236; fax: +44-1752-

764234.

E-mail address: ademaine@plymouth.ac.uk (A.G. Demaine).

Journal of Diabetes and Its Complications 17 (2003) 1–6

imbalance in the cell (Shweki, Itin, Soffer, & Keshet, 1992).

VEGF induces vascular endothelial cell proliferation,

migration and vasopermeability in many types of tissues

including glomerular capillaries (Aillo & Wong, 2000). In

situ hybridisation and immunohistochemical studies have

shown that VEGF is expressed in the kidney at all ages in

man and is mainly distributed in the visceral glomerular

epithelial cells or podocytes (Brown et al., 1992; Simon

et al., 1995). Human mesangial cells and peripheral mono-

nuclear cells produce VEGF in vitro as well (Lijima,

Yoshikawa, Connoly, & Nakamura, 1993). Consequently,

it is thought to contribute to the susceptibility to diabetic

microvascular complications.

Several studies have shown VEGF expression is in-

creased in patients with diabetic retinopathy as well as those

with nephropathy (Cha et al., 2000; Chiarelli et al., 2000;

Hovind, Tarnow, Oestergaard, & Parving, 2000; Murata

et al., 1995). In experimental models of diabetic complica-

tions, there is increased expression of VEGF and its recep-

tors with elevation of the protein in the kidney of

experimental animals with diabetes and the vascular dys-

function may also be mediated by VEGF (Cooper et al.,

1999; Tilton et al., 1997). Increased expression of VEGF is

also found in epithelial or mesangial cell lines exposed to

high concentrations of glucose (Gilbert et al., 1998; Kim,

Jung, Cha, & Choi, 2000; Sone et al., 1996). These studies

suggest that VEGF could be a potential mediator of glom-

erular hyperfiltration and proteinuria in diabetic nephrop-

athy and antibodies against VEGF in the early stages of

experimental diabetes can ameliorate the renal dysfunction

(De Vriese et al., 2001).

In hypoxic conditions, expression of VEGF is regulated

via hypoxia response elements (HRE) in a redox-sensitive

manner (Liu, Cox, Morita, & Kourembanas, 1995; Tsuzuki

et al., 2000). The expression of VEGF in diabetes may be

mediated through the HRE due to the redox imbalance that

is thought to occur in this condition (Willimson et al., 1993).

Several novel polymorphisms in the promoter region and

exon 1 of the VEGF-A gene have recently been identified

(Brogan et al., 1999; Watson, Webb, Bottomley, & Brench-

ley, 2000). Of particular interest is a deletion/insertion (D/I)

of an 18 base pair (bp) fragment at � 2549 of the promoter

region. This D/I polymorphism was in complete linkage

with a single nucleotide polymorphism at � 2578. Individ-

uals with the A(� 2578) had the 18 bp insert, whilst those

with the C(� 2578) did not. The aim of this study was to

investigate these polymorphisms in patients with type 1

diabetes mellitus (T1DM) with clinically well-defined

microvascular complications.

2. Materials and methods

2.1. Subjects

Two hundred and thirty-two British Caucasoid patients

with T1DM who attended the Diabetic Out-Patient Clinic

of Derriford Hospital (Plymouth, England) were recruited

for this study. The patients were classified according to

their microvascular complications as previously described

(Demaine, Cross, & Millward, 2000; Heesom, Hibberd,

Millward, & Demaine, 1997). These categories are sum-

marised below:

2.1.1. Uncomplicated (n = 66)

These patients have had T1DM for at least 20 years but

remain free of retinopathy (fewer than five dots or blots per

fundus) and proteinuria (urine Albustix negative on the con-

secutive occasions over 12 months).

2.1.2. Nephropaths (n =102)

These patients have had diabetes for at least 10 years

with persistent proteinuria (urine Albustix positive on at

least three consecutive occasions over 12 months or three

successive total urinary protein excretion rates > 0.5 g/24 h)

in the absence of hematuria or infection on midstream urine

samples. All these patients had coexistent retinopathy.

2.1.3. Retinopaths (n =64)

These patients had retinopathy defined as more than five

dots or blots per eye, hard or soft exudates, new vessels or

fluorescein angiographic evidence of maculopathy or pre-

vious laser treatment for proliferative or proliferative retin-

opathy and maculopathy or vitreous hemorrhage. None of

these patients had proteinuria. The clinical features of the

patients are shown in Table 1.

The normal controls consisted of 141 sequential Cau-

casoid cord blood samples obtained following a normal

healthy obstetric delivery on the Maternity Unit, Derriford

Hospital. Two hundred and seven of the patients had been

Table 1

Clinical characteristics of patients with T1DM and normal healthy controls

Uncomplicated (n= 66) Nephropaths (n= 102) Retinopaths (n= 64) Normal controls (n= 141)

Male:female 43:23 46:56 32:32 78:63

Age at onset of diabetes (years) 16.1 (1–42) 16.3 (1–56) 18.0 (1–45)

Duration of diabetes (years) 32.0 (18–57) 32.0 (6–61) 31 (14–57)

The results are shown as mean and range (in parentheses) in years. Uncomplicated: patients who have had TIDM for more than 20 years but have no

microalbuminuria, background retinopathy or overt neuropathy. Nephropaths: patients who have had TIDM for more than 10 years and have persistent

proteinuria (0.5g/24 h) and retinopathy. Retinopaths: patients with retinopathy but no microalbuminuria.

B. Yang et al. / Journal of Diabetes and Its Complications 17 (2003) 1–62

previously typed for the 50 aldose reductase (ALR2) micro-

satellite in the 50 promoter region of the ALR2 gene (De-

maine et al., 2000; Heesom et al., 1997). The 50ALR2 gene

is been shown to be associated with the susceptibility to

diabetic microvascular complications.

2.2. Extraction, amplification and detection of the VEGF

D/I polymorphism

The blood samples from all subjects were collected into

5% EDTA and stored at � 20 �C. High molecular weight

genomic DNA was prepared from these samples using the

Nucleon II extraction kit (Scotlab, Lanarkshire, UK) follow-

ing the manufacturer’s instructions. An aliquot of this DNA

was used to amplify across position � 2705 to � 2494 of

the promoter region of the human VEGF gene (AF098331)

using the following amplimers: sense 50-GCTGAGAGTGG-

GGCTGACTAGGTA-30 and antisense 50-GTTTCTGACCT-

GGCTATTTCCAGG-30.

The amplification was carried out in a volume of 25 mlwith 100–200 ng of template DNA using the following

conditions: 95 �C for 5 min followed by 30 cycles at 95 �Cfor 1 min, 57 �C for 1.5 min and 72 �C for 2 min and a final

10 min extension at 72 �C. The amplification products were

separated by electrophoresis through a 2.5% agarose gel

containing ethidium bromide. Two bands of either 211 or

229 bp were detected.

2.3. Analysis of functional role of the �2705 to �1728

region of the VEGF gene using luciferase reporter assays

The 50 promoter region of the VEGF gene from position

� 2705 to � 1728 was amplified using the following

primers: 50-CTAACGCGTGCTGAGGATGGGGCTGAC-

TAGG-30 and 50-CGGCTCGAGTGCAGACATCAAAGT-

GAGCGGC-30.

The template DNA consisted of samples from individuals

who have been genotyped for the D/I polymorphism. The

amplification products were cloned into the plasmid pCR-

XL-TOPO vector (Invitrogen, The Netherlands) following

the manufacturer’s instructions. The insert sequences of the

recombinants were confirmed by sequencing (MWG, Mil-

ton Keynes, UK). Recombinants containing either the D or

the I allele were prepared and the inserts were excised using

restriction endonucleases XhoI and MluI. The inserts were

then subcloned into the XhoI and MluI sites of the plasmid

pGL3 enhancer luciferase reporter vector following the

manufacturer’s instructions (Promega, Southampton, UK).

The recombinant plasmids containing either the D or I

allele were purified using QIAGEN-Tip100 columns (QIA-

GEN, Dorking, UK) and then used to transfect HepG2 cells

(ECACC, Salisbury, UK). Briefly, the HepG2 cells were

maintained in Eagle’s minimal essential medium supple-

mented with 10% fetal calf serum (FCS), 2 mM glutamine,

1% nonessential amino acids and antibiotics, seeded into

24-well plates at 105 cells/well and cultured for 2 days until

80% confluent in a 5% CO2 incubator at 37 �C. The trans-

fections were performed by adding 0.675 mg of the test

plasmid together with 0.075 mg of pRL-TK control plasmid

together in an Eppendorf tube containing tissue culture

medium without FCS to a final volume of 197.8 ml.Tfx-20 transfectant reagent (Promega) (2.2 ml) was added

to make final volume of 200 ml. The tubes were incubated

at room temperature for 15 min before adding to each of the

wells of the 24-well plates. After 24 h, the wells were

divided into three groups. Group A consisted of control

transfectants that were kept at 37 �C for a further 20 h.

Group B were supplemented with D-glucose to a final con-

centration of 24 mM and incubated at 37 �C for a further

20 h. Group C were exposed to hypoxia by incubating the

cells for a further 20 h in a 1% O2, 5% CO2 environment at

37 �C. At the end of incubation time, all cells were lysed by

using 100 ml of passive lysis buffer (Promega). The lysates

were transferred to fresh microcentrifuge tubes and stored at

� 80 �C. For each group, a minimum of three transfections

were performed.

The luciferase activity in the transfected cells was meas-

ured using dual-luciferase reporter assay system (Promega)

Table 2

Frequency of the D/I polymorphism in the promoter region of the VEGF gene in patients with diabetic microvascular complications

Uncomplicated (n= 66) Nephropaths (n= 102) Retinopaths (n= 64) Normal controls (n= 141)

Genotype

I/I 13 (19.7) 14 (13.7) 12 (18.8) 24 (17.0)

D/D 15 (22.7) 41 (40.2)a 21 (32.8) 37 (26.2)b

I/D 38 (57.6) 47 (46.1) 31 (48.4) 80 (56.7)

Allele

I 64 (0.48) 75 (0.37) 55 (0.43) 128 (0.45)

D 68 (0.52) 129 (0.63)c 73 (0.57) 154 (0.55)

Uncomplicated: those patients with no microvascular complications after 20 years duration of T1DM. Nephropaths: patients with T1DM for more than 10 years

and persistent proteinuria with coexistent retinopathy. Retinopaths: patients with retinopathy but no proteinuria. I = presence of the 18 bp insert. D = absence of

the 18 bp insert.a vs. frequency in uncomplicated group (c2 = 5.5, P < .025, Pc = ns).b vs. frequency in nephropaths (c2 = 5.3, P< .05, Pc = ns).c vs. frequency in uncomplicated group (c2 = 4.6, P < .05, Pc = ns).

B. Yang et al. / Journal of Diabetes and Its Complications 17 (2003) 1–6 3

in a MLX luminometer (Dynex Technologies, USA). The

intensity of light emission was used to determine the

transcriptional activity of the promoter region sequence.

2.4. Statistical analysis

The frequency of alleles and genotypes in the patient

subgroups and normal control groups were compared using

c2 test. The P-values were corrected for the number of

comparisons (Tiwari & Terasaki, 1985). A Pc value of < .05

was considered to be significant. The transcriptional activ-

ities were expressed as mean values. The activities of the

promoter region of the VEGF gene in the luciferase assays

were compared using analysis of variance (ANOVA). A

value of P < .05 was considered to be significant.

3. Results

There were no differences in either the age at onset or

mean duration of diabetes between any of the patient

subgroups. The frequency of the genotype and alleles in

the patient subgroups and normal controls are summarised

in Table 2. The frequency of the D and I genotypes in all

groups conformed to the Hardy–Weinberg equilibrium.

The allele and genotype frequencies were similar

between uncomplicated and normal control groups. How-

ever, there was a significant increase in the frequency of the

D/D genotype in the nephropaths compared to both the

uncomplicated and the normal control groups (40.2% vs.

22.7% and 26.2%, respectively, P < .05). There was also an

increase in the frequency of the D allele in the nephropaths

compared to the uncomplicated and normal control groups

(0.63 vs. 0.52 and 0.55, respectively). The increase of the

D/D genotype and allele in the retinopaths was not signifi-

cantly different from the uncomplicated or normal control

groups. There was no association between the D and I

alleles and either age at onset of diabetes or gender (data

not shown).

Table 3 shows the frequency of the VEGF D/I together

with the 50ALR2 genotypes in the patient subgroups. The

VEGF I together with the Z + 2 50ALR2 alleles were found

in 27/62 uncomplicated patients compared with only 6/83

nephropaths (c2 = 26.7, P < .000001). In contrast, the VEGF

D allele and the Z-2 50ALR2 allele was found in 28/83

nephropaths and 12/62 uncomplicated patients. These dif-

ferences were less pronounced in the retinopaths. The

frequency of the combinations in the normal control popu-

lation was different to all the patient subgroups.

To determine whether the presence (I) or absence (D) of

the 18 bp insertion in the promoter region of the VEGF gene

altered transcriptional rate the two variants and the flanking

region (from � 2705 to � 1728) was cloned into a lucifer-

ase reporter plasmid. The construct containing D allele

had an elevated activity of 1.95-fold compared to the con-

struct containing the I allele (P < .01). Further, there was a

1.76-fold higher activity compared to transfectants with

both the D and the I allele constructs (P < .01). Whilst

hyperglycaemia did not change the rate of transcription

compared to the normal culture conditions, hypoxia de-

creases the level of activities in all the constructs.

4. Discussion

The results presented here show that the D allele and D/D

genotype of the VEGF may be associated with susceptibility

to diabetic nephropathy. Further, there may be an interaction

between VEGF and the ALR2 loci. The in vitro functional

studies suggest that the presence of the D allele at � 2549 in

the promoter region of the VEGF gene will lead to enhanced

expression of the gene. Previous studies have also shown

that polymorphisms in the promoter as well as the 30

untranslated regions of the VEGF gene are associated with

the production of the VEGF. For instance, a significant

correlation was found between the VEGF protein produced

by lipopolysaccharide-stimulated peripheral blood mononu-

clear cells and the + 405 polymorphic site (Watson et al.,

Table 3

Frequency of the combination of the D/I VEGF polymorphism and 50ALR2 polymorphism in patients with diabetic microvascular complications

VEGF/50ALR2 genotype Uncomplicated (n= 62) Nephropaths (n= 83) Retinopaths (n= 62) Normal controls (n= 83)

D/I Z + 2/X 18 (29.0)a 6 (7.2) 5 (8.1) 6 (7.2)

D/I Z� 2/X 7 (11.3) 5 (6.0) 13 (21.0) 19 (22.9)

D/I Z + 2/Z� 2 2 (3.2) 2 (2.4) 3 (4.8) 2 (2.4)

D/I X/X 8 (12.9) 10 (12.0) 8 (12.9) 10 (12.0)

I/I Z + 2/X 9 (14.5)a 0 (0.0) 2 (3.2) 0 (0.0)

I/I Z� 2/X 1 (1.6) 7 (8.4) 5 (8.1) 7 (8.4)

I/I (Z + 2/Z� 2) 0 (0.0) 1 (1.2) 1 (1.6) 1 (1.2)

I/I X/X 3(4.8) 5 (6.0) 4 (6.5) 5 (6.0)

D/D Z+ 2/X 5 (8.1) 5 (6.0) 9 (14.5) 5 (6.0)

D/D Z� 2/X 4 (6.5) 18 (21.7) 8 (12.9) 18 (21.7)

D/D (Z + 2/Z� 2) 1 (1.6) 1 (1.2) 3 (4.8) 1 (1.2)

D/D X/X 4 (6.5) 9 (10.8) 1 (1.6) 9 (10.8)

a vs. frequency in nephropaths group (c2 = 26.7, P< .000001, Pc = .000014).

B. Yang et al. / Journal of Diabetes and Its Complications 17 (2003) 1–64

2000). It has also been suggested that the C( + 936)T site is

associated with VEGF plasma levels (Renner, Kotschan,

Hoffmann, Obermayer-Pietsch, & Pilger, 2000). Our results

concur with these studies. Together, they suggest that poly-

morphisms of the VEGF gene may influence its expression.

The association of the D allele with the susceptibility to

diabetic nephropathy can be explained by the enhanced rate

of transcription compared with the I allele. This would

likely lead to elevated levels of VEGF in these patients

compared to the uncomplicated subjects who possess the I

allele. This was a cross-sectional study and it was not

possible to measure plasma VEGF levels. However, there

are numerous reports suggesting that patients with micro-

vascular complications have elevated levels of VEGF.

Further, the level and local production of VEGF may vary

between tissues. There may also be differences in the

expression of the VEGF isoforms. It is not known whether

these polymorphisms may correlate with low or high

expression of the gene.

In this study, we also found a highly significant inter-

action between the VEGF and the ALR2 loci. It is becoming

clear that polymorphisms in the promoter region of ALR2

are associated with susceptibility to diabetic microvascular

complications (Demaine et al., 2000; Heesom et al., 1997;

Moczulski et al., 2000). It has also been shown that the Z-2

ALR2 susceptibility allele is associated with increased

mRNA expression in those patients with diabetic nephrop-

athy (Hodgkinson et al., 2001; Shah et al., 1998). The excess

flux through the polyol pathway in these patients is likely to

generate major metabolic disturbances leading to the

increased generation of free radicals and depletion of the

intracellular cofactors NAD +, NADPH and possibly ATP.

This pseudohypoxic state in the cell is likely to lead to

enhanced expression of VEGF in subjects who already have

an increased expression of the gene. It has been demonstra-

ted that vascular dysfunction induced by elevated glucose

levels in rats is mediated by VEGF and is linked to increased

flux of glucose via the polyol pathway (Tilton et al., 1997).

The elevated VEGF transcription and protein levels

would cause the proliferation of endothelial cells, increased

vascular permeability and enhanced extracellular matrix

component accumulation (Del Prete et al., 1998). The

relentless decline of renal function in diabetic nephropathy

varies considerably between individuals. At the present

time, it is impossible to determine whether polymorphisms

of the VEGF or indeed the ALR2 gene is associated with the

initiation of the damage or with the rate of decline of

function. The Z-2 ALR2 susceptibility allele has recently

been shown to be associated with a fast progression form of

diabetic retinopathy (Olmos et al., 2000). Further work is

now required to ascertain whether similar a association

exists for diabetic nephropathy.

In conclusion, we have shown that a D/I polymorphism

in the promoter region of the VEGF is associated with the

susceptibility to diabetic nephropathy and the presence of

the deletion is linked to increased transcriptional activity.

The possible interaction of the VEGF and ALR2 loci in the

susceptibility to nephropathy.

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