Apoptotic factors (Bcl2 and Bax) and diabetic retinopathy in type 2 diabetes

ORIGINAL PAPER

Apoptotic factors (Bcl-2 and Bax) and diabetic retinopathy in type2 diabetes

Taoufik Khalfaoui • Nuria Basora •

Amel Ouertani-Meddeb

Received: 29 March 2010 / Accepted: 25 May 2010 / Published online: 8 June 2010

� Springer Science+Business Media B.V. 2010

Abstract The expression of apoptotic factors Bcl-2 and

Bax were studied in the conjunctiva of diabetic patients

with and without retinopathy. All patients underwent a

complete ophthalmic examination including ocular fundus

and retinal fluorescein angiography. The indirect immun-

operoxidase method was performed on 15 normal con-

junctiva taken during cataract surgery (group 1), on 40 eyes

of 40 patients with type 2 diabetes without diabetic reti-

nopathy (group 2) and 13 eyes of 13 patients with diabetic

retinopathy (group 3). In normal human conjunctiva, Bax

showed positive expression in epithelial, vascular and

stromal cells whereas Bcl-2 staining was negative. In the

conjunctiva of diabetic patients without diabetic retinopa-

thy, Bax was widely, and strongly, expressed in epithelial

cells, vascular endothelial cells, fibroblasts and infiltrating

cells such as macrophages. For patients with diabetic reti-

nopathy, Bax was consistently strong to very strong. Bcl-2

protein expression became weak to negative for diabetic

patients both with and without diabetic retinopathy.

Immunoreactivity was not correlated between Bcl-2 and

Bax in the conjunctiva of diabetic patients. Bax was always

localized in tissues characterized by a high rate of apoptosis,

whereas, Bcl-2 was absent. Our results suggest that diabetic

human conjunctiva, with its inflammatory phenomena, is

considered as a privileged target for programmed cell death.

Keywords Apoptosis � Bax � Bcl-2 � Conjunctiva �Diabetes � Diabetic retinopathy (DR)

Introduction

Type 2 diabetes is a major public health problem and its

prevalence is increasing in the world population (Braun-

stein and White 2005; Hasnan et al. 2010). Type 2 diabetes

is characterized by hyperglycemia which has been docu-

mented to contribute to the development of microvascular

complications and macrovascular lesions.

Hyperglycemia initiates a sequence of events that leads

to the development of diabetic retinopathy (DR) which

remains one of the leading causes of blindness worldwide

(Yam and Kwok 2007; Xin-Yuan et al. 2009). The duration

of diabetes and the severity of hyperglycemia are the major

risk factors that accelerate development of the disease

(Klein et al. 1984; Tapp et al. 2003; Fong et al. 2004;

Santos et al. 2005). DR progresses through various stages

from non-proliferative diabetic retinopathy (NPDR) to

proliferative diabetic retinopathy (PDR). Strict metabolic

control, tight regulation of blood pressure and early-con-

tinued glycemia management can significantly reduce the

risk of DR development and progression (Yam and Kwok

2007; Xin-Yuan et al. 2009). It appears that the most

effective and beneficial act for DR is glycemic control

(Xin-Yuan et al. 2009).

Apoptosis has become a new focus in the field of oph-

thalmic research. It has been shown that programmed cell

death was implicated in several pathologies including those

affecting the retina (Toshiyuki and Sayon 2005; Sennlaub

et al. 2002; Yamashiro et al. 2003; Abu-El-Asrar et al. 2004;

Bai-hua et al. 2006), the cornea (Yew et al. 2001), the

conjunctiva (Yew et al. 2001; Weinstein et al. 2002), and the

T. Khalfaoui � A. Ouertani-Meddeb

Service d’ophtalmologie, hopital Charles-Nicolle, boulevard du

9-Avril, 1006 Tunis, Tunisia

T. Khalfaoui (&) � N. Basora

CIHR Team on the Digestive Epithelium, Departement

d’anatomie et biologie cellulaire, Faculte de medecine et des

sciences de la sante, Universite de Sherbrooke, Sherbrooke, QC

J1H 5N4, Canada

e-mail: [email protected]

123

J Mol Hist (2010) 41:143–152

DOI 10.1007/s10735-010-9271-9

lens (Takamura et al. 2001). Recent in vivo and in vitro,

studies have demonstrated that many pathways induced by

hyperglycemia, such as oxidative stress (Abu El-Asrar et al.

2007), advanced glycation end products (Bai-hua et al.

2006) and the polyol pathways (Veronica et al. 2003) lead

to oxidative injury of neurons, which in turn activate the

death pathways implicated in neuronal apoptosis.

Apoptosis, as a physiological and highly regulated

process, is a key feature of normal development, morpho-

genesis, and tissue repair (Yew et al. 2001; Khalfaoui et al.

2007; Abu El-Asrar et al. 2007). In vertebrates, apoptosis is

a conserved evolutionary process which plays an essential

role in the homeostasis and function of immune cells by

maintaining adequate cell numbers and in the deletion and

removal of unwanted and auto-reactive cells (Tan et al.

2000; Wassim et al. 2004; Khalfaoui et Al. 2007). Pro-

grammed cell death is an active phenomenon playing a

central role on the tissue regulation and differentiation

(Brignole et al. 2003) and is necessary for the survival of

multi-cell organisms (Tang and Porter 1996). It implicates

individual cells habitually in a tissue and does not cause

inflammation (Kajta 2004) and is found in physiological or

pathological situations in several aspects of biological

processes (Yee et al. 1994; Brignole et al. 2003). Apoptosis

is a spontaneous process of cell elimination involved in the

maintenance of cellular homeostasis (Yee et al. 1994,

Noble et al. 1999) and when uncontrolled leads to cell loss,

tissue hypoplasia and atrophy of organs.

The Bcl-2 gene was originally discovered at the junction

of the chromosomal translocation t (14;18) found in can-

cerous B cells and is an anti-apoptosis gene expressed by

many cell types (Masao 2002). Excessive proliferation of

these cells has been explained by the over-expression of

Bcl-2 which prevents their natural death (Camillari-Brote

and Molina 2000). It has been shown that Bcl-2 protects the

cell against apoptosis with the notable exception of apop-

tosis induced by cytotoxic lymphocytes (Camillari-Brote

and Molina 2000; Sophie and Molina 2000; Israels and

Israels 1999; Krajewski et al. 1994). In 1993, the first pro-

apoptotic member of the Bcl-2 family was reported

(Korsmeyer et al. 1993; Reed 2006). Bax (Bcl-2 Antagonist

X) was identified as a Bcl-2-interacting protein that

opposed Bcl-2 and promoted apoptotic cell death (Kors-

meyer et al. 1993; Zhang et al. 2004) by forming a het-

erodimer with Bcl-2. When overexpressed Bax formed

homodimers, accelerated apoptotic death and eliminated

the protective effect conferred by overexpression of Bcl-2.

These data have led to a model proposing that the ratio of

pro/anti-apoptotic proteins determines cell fate (Tan et al.

2000; Krajewski et al. 1994).

It has been shown that accelerated apoptosis preceded

the appearance of pathological vascular abnormalities in

the retina of diabetics (Podesta et al. 2000) and also

appeared to play a key role in the installation and devel-

opment of diabetic microangiopathy (Podesta et al. 2000).

The aim of our study was to determine and compare the

expression of apoptotic factors Bax and Bcl-2 in the bulbar

conjunctiva of diabetic patients with and without

retinopathy.

Materials and methods

Patients

Biopsies were taken from the bulbar conjunctiva of 40 eyes

from 40 type 2 diabetes patients without DR and from 13

eyes of 13 type 2 diabetes patients with DR, including 8

with nonproliferative diabetic retinopathy (NPDR) and 5

with proliferative diabetic retinopathy (PDR). Formal

consent was obtained from all patients selected for our

study in compliance with the Declaration of Helsinki. All

patients underwent a complete ophthalmic examination,

including ocular fundus and retinal fluorescein angiogra-

phy. The normal conjunctivas were taken from 15 patients

undergoing senile cataract surgery.

We have excluded any patient with other ocular, fibro-

vascular, and/or general pathology. We have also excluded

those who underwent surgery less than 1 year from the date

of their first recruitment. Similarly, we have excluded

diabetic patients with uncontrolled hypertension and with

type 1 diabetes. Finally we have excluded any patient with

any allergic predisposition or any surface disease.

Demographic data

A control study, from six men and nine women, was con-

ducted on 15 normal conjunctivas (group 1) biopsied during

cataract surgery. Age ranged from 53 to 83 years, with a

mean of 67.77 ± 8.46 years. Of the 53 patients with type 2

diabetes included in the study, 40 did not have clinical DR

(group 2): 21 men and 19 women; mean age, 53.51 ±

10.51 years (range, 30–73 years); mean duration of diabe-

tes, 5.97 ± 5.65 years (range, 1–20 years). The remaining

13 diabetic patients had DR (group 3): six men and seven

women; mean age, 56.69 ± 8.63 years (range, 35–68

years); mean duration of diabetes, 10.30 ± 4.91 years

(range, 3–21 years).

Biopsy tissue collection

One eye was randomly selected. The eye was prepared and

draped in the usual sterile fashion. After insertion of a lid

speculum, the conjunctiva was instilled with local anaes-

thesia, combining topical and subconjunctival 2% Xylo-

caine. Small biopsies (2–3 mm2) were taken approximately

144 J Mol Hist (2010) 41:143–152

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4 mm posterior to the nasal limbus. The normal conjunc-

tiva tissue was obtained at the same site during cataract

surgery.

Antibodies and immunohistochemistry

Sections were stained with a rabbit polyclonal anti-human

Bax (Dako Corporation, Carpinteria, CA), diluted to 1:20,

and a mouse monoclonal anti-human Bcl-2 antibody

(DakoCytomation, Glostrup, Denmark), diluted to 1:10.

Detection of the primary antibodies was carried out by

means of a three-step immunoperoxidase method: labelled

streptavidin biotin (LSAB�, Kit Universal, Dako).

Paraffin-embedded conjunctiva sections were fixed with

10% neutral buffered formalin. After deparaffinization and

rehydration, tissue sections were incubated with 3%

hydrogen peroxide in methanol to quench endogenous

peroxidise activity. Antigen retrieval was carried out by

microwaving sections in citrate buffer (0.01 mol/, pH = 6)

(Shi et al. 1991). This is a high-temperature heating method

to recover the antigenicity in tissue sections that have been

masked by formalin fixation. The sections were blocked for

30 min with normal mouse serum at room temperature and

incubated overnight with the primary antibody at 4�C. The

sections were then washed with phosphate buffered saline,

PBS (HCl 0.05 mol/L, pH 7–7.6) and incubated with a

biotinylated secondary antibody (DakoCytomation) for

60 min. After several washes with PBS, products were

visualized with the streptavidin horseradish peroxidase and

3-amino-9-ethyl-carbasole (AEC) was used as a chromo-

gen. Hematoxylin (DakoCytomation) was used as a coun-

terstain. Negative controls were carried out with normal

mouse and rabbit serum diluted to the same concentration

as the primary antibody. Immunohistochemical detection

of the different samples was carried out with the same

incubation times, and analyzed in bright field microscopy

with a Nikon Eclipse E600 microscope (Nikon, Tokyo,

Japan).

Semi-quantitative analysis of Bax and Bcl-2 expression

The number of positive cells was counted in five repre-

sentative fields. We used an eyepiece calibrated grid with

4009 magnification. At this magnification and calibration,

we counted the total number of cells present in a 10 9 10-

mm area and determined the percentage of positively

stained cells. The scoring system is based on a scale of

0–4? (Vorkauf et al. 1993). 41: very high (75–100%

positive cells), 31: high (50–75% positive cells), 21:

moderate (25–50% positive cells), 11: low (\25% positive

cells), 0: negative (0 positive cells). Means were calculated

for each cell type in diabetic and control specimens.

Statistical analysis

The Statistical Package for the Social Sciences for Win-

dows (SPSS Inc., version 10.0, Chicago, IL) was used for

statistical analysis and P \ 0.05 was considered to be

significant. The difference in expression of Bax and Bcl-2

in epithelial, vascular endothelial cells and stromal cells

(fibroblasts and macrophages) between controls, diabetic

patients without DR, and diabetic patients with DR was

analyzed using Pearson’s test and the results were

expressed as mean ± SD.

Results

Distribution of Bax and Bcl-2 in normal human

conjunctiva (group 1)

Normal human conjunctiva showed positive immuno-

staining for Bax in 12 (80%) of the 15 controls. Staining

was strong particularly in epithelial and endothelial cells

while stromal cells were classified as weak to negative

(Fig. 1a). In contrast, Bcl-2 (Fig. 1b) was negative in 15

(100%) of the 15 controls (Table 1).

Distribution of Bax and Bcl-2 in diabetic conjunctiva

without DR (group 2)

The results of expression and distribution scores compiled

for Bax and Bcl-2 in the conjunctiva of diabetic patients

without DR are summarized in Table 2. Bax immunohis-

tochemical scores were high (3?) to very high (4?) in 27/40

(68%) of diabetic subjects, weak (1?) to moderate (2?) in

9/40 subjects (22%) and negative (0) in 4/40 (10%) of the

subjects. Bax scores varied over the entire range of possible

scores and expression was predominantly found in epi-

thelial and endothelial cells, but was also observed in

stromal cells such as fibroblasts and macrophages (Figs. 2a

and 3a). Expression scores for Bcl-2 remained negative in

25 patients (63%) (Fig. 2b) but was weak in 14 patients

(35%) (Fig. 3b) and was high to very high in 1 patient

(2%). Our data showed a significant increase in Bax

expression for each of the different cell types (P = 0.001)

(Table 4), but not for Bcl-2 expression in the conjunctiva

of diabetic patients without retinopathy in comparison to

those with normal conjunctiva.

Distribution of Bax and Bcl-2 in diabetic conjunctiva

with DR (group 3)

Table 3 summarizes Bax and Bcl-2 immunoreactivity

staining scores for group 3. In diabetic patients with DR

(NPDR and PDR) the expression scores of Bax were high

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(3?) to very high (4?) in 12 of the 13 patients (92%) and

moderate only for one case (8%). In contrast, scoring for

Bcl-2 was weak in 7/13 patients (54%) and negative in 6

patients (46%).

Regardless of the duration of diabetes, Bax scores were

always high (3?) to very high (4?) for different cell types

in the conjunctiva of diabetic patients with DR, especially

for epithelial and endothelial cell layers (Fig. 4a). The

increased expression scores for Bax in diabetic conjunctiva

with DR in comparison with those without DR was sta-

tistically significant (P = 0.001) (Table 4). The expression

of Bcl-2, however, did not significantly change and was

found to remain negative (0) to weak (1?) (Fig. 4b).

Immunohistochemical expression of Bax and Bcl-2

and disease progression

Table 4 summarizes differences in expression scores for

Bcl-2 and Bax between the 3 different groups. Bcl-2

expression was entirely and systematically negative for

group 1 and weak to negative for groups 2 and 3 and these

differences did not attain statistical significance (P [ 0.05)

for the different cell types. On the other hand, expression of

Fig. 1 Normal human conjunctiva: negative expression of Bcl-2 (a) and positive expression of Bax (b) in epithelial cells (Ep), vascular

endothelial cells (Vx), and stromal cells [fibroblasts (F), macrophages (Mcp)]

Table 1 Immunohistochemical scoring for apoptotic factors Bax and

Bcl-2 in the normal human conjunctiva of non diabetic controls

N Gender Age Epithelium Vessels Stromal cells

Bax Bcl-2 Bax Bcl-2 Bax Bcl-2

1 F 53 2? 0 1? 0 1? 0

2 M 58 0 0 0 0 0 0

3 F 58 3? 0 3? 0 2? 0

4 F 58 1? 0 3? 0 1? 0

5 M 63 1? 0 1? 0 2? 0

6 F 68 0 0 0 0 0 0

7 M 68 1? 0 1? 0 0 0

8 M 68 2? 0 2? 0 2? 0

9 F 70 3? 0 3? 0 1? 0

10 F 71 1? 0 2? 0 0 0

11 F 71 2? 0 2? 0 1? 0

12 F 74 2? 0 3? 0 2? 0

13 M 75 0 0 0 0 0 0

14 M 79 2? 0 2? 0 1? 0

15 F 83 2? 0 1? 0 1? 0

Stroma includes cells with the morphological aspect of fibroblasts

(spindle shapes) and macrophages (spheric shapes)

M male, F female, N normal

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Bax was affected by diabetes. Scores were weak to mod-

erate and sometimes negative for group 1, high to very high

for group 2, and always very high for group 3. Differences

in expression scores for Bax between the 3 groups were

statistically significant (P \ 0.05) between the different

cell types (Table 4).

Risk factors influencing DR progression

Table 5 shows the comparison of certain risk factors for the

development and progression of DR within our two dia-

betic groups. In the current study a variety of biochemical

data was collected including blood glucose levels, glycated

Table 2 Immunohistochemical scoring for apoptotic factors Bax and Bcl-2 in the conjunctiva of diabetic patients without DR

N Gender Age Duration

of diabetes

Eye fundus Epithelium Vessels Stroma cells

Bax Bcl-2 Bax Bcl-2 Bax Bcl-2

1 M 57 1 N 4? 1? 1? 0 2? 0

2 M 57 1 N 1? 1? 1? 0 1? 0

3 F 43 1 N 4? 3? 2? 0 2? 0

4 M 61 1 N 3? 0 2? 0 1? 0

5 M 53 1 N 4? 1? 4? 0 4? 1?

6 F 47 1 N 2? 0 1? 0 1? 0

7 M 49 1 N 0 0 0 0 0 0

8 F 48 1 N 3? 0 0 0 1? 0

9 M 52 2 N 4? 1? 4? 0 2? 0

10 F 53 2 N 4? 0 4? 0 2? 0

11 F 60 2 N 4? 0 4? 0 2? 0

12 F 47 2 N 3? 0 3? 0 3? 0

13 F 42 2 N 1? 1? 0 0 0 0

14 M 51 2 N 0 0 0 0 0 0

15 F 52 3 N 1? 0 0 0 0 0

16 F 44 3 N 4? 0 0 0 1? 0

17 F 36 3 N 4? 0 1? 0 2? 0

18 M 55 3 N 0 1? 0 0 0 0

19 F 66 3 N 4? 1? 4? 0 2? 1?

20 M 53 4 N 4? 0 4? 0 4? 0

21 M 48 4 N 3? 0 0 0 1? 0

22 M 39 4 N 2? 1? 4? 0 1? 0

23 M 65 4 N 1? 0 0 0 0 0

24 M 65 4 N 3? 1? 2? 0 0 0

25 F 61 5 N 3? 0 3? 0 3? 0

26 F 41 5 N 4? 0 4? 0 4? 0

27 M 54 7 N 1? 1? 1? 0 1? 1?

28 M 61 7 N 3? 1? 0 0 0 0

29 F 66 8 N 1? 0 0 0 0 0

30 F 36 9 N 4? 0 3? 0 1? 0

31 F 49 10 N 4? 1? 3? 0 1? 1?

32 F 57 10 N 3? 0 4? 0 2? 0

33 F 44 10 N 4? 0 4? 0 2? 0

34 M 73 11 N 3? 0 1? 0 3? 0

35 F 61 12 N 3? 0 3? 0 2? 0

36 M 30 13 N 4? 1? 4? 0 2? 0

37 M 65 17 N 4? 1? 4? 0 4? 0

38 M 71 20 N 4? 0 4? 0 4? 0

39 M 64 20 N 0 0 0 0 0 0

40 M 72 20 N 2? 0 0 0 0 0

Stroma includes cells with the morphological aspect of fibroblasts (spindle shapes) and macrophages (spheric shapes)

J Mol Hist (2010) 41:143–152 147

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haemoglobin (HbA1C), microalbuminuria, triglycerides,

cholesterol, HDL and LDL. Statistically significant differ-

ences were found for only two parameters: HbA1C levels

and duration of diabetes. These two risk factors are

the most known, studied and the most associated with the

development and aggravation of DR, confirming the

validity of our groups.

Discussion

Our present study identified the changes in expression of

Bax and Bcl-2 in the conjunctiva of diabetic patients with

and without DR compared to normal conjunctiva. Bax and

Bcl-2, mitochondrial membrane proteins, are involved in

the induction and in the inhibition of apoptotic mecha-

nisms, respectively. The conjunctiva is a dynamic tissue

that relies on active renewal by stem cells (Daniels et al.

2001; Yew et al. 2001) and homeostasis in this tissue is

maintained through a delicate balance between cellular

proliferation and cellular apoptosis.

Apoptosis has been increasingly shown to be involved in

ophthalmic diseases, particularly those of the retina

(Brignole et al. 2003; Habn et al. 2003; Abu El-Asrar et al.

2007) such as glaucoma, macular degeneration related to

age (Haddad et al. 2003), retinitis pigmentosa and detached

Fig. 2 Conjunctiva of a 41-year-old diabetic patient with diabetes and without DR (No 26 in Table 1): High expression of Bax (a) and negative

expression of Bcl-2 (b) in epithelial cells (Ep), vascular endothelial cells (Vx), and stromal cells

Table 3 Immunohistochemical scoring for apoptotic factors Bax and Bcl-2 in the conjunctiva of diabetic patients with DR

N Age Gender Duration

of diabetes

Eye fundus Epithelium Vessels Stroma cells

Bax Bcl-2 Bax Bcl-2 Bax Bcl-2

1 56 F 3 NPDR 4? 1? 1? 0 1? 0

2 56 F 6 NPDR 3? 1? 2? 0 2? 0

3 60 F 7 NPDR 4? 0 1? 0 3? 0

4 48 M 10 NPDR 2? 1? 1? 0 1? 0

5 66 M 10 NPDR 3? 1? 4? 0 4? 1?

6 52 F 12 NPDR 4? 0 4? 0 4? 0

7 63 F 15 NPDR 4? 1? 4? 0 4? 0

8 55 F 21 NPDR 4? 0 4? 0 3? 0

9 68 F 4 PDR 4? 0 4? 0 4? 0

10 56 M 9 PDR 4? 1? 4? 0 3? 0

11 58 M 10 PDR 4? 0 4? 0 4? 0

12 35 M 12 PDR 4? 1? 4? 0 4? 0

13 64 M 15 PDR 4? 0 4? 0 4? 0

Stroma includes cells with the morphological aspect of fibroblasts (spindle shapes) and macrophages (spheric shapes)

NPDR non proliferative diabetic retinopathy, RDP proliferative diabetic retinopathy

148 J Mol Hist (2010) 41:143–152

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retina (Barber et al. 1998; Podesta et al. 2000; Doonan

et al. 2003; Cullinan and Brandt 2004). It has been shown

that apoptosis is involved in cell death of different layers of

the retina (Barber et al. 1998; Doonan et al. 2003) princi-

pally affecting the photo-receptor cell layer and the gan-

glion cell layer (Takamura et al. 2001). The treatment of

neurodegenerative diseases of the eye is not always

effective. Since apoptosis has been shown to be involved in

the pathophysiology of these diseases, the prevention of

apoptosis could inhibit the progression of the disease and

the removal of its symptoms.

In this study, we described for the first time, to our

knowledge, the in vivo distribution of key players associ-

ated with apoptosis in the bulbar conjunctiva of diabetic

patients by using an immunohistochemical approach

(Khalfaoui et al. 2007). Our results extend previous studies

which showed that TUNEL-positive apoptotic cells were

distributed throughout the whole structure of normal con-

junctiva obtained by surgical biopsies (Tan et al. 2000).

Our immunohistochemical study showed that Bax was

more widely expressed than Bcl-2 in the conjunctiva of

diabetic patients. These results support numerous studies

(Podesta et al. 2000; Mohr et al. 2002; Oshitari and Roy

2005; Hasnan et al. 2010) that suggest that high expression

of Bax and low expression of Bcl-2 occur in the conjunctiva

of diabetic patients with and without DR in response to high

glucose levels and are conducive to apoptosis. Although

previous studies found that increased apoptosis in the nor-

mal conjunctiva was age dependent (Zhang and Herman.

2002; Jurgen et al. 2005), Bcl-2 was negative in 100% of the

controls despite a higher mean age, 68 years, than that of

the diabetic patients. Indeed, it has been reported that nor-

mal conjunctiva specimens displayed no Bcl-2 expression

and apoptotic cells were seen throughout the entire width of

Fig. 3 Weak expression of Bax in the conjunctiva of a 47-year-old diabetic patient with diabetes without DR (No 6 in Table 1) (a).Weak

expression of Bcl-2 in the conjunctiva of a 54-year-old diabetic patient with diabetes without DR (No 27 in table 1) (b)

Table 4 Increased expression of Bax and not of Bcl-2 in diabetic conjunctiva in comparison with normal conjunctiva: immunohistochemical

analysis

Apoptotic factors Cell types % of positive cells

Group 1 Group 2 Group 3 P value

Bax Epithelium 26.00 ± 19.47 63.52 ± 33.60 83.53 ± 16.50 0.000

Vessels 22.82 ± 29.33 44.07 ± 38.45 71.38 ± 38.78 0.025

Stroma 15.33 ± 13.94 25.87 ± 31.31 52.30 ± 36.64 0.014

Bcl-2 Epithelium 0.00 ± 0.00 6.75 ± 11.22 8.75 ± 7.72 0.816

Vessels 0.00 ± 0.00 0.000 ± 0.00 0.000 ± 0.00 a

Stroma 0.00 ± 0.00 1.32 ± 4.06 1.15 ± 4.16 0.721

Stroma includes cells with the morphological aspect of fibroblasts (spindle shapes) and macrophages (spheric shapes)

Data are shown as mean of positive cells (%) ± standard deviation (SD). Group 1: control, Group 2: diabetic patients without DR, Group 3:

diabetic patients with DRa It was impossible to calculate p because Bcl-2 was always negative in vessels

p \ 0,05 is significant

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the epithelial layer, coupled with high levels of Bax

expression (Tan et al. 2000). Bax was a target of diabetes in

the diabetic retina in vivo and high glucose in the culture of

retinal pericytes in vitro (Dubois-Dauphin 2003).

Several other studies have shown differences in the

expression profiles between these two proteins in thyroid

glands with subacute thyroiditis (Koga et al. 1999), in

lymph nodes of patients with oral squamous cell carcinoma

(Zhang et al. 2009), in prostate cancer (Reagan-Shaw et al.

2008) and in human small intestinal adenocarcinoma (Gao

and Wang 2009). In the study presented here the expression

of the pro-apoptotic factor Bax, appeared to be tightly

related to disease aggravation in which apoptotic processes

play a central role (Table 4).

Transcription of both Bcl-2 and Bax genes is regulated

by the tumor suppressor gene p53, and p53 inhibited

expression of Bcl-2 and activated the expression of Bax

(Israels and Israels 1999; Tan et al. 2000; Dubois-Dauphin

2003). Recently, it has also been shown to be involved in

the diabetic process (Adamis 2002). In a previous study

from our laboratory using the same samples (Kria et al.

2005) we found that p53 showed an identical profile of

expression as Bax suggesting a mechanism for increased

Bax expression.

As shown in Table 5, which compares the values of the

risk factors for DR within our diabetic patients with and

without DR, significant differences are found only for the

duration of diabetes and for HbA1C. According to the

Fig. 4 Conjunctiva of a 52-year-old diabetic patient with diabetes with DR (No 6 in Table 2): Very high expression of Bax (a) and negative

expression of Bcl-2 (b) in epithelial cells (Ep), vascular endothelial cells (Vx), and stromal cells. The scale bar is 50 lm

Table 5 Correlation between DR progression and its risk factors

Variable Group 2 (n = 40) Group 3 (n = 13) P value

Age (years) 53.51 ± 10.51 56.96 ± 8.63 0.358

Diabetes duration (years) 5.97 ± 5.65 10.30 ± 4.91 0.017

Glycemia (mmol/L) 11.08 ± 4.24 11.11 ± 4.26 0.983

HbA1c (%) 8.87 ± 2.55 12.72 ± 1.52 0.046

Triglycerides (mmol/L) 1.41 ± 0.51 1.74 ± 0.46 0.117

Microalbuminuria (lmol/L) 43.19 ± 52.20 47.87 ± 20.56 0.815

Cholesterol (mmol/L) 5.18 ± 0.80 4.75 ± 1.35 0.292

HDL (mmol/L) 1.16 ± 0.48 1.02 ± 0.29 0.560

LDL (mmol/L) 3.84 ± 1.16 2.90 ± 0.60 0.224

Data are shown as mean ± standard deviation (SD). Group 2: diabetic patients without DR, Group 3: diabetic patients with DR

P \ 0.05 is significant

150 J Mol Hist (2010) 41:143–152

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abundant literature, duration of diabetes, severity of

hyperglycaemia and poor control of blood pressure are the

major risk factors that accelerate development of the dis-

ease (Klein et al. 1984; Tapp et al. 2003; Fong et al. 2004;

Manaviat et al. 2004; Santos et al. 2005). Glycemic control

remains the main parameter on which we can act to prevent

or at least delay the apparition of microvascular complica-

tions of diabetes (Altannavch et al. 2004; Fong et al. 2004).

It has been admitted that Bcl-2 family genes can nega-

tively or positively regulate apoptosis and this regulation

depended specifically on the ratio of Bax/Bcl-2 (Wassim

et al. 2004; Murphy et al. 2000; Del Poeta et al. 2003).

More recently, it was shown that this ratio determined to a

large extent whether the cell initiated apoptosis or alter-

natively, re-entered the cell cycle (Murphy et al. 2000; Del

Poeta et al. 2003). Increased Bax levels may played a role

in the apoptosis of conjunctival cells by tilting the cellular

balance of apoptosis regulators in a direction that increased

susceptibility to stressful stimuli, but could be sufficient to

kill cells directly (Kria et al. 2005).

Exploitation of the knowledge acquired on the regula-

tion of apoptosis has and will permit us to better identify

not only the factors involved in the aetiology of certain

diseases, but also to identify targets for the development of

innovative therapies for diabetics. Research on apoptosis

concerns so many different fields of medicine. Several

acute or chronic diseases are the result of excessive loss of

certain cell populations, but many others diseases are

characterized by abnormal inhibition of cell death. In the

first case, therapies aimed to block the activity of anti-

apoptotic proteins, and in the second case to activate the

executors of self auto destruction (Burmester and Pezzutto

1998; Melino et al. 1994).

These results may lead to an additional approach in the

study of DR pathogenesis. More extensive molecular

studies are needed to support and confirm these hypotheses

and to better understand the possible impact of diabetes,

inflammation, angiogenesis and apoptosis in conjunctiva

haemostasis and remodelling.

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