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Expression modification of uncoupling proteins and MnSOD inretinal endothelial cells and pericytes induced by high glucose:

The role of reactive oxygen species in diabetic retinopathy

Yan Cui a,b, Xun Xu a,*, Hongsheng Bi b, Qi Zhu a, Jianfeng Wu b,Xin Xia a, Qiushi Ren c, Patrick C.P. Ho d

a Department of Ophthalmology, Shanghai First People’s Hospital, 85 Wu Jin Road, Shanghai 200080, P.R. Chinab Jinan Shierming Eye Hospital, 48 Jinan Ying Xiong Shan Road, Jinan 250002, P.R. China

c Department of Biomedical Engineering, Shanghai Jiao-Tong University, 800 Dong Chuan Road, Shanghai 200240, P.R. Chinad 8/F Kailey Tower, 16 Stanley Street, Central, Hong Kong, P.R. China

Received 9 November 2005; accepted in revised form 31 March 2006

Available online 5 June 2006

Abstract

Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They belong to the family of anion

mitochondrial carriers. UCPs could act as proton carriers activated by metabolites and create a shunt between complexes of the respiratory chain

and ATP synthase. The increased leakiness of the mitochondrial inner membrane to protons may be to minimize superoxide production by lim-

iting the maximum DmHþ. The purpose of this study was to detect UCP expression in retinal capillary cells and their modification in high levels

of glucose. The role of reactive oxygen species (ROS) of mitochondria and UCPs in pathogenesis of diabetic retinopathy was investigated. Bo-

vine retinal capillary endothelial cells and pericytes were cultured with selective culture media, respectively. Passage cells were cultured in three

different glucose concentrations (5, 23, 30 mM) until passage four. ROS changes in mitochondria of these cells in different glucose concentra-tions were detected with scanning laser confocal microscopy (SLCM). The mitochondria membrane potential (Dj), cell death rate and apoptosis

rate were measured with flowing cytometry. UCP expression in retinal capillary cells was detected by immunocytochemistry. Expression and

modification of MnSOD and uncoupling proteins (UCPs) in different concentrations of glucose were detected by means of semi-quantitative

RTePCR. ROS in mitochondria of both endothelial cells and pericytes increased as the glucose concentration of media increased. Dj and

cell death rate of endothelial cells increased also. ROS was correlated to Dj and cell death rate positively in endothelial cells. No difference

in Dj and cell death rate among different glucose levels was found in pericytes. Apoptosis rate of endothelial cells and pericytes in high glucose

levels was higher than that in lower glucose levels. UCP1 and UCP2 were expressed in cultured retinal capillary cells whereas UCP3 was not. At

high levels of glucose, expression of UCP1, UCP2 and MnSOD increased to accommodate ROS production compensatively. The compensative

mechanism disappeared when glucose concentration was too high (30 mM). The results of this study showed that increasing mitochondrial ROS

could be induced by high glucose concentration. Those proteins related to antioxidation mechanism, such as MnSOD and UCPs, could exert

compensative action to a certain extent. This compensative action was insufficient when the glucose concentration was too high.

Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: diabetic retinopathy; reactive oxygen species; mitochondria; uncoupling proteins; MnSOD

1. Introduction

Oxidative stress induced by hyperglycemia is an import-

ant pathway of diabetic microvascular complications. An in-

creasing number of studies place mitochondrial ROS

overproduction at the heart of pathogenesis of diabetic

* Corresponding author. Tel.: þ86 21 6324 0090x3162; fax: þ86 21 6324

0825.

E-mail address: [email protected] (X. Xu).

0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.exer.2006.03.024

Experimental Eye Research 83 (2006) 807e816www.elsevier.com/locate/yexer

mailto:[email protected]


http://www.elsevier.com/locate/yexer

http://www.elsevier.com/locate/yexer




microvascular complications. Normalizing mitochondrial su-

peroxide production can block other pathways of hyperglyce-

mic damage (Brownlee, 2001; Nishikawa et al., 2000).

Mitochondrial electron transport chain is the main source of

reactive oxygen species (ROS), such as superoxide anion

and hydrogen peroxide. ROS production is associated with

the activity of respiratory complexes I and III and with ubise-miquinone generated in the course of electron transport reac-

tions in the respiratory chain. Mild uncoupling of respiration

diminishes mitochondrial ROS formation by complexes I

and III, because ROS formation depends on the mitochondrial

proton gradient and the mitochondrial potential. That is, a mild

uncoupling of respiration may participate in antioxidant de-

fense and the uncoupling proteins (UCPs) may be the effectors

of such a defense mechanism.

Uncoupling proteins (UCPs) belong to the mitochondrial

anion carrier gene family and, as suggested by their name,

can uncouple ATP production from mitochondrial respiration

by causing proton leak (Adams, 2000; Erlanson-Albertsson,

2003). As an elevated proton electrochemical potential gradi-ent (DmHþ) favors superoxide production, limiting the magni-

tude of this gradient should decrease superoxide production

(Skulachev, 1996). The increased leakiness of the mitochon-

drial inner membrane to protons may be to minimize superox-

ide production by limiting the maximum DmHþ. Causing

proton leak is the essential function of UCPs, which are mito-

chondrial transporters present in the inner membrane of mito-

chondria (Erlanson-Albertsson, 2002). Five different UCPs

have already been identified: UCP1e4 and UCP5, or Brain

mitochondrial carrier protein 1 (BMCP1). These proteins are

expressed in different tissues and play different roles in

cellular metabolism. UCPs are related to ROS production(Skulachev, 1998).

Which kinds of UCPs isomers are expressed on retinal cap-

illary cells? Does UCPs and MnSOD expression change in

high glucose? To answer these questions, in this study we cul-

tured bovine retinal endothelial cells and pericytes in different

concentrations of glucose. Mitochondrial ROS quantification,

its effects and possible causes were studied. The expression

of UCPs and MnSOD in the retinal capillary cells and their

modification in high glucose concentration were also studied.

The role of ROS and UCPs on the pathogenesis of diabetic ret-

inopathy was investigated.

2. Materials and methods

2.1. Cell culture, identification and UCP expression

The primary culture approach of retinal capillary endothe-

lial cells and pericytes was similar to the previous reports

(Capetandes and Gerritsen, 1990; Kim et al., 2002). In brief,

the retina of fresh bovine eyes without pigment cells was ho-

mogenized and digested in 0.05% collagenase I (Sigma Chem-

ical, St. Louis, MO) with 0.025% BSA at 37 C for 45 min.

The digestive material was filtered with 88-mm mesh and col-

lected. After centrifugation, the pellet tissue was used for both

endothelial cells and pericyte culture. For endothelial cell cul-

ture, the pellet was maintained in low-glucose Dulbecco’s

modified Eagle medium (DMEM; GIBCO, Grand Island,

NY) supplemented with 10% human serum (Sigma), 100 mg/

ml heparin (Sigma) and 10 mmol/l HEPES (GIBCO). The cul-

ture plate was coated with Fibronectin (FN, GIBCO, Invitro-

gen, CA) beforehand. The medium for pericytes waslow-glucose DMEM supplemented with 10% FBS and the

plate did not need to be pretreated.

After reaching confluency, bovine endothelial cells and

pericytes were maintained in DMEM containing 10% human se-

rum and 10% FBS, respectively, for passage. The passage endo-

thelial cells and pericytes were incubated with different glucose

concentrations, i.e. 5 mM, 23 mM and 30 mM. Equimolar man-

nitol was supplemented for osmotic controls. Lab-Tek II Cham-

ber Slide System (Nalge Nunc International, USA) was used for

later confocal microscopy examination.

The endothelial cells and pericytes generated after four pas-

sages were used in the latter experiments including identifica-

tion with Von Willebrand polyclonal antibody (Santa Cruz)and Actin, Smooth Muscle Ab-7 (Neo Markers).

UCP expression of endothelial cells and pericytes was de-

tected by an immunocytochemistry test. The process followed

the staining protocol of Histostain-Plus Kits (Zymed Laborato-

ries Inc., South San Francisco, CA). The primary antibody in-

cluded UCP1, -2 and -3 (20 mg/ml rabbit polyclonal affinity

purified IgG, Alpha Diagnostic International, TX, USA). Fi-

nally, color was developed with DAB chromogen (Reagent

D of Histostain-Plus Kits). For negative controls, primary an-

tibody was omitted.

2.2. Localization and quantification of ROS

Localization and quantification of ROS production was in-

vestigated in cultured cells double-labeled with 20,70-dichloro-

dihydrofluorescein diacetate (H2DCFDA; Molecular Probes,

Eugene, OR) to detect ROS production and MitoTracker

Red CM-H2XRos (MTR; Molecular Probes) to visualize mito-

chondria. MTR is a cationic lipophilic compound that readily

accumulates in mitochondria that possess a membrane poten-

tial, and it becomes fluorescent when oxidized. MTR has a lon-

ger emission wavelength (608 nm), which is well resolved

from the 525-nm emission wavelength of green fluorescein an-

alogues such as DCF. Confluent bovine retinal endothelial

cells and pericytes cultured in different glucose concentrations

were rinsed with PBS two times. After adding H2DCFDA and

MTR into culture media with final concentrations of 2 mM and

500 nM, cells were incubated at 37 C for 1 h, then washed

twice with fresh pre-warmed medium and imaged using a Zeiss

LSM 510 laser scanning confocal microscope. Green fluores-

cence of DCF was excited at 488 nm and excitation of MTR

was achieved at 543 nm using a helium/neon laser.

To determine the site of high glucose-induced intracellular

ROS production, endothelial cells and pericytes cultured in

23 mM and 30 mM glucose were first incubated with carbonyl

cyanide m-chlorophenylhydrazone (CCCP; Sigma), the

808 Y. Cui et al. / Experimental Eye Research 83 (2006) 807 e816



protonophoric uncoupler with final concentration 0.5 mM at

37 C for 5 min. The foregoing steps were then repeated.

Images were background-corrected before analysisand theav-

erage fluorescence intensity per mm2 of cell area was calculated

using the analysis software package supplied by Zeiss.

2.3. Assessment of mitochondrial membrane potential ( Dj), cell death rate and apoptosis rate

Mitochondrial membrane potential (Dj) changes were de-

tected using the mitochondrial membrane potential-sensitive

fluorescent dye 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzi-

midazolyl-carbocyanine iodide (JC-1; Molecular Probes).

The endothelial cells and pericytes cultured in different glu-

cose were collected, pelleted, washed and resuspended in

PBS. For each sample, cells were suspended in 1 ml warm

PBS at approximately 1 Â 106 cells/ml containing 3 mM JC-1

and the cellsincubated at 37 C,5%CO2 for 30 min. After load-

ing, the cells were washed twice with PBS and analyzed by flow

cytometry (Beckman Coulter XL-4) (excitation at 488 nm, witha 525 nm band-pass filter to collectgreen emission and a 590 nm

band-pass filter to collect orange emission). For the control tube,

CCCP (Sigma) was added with final concentration 50 mM and

the cells incubated at 37 C for 5 min. Using the CCCP-treated

sample, standard compensation was performed. Mitochondrial

membrane potential was indicated by the orange/green fluores-

cence intensity ratio.

Apoptosis and cell death rate were measured using Alexa

Fluor 488-conjugated annexin V and propidium iodide (PI)

labeling (Molecular Probes), respectively. The endothelial cells

and pericytes cultured in different glucose concentrations were

harvested and washed twice in cold PBS and suspendedin 0.5 ml annexin-binding buffer with Alexa Fluor 488-

conjugated annexin V (1:10 final dilution) and 10 mg/ml PI at

room temperature for 15 min. Fluorescence was measured by

a flow cytometer (Beckman Coulter XL-4) as soon as possible.

The emission wavelength was 530 nm and 675 nm, respectively.

Apoptotic cells show green fluorescence and dead cells show

both red and green fluorescence. Final analysis was accom-

plished on a PC computer with WinMDI 2.9 software.

Six samples in each group were tested. Correlation between

Dj, cell death rate and ROS was analyzed.

2.4. Detection of UCPs and MnSOD mRNA by

semi-quantitative RT e PCR

Total RNAs were extracted from each 3.5 mm culture plate

of passage 4 endothelial cells and pericytes using a TRIzol

Max Kit (Invitrogen, CA, USA). Five micrograms of RNA

was reverse transcribed using a commercial kit (Promega,

Madison, WI). Samples of cDNA were subjected to UCP1,

UCP2, UCP3 and MnSOD amplification with b-actin as

a housekeeping gene. The primers were designed using Primer

Express software (PerkineElmer) as shown in Table 1. Reac-

tions were performed in 50 ml volume containing 5 ml buffer,

1 ml of 10 mM of dNTP, 25 mmol each amplification primer,

5 U Taq polymerase and 10 ml RT product. Subsequent cycles

of PCR were performed using the following conditions: 95 Cfor 5 min, then 30 cycles of 1 min denaturation at 94 C,

1 min annealing at each T m (Table 1), 1 min extension at

72 C, and a 10-min terminal extension at 70 C. PCR prod-

ucts were electrophoresed on 1.5% agarose gel and imaged

by Alphaimager 2200 (Alpha Innotech, CA, USA). Quantita-

tive analysis was achieved by measuring the integrated density

values (IDV) of PCR products in gel photographs using Al-

phaEase FC 3.12 Image software.

2.5. Statistics

Data are reported as the mean Æ SE. Data in two groupswere analyzed with an independent-samples t -test. Multiple

comparisons used one-way ANOVA. The relationship between

two factors was analyzed by bivariate correlate analysis. The

minimum level of significance was set at P < 0.05.

3. Results

3.1. Cell culture, identification and UCP expressions

Pericytes and endothelial cells grew well in the selective

media with their morphology characteristics (Wong et al.,

1987; Porta et al., 1994). In these selective media, preparations

became pure gradually after 3e4 passages. Pericytes were ho-

mogeneously positive for smooth muscle actin antigen and

negative for Von Willebrand (Fig. 1A,C), whereas a finely

granular cytoplasmic staining of Von Willebrand was detected

in endothelial cells and was negative for smooth muscle actin

antigen (Fig. 1B,D).

At the fourth passage, pericytes were cultured in different

glucose concentrations for 24e25 days, while endothelial cells

were cultured for 45e46 days.

UCP1 and UCP2 were detected positively by the immuno-

cytochemical method in both endothelial cells (Fig. 2A,B) and

pericytes (Fig. 2D,E). For these two kinds of cells, UCP3 ex-

pression was negative (Fig. 2C,F).

Table 1

Sequences of primers and T m values for RTePCR of bovine b-actin, UCP1e3

and MnSOD

Gene (GenBank

accession #)

Primer sequence Length

(bp)

T m(C)

b-Actin

(AY141970)

50-TCGTGATGGACTCCGGTGAC-30

and

445 61.9

50-AGCACCGTGTTGGCGTAGAG-30

UCP1

(RNUCPB24)

50-CTGGGAAGAAGACGGAACAC-30

and

221 59.9

50-ACATTTGCCAGGGTCTACAC-3 0

UCP2

(AF127029)

50-CAGTTCTACACCAAGGGCTC-30

and

332 59.9

50-GGGAGGTCGTCTGTCATTAG -30

UCP3

(NM_174210)

50-ATCATTACCCGGATTTTGGC-30

and

550 59.9

50-CCCGTTTCATCTGCTCGTAG-30

MnSOD

(S67818)

50-GGACAAATCTGAGCCCTAAC-30

and

161 59.9

50-CTCCTTATTGAAGCCGAGCC-30

809Y. Cui et al. / Experimental Eye Research 83 (2006) 807 e816

http://www.ncbi.nlm.nih.gov/












3.2. Localization of high glucose-induced

ROS to mitochondria

Confocal microscopy was used to localize the high concen-

tration glucose-induced increase of ROS production to

mitochondria. The intensity of DCF fluorescence was more in-

tense in endothelial cells (Fig. 3B,E,H) and pericytes

(Fig. 4B,E,H) that had been exposed to high levels of glucose.

Quantitative analysis of the DCF fluorescence indicated an in-

crease in the relative fluorescence intensity per mm2 of cell area

Fig. 1. Identification of cultured capillary cells. Pericytes were homogeneously positive for smooth muscle actin antigen (A) and negative for Von Willebrand (C),

whereas a finely granular cytoplasmic staining of Von Willebrand was detected in endothelial cells (B) and negative for smooth muscle actin antigen (D).

Fig. 2. UCP1 and UCP2 were detected positively by immunocytochemical method in both endothelial cells and pericytes. For these two kinds of cells, UCP3

expression was negative. (A), (B) and (C) are for endothelial cells UCP1, UCP2 and UCP3, respectively whereas (D), (E) and (F) are for pericytes UCP1,

UCP2 and UCP3, respectively.




in higher glucose as compared to the lower glucose both in en-

dothelial cells (n ¼ 6, P ¼ 0.00) and in pericytes (n ¼ 6,

P ¼ 0.01) (Fig. 5A). In contrast, there was no difference in

the MTR relative fluorescence intensity per mm2 of cell area

in three-glucose concentration both in endothelial cells

(n ¼ 6, P ¼ 0.20) and in pericytes (n ¼ 6, P ¼ 0.14)

(Fig. 3A,D,G; Fig. 4A,D,G; Fig. 5B). To determine whether

DCF fluorescence (i.e. ROS production) was localized to mito-

chondria, the two images were superimposed. Thus, those re-

gions where MTR and DCF fluorescence coincide would

appear yellow due to the combination of red and green pixels

and provide direct evidence that mitochondria are a site of

ROS production. There are significantly more distinct subcellu-

lar yellow structures in cells exposed to higher glucose as com-

pared to lower glucose, which indicates that mitochondria are

the major source of ROS production after exposure to high-

concentration glucose (Fig. 3C,F,I; Fig. 4C,F,I). DCF fluores-

cence intensity of cells cultured in 23 mM or 30 mM glucose

that were first incubated with CCCP decreased to the level of

baseline conditions (5 mM glucose) (n ¼ 6, P > 0.05). CCCP

completely prevented the ROS-inducing effect by high glucose

(Fig. 5A).

3.3. Mitochondria membrane potential ( Dj),

cell death rate and apoptosis rate

The lipophilic cationic compound 5,50,6,60-tetrachloro-

1,10,3,30-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)

possesses the unique ability to differentially label mitochon-

dria with low and high membrane potential. In mitochondria

with high membrane potential, JC-1 forms aggregates emitting

in the high orange wavelength of 590 nm. In mitochondria

with low membrane potential, JC-1 forms monomers, which

emit in the green wavelength (525e530 nm). FL1 (green fluo-

rescence from JC-1) and FL2 (orange fluorescence from JC-1)

were collected after compensation for spectral overlap. The ra-

tio of orange/green fluorescence was regarded as an index for

the mitochondrial membrane potential (Dj).

The mitochondrial membrane potential (Dj) of endothelial

cells increased with increasing glucose in media (n ¼ 6,

Fig. 3. Localization of high glucose-induced ROS production to the mitochondria using confocal microscopy. Endothelial cells were incubated in different glucose

in the presence of H2DCFDA (2 mM) and MTR (500 nM) for 1 h. (A), (B) and (C) were for 5 mM glucose; (D), (E) and (F) were for 23 mM glucose; (G), (H) and

(I) were for 30 mM glucose. Red fluorescence was MTR, which indicated mitochondria; green fluorescence was DCF, which indicated ROS sites; yellow fluores-

cence was those regions where MTR and DCF fluorescence coincide.




P ¼ 0.01), as did the cell death rate of endothelial cells (n ¼ 6,

P ¼ 0.02). But for pericytes, no difference in Dj (n ¼ 6,

P ¼ 0.65) and cell death rate (n ¼ 6, P ¼ 0.94) was detected

in media of different glucose concentrations (Fig. 6A,B). As

for apoptosis rate, both endothelial cells and pericytes showed

the same features. The apoptosis rate in high glucose concen-

trations (23 mM and 30 mM) was higher than that in low glu-

cose concentration (5 mM) (n ¼ 6, P < 0.05). There was no

significant difference between apoptosis rates in 23 mM and

30 mM glucose (n ¼ 6, P > 0.05) (Fig. 6C).

For endothelial cells, Dj was positively correlated to ROS

(r ¼ 0.98, P ¼ 0.00). Cell death rate was positively correlated

to ROS (r ¼ 0.97, P ¼ 0.00). For pericytes, no correlation re-

lationship between Dj or cell death rate and ROS was found.

3.4. UCPs and MnSOD mRNA expression in different

glucose concentrations

The RTePCR data revealed two main findings. First, UCP1

and UCP2 mRNA was expressed in passage 4 endothelial cells

and pericytes. Neither endothelial cells nor pericytes expressed

UCP3 mRNA(Fig. 7A,C). Secondly, UCP1, UCP2 and MnSOD

mRNA expression levels were affected by glucose concentra-

tion. The changing pattern of UCP1 and UCP2 mRNA expres-

sion of endothelial cells and pericytes were similar. The

expression level was greatest when the glucose concentration

was 23 mM. As glucose concentration increased to 30 mM,

UCP1 and UCP2 mRNA expression level decreased to the level

of low glucose. MnSOD mRNA expression level of endothelial

cells in 23 mM glucose was 2.22-fold the level in 5 mM glucose.

In 30 mM glucose, it decreased to the level in 5 mM glucose

(Fig. 7B). MnSOD mRNA expression level of pericytes in

23 mM glucose was 2.55-fold the level in 5 mM glucose. In

30 mM glucose, it decreased to a certain extent. But the level

was significantly greater than expression level in 5 mM glucose.

It was 1.86-fold the level in 5 mM glucose (Fig. 7D).

4. Discussion

Uncoupling proteins (UCPs) are mitochondrial transporters

present in the inner membrane of mitochondria. They belong

Fig. 4. Localization of high glucose-induced ROS production to the mitochondria using confocal microscopy. Pericytes were incubated in different concentrations

of glucose in the presence of H2DCFDA (2 mM) and MTR (500 nM) for 1 h. (A), (B) and (C) were for 5 mM glucose; (D), (E) and (F) were for 23 mM glucose;

(G), (H) and (I) were for 30 mM glucose. Red fluorescence was MTR, which indicated mitochondria; green fluorescence was DCF, which indicated ROS sites;

yellow fluorescence was those regions where MTR and DCF fluorescence coincide.




to the family of anion mitochondrial carriers. UCPs could act

as proton carriers activated by metabolites and create a shunt

between complexes of the respiratory chain and ATP synthase.

The increased leakiness of the mitochondrial inner membrane

to protons may occur to minimize superoxide production by

limiting the maximum DmHþ. Five different UCPs have al-

ready been identified: UCP1e4 and UCP5, or Brain mitochon-

drial carrier protein 1 (BMCP1). These proteins are expressed

in different tissues and play different roles in cellular metabo-

lism. Brain-specific UCP4 and UCP5 has been suggested to

play a role in apoptosis in the brain (Mattson and Kroemer,

2003; Mattson and Liu, 2003). UCP1, UCP2 and UCP3 are in-

volved in the limitation of free radical level in cells. Immuno-

cytochemistry of UCP expression in endothelial cells and

pericytes showed that UCP1 and UCP2 expression was posi-

tive in both kinds of cells, and UCP3 was negative. In these

two kinds of cells, perhaps UCP1 and UCP2 regulate the pro-

duction of ROS through proton leakage in different situations.

It is well known that ROS can damage the structure and

function of cells directly. As this study showed, ROS of endo-

thelial cells and pericytes increased following an increase in

glucose concentration of media. These ROS were mitochon-

drial in origin. DCF fluorescence intensity of cells cultured

in 23 mM or 30 mM glucose that were first incubated with

CCCP, the protonophoric uncoupler, decreased to the levelof baseline conditions. CCCP completely prevented the

ROS-inducing effect by high glucose. Thus we concluded mi-

tochondrial ROS increased inducing by high glucose levels.

ROS overproduction could activate the other pathways to-

ward diabetic microvascular complications. The intracellular

AGE formation probably reflects increased triose phosphate

levels resulting from inhibition of GAPDH by mitochondrial

overproduction of ROS (Anderson et al., 1999). Resulting

from inhibition of GAPDH, dihydroxyacetone phosphate

levels increased and PKC was activated by increasing the de

novo synthesis of DAG (Koya and King, 1998). Hyperglyce-

mia increases hexosamine pathway flux by providing more

fructose-6-phosphate for GFATd

the rate-limiting enzyme of the pathwaydand the increasing level of fructose-6-phosphate

was due to inhibition of GAPDH by ROS (Du et al., 2000).

Therefore, mitochondrial ROS overproduction is likely to be

the central factor that induced other pathways of diabetic

retinopathy.

For endothelial cells, Dj and cell death rate increased fol-

lowing the increase of glucose concentration in media. Both

Dj and cell death rate were positively correlated with mito-

chondrial ROS.

When the electrochemical potential difference generated by

the proton gradient across the inner mitochondrial membrane

is high, the lifetime of superoxide generating electron trans-port intermediates such as ubisemiquinone is prolonged. There

seems to be a threshold value above which superoxide produc-

tion is markedly increased (Young et al., 2002). Hyperglyce-

mia increases the proton gradient above this threshold value

as a result of overproduction of electron donors by the TCA

cycle (Du et al., 2001). This, as a result, causes a marked in-

crease in the production of superoxide by pericytes and endo-

thelial cells. In turn, the increase of ROS can damage

mitochondrial membrane directly or indirectly and decrease

the Dj and ROS production. So a kind of feedback accommo-

dation is formed.

For pericytes, there was no obvious difference in Dj and

cell death rate among different glucose media at the fourth

passage. The relationship between Dj and ROS could not be

simply explained by the former feedback accommodation the-

ory. Perhaps other factors are involved between them. The cell

death rate features of pericytes reflect the ageing phenomenon

of pericytes after several passages. As the component cells of

retinal capillary, pericytes and endothelial cells exhibit differ-

ent characteristics.

Apoptosis of endothelial cells and pericytes was increased

at high levels of glucose after 3 days of culture (Beltramo

et al., 2004). Several groups (Li et al., 1998; Naruse et al.,

2000; Romeo et al., 2002) have reported that cultured retinal

pericytes exposed to high levels of glucose (25e

30 mM) for

Fig. 5. Quantitative analysis of the DCF and MTR fluorescence intensity per

mm2 of cell area in endothelial cells and pericytes. Data in (A) indicated an

increased tendency of DCF intensity per mm2 of cell area following increasing

glucose concentration, both in endothelial cells (n ¼ 6, P ¼ 0.00) and in peri-

cytes (n ¼ 6, P ¼ 0.01). DCF fluorescence intensity of cells cultured in 23 mM

or 30 mM glucose that was first incubated with CCCP decreased to the level of

baseline conditions (5 mM glucose) (n ¼ 6, P > 0.05). (B) There was no dif-

ference in the MTR relative fluorescence intensity per mm2 of cell area in

three-glucose concentrations both in endothelial cells (n ¼ 6, P ¼ 0.20) and

in pericytes (n ¼ 6, P ¼ 0.14).




a period of 7 days or more show a higher rate of apoptosis than

cells grown at 5.5 mM glucose. Cultured in high glucose for

24e25 days and 45e46 days, apoptosis rates of pericytes

and endothelial cells in high glucose concentration (23 mM

and 30 mM) were higher than that in low glucose concentra-

tion (5 mM). There was no significant difference between ap-

optosis rates in 23 mM and 30 mM glucose. Variation of apoptosis rate of capillary cells exposed to high levels of glu-

cose during different periods needs further study.

Immunocytochemistry and RTePCR proved that UCP1 and

UCP2 were expressed in endothelial cells and pericytes of ret-

inal capillaries. UCP3 was not expressed. What is the signifi-

cance of the UCP expression? Mitochondria are the cellular

organelles where respiration occurs. They contain two com-

partments bound by inner and outer membranes. The outer

membrane is permeable to small metabolites, whereas the per-

meability of the inner membrane is controlled to maintain the

high electrochemical gradient created by the mitochondrial re-

spiratory chain that is necessary for energy conservation and

ATP synthesis in mitochondria. Energy produced by mito-chondrial respiration is used for ATP synthesis by oxidative

phosphorylation. Although respiration is coupled with ADP

phosphorylation, this coupling is less than perfect and may

be partially or very partially loose. The uncoupling proteins

are particular mitochondrial transporters of the inner mem-

brane that appear to be controlling the level of respiration cou-

pling. The term ‘‘uncoupling protein’’ was originally used for

UCP1, which is mainly present in mitochondria of brown ad-

ipocytes. In these cells, UCP1 acts as a proton carrier activated

by free fatty acids and creates a shunt between complexes of

the respiratory chain and ATP synthase. Activation of UCP1

enhances respiration, and the uncoupling process results ina futile cycle and dissipation of oxidation energy as heat. In

comparison to the established uncoupling and thermogenic ac-

tivities of UCP1, UCP2 and UCP3 appear to be involved in the

limitation of free radical levels in cells rather than in physio-

logical uncoupling and thermogenesis (Skulachev, 1998).

Respiration is associated with production of ROS because

the oxygen molecule is capable of accepting an additional

electron to create the superoxide ion, a more reactive form

of oxygen (Raha and Robinson, 2000). Mitochondria can pro-

duce a large part of the total ROS made in cells. Mitochondrial

ROS increased in retinal capillary cells in high-level glucose.

Being an effector of ROS production regulation by proton leak

mechanism, uncoupling protein expression was modified in

different levels of glucose. It is known that mild uncoupling

of respiration diminishes mitochondrial ROS formation. The

explanation for the control of ROS production by respiration

uncoupling is that ROS formation depends on the mitochon-

drial proton gradient and the mitochondrial potential. In other

words, a mild uncoupling of respiration may participate in an-

tioxidant defense and the UCPs are just the effectors of such

a defense mechanism (Arsenijevic et al., 2000).

Fig. 6. The mitochondrial membrane potential (Dj), cell death rate and apo-

ptosis rate of endothelial cells and pericytes in different glucose media. (A)

Dj of endothelial cells increased with increasing glucose in media (n ¼ 6,

P ¼ 0.01). But for pericytes, no change in Dj (n ¼ 6, P ¼ 0.65) was detected

in media of different glucose concentrations. (B) Cell death rate of endothelial

cells increased following the increase of glucose concentration in media

(n ¼ 6, P ¼ 0.02). For pericytes, no difference in cell death rate (n ¼ 6,

P ¼ 0.94) was detected in media of different glucose concentrations. (C) Ap-

optosis rate of endothelial cells and pericytes at high glucose concentration

(23 mM and 30 mM) was higher than that at low glucose concentration

(5 mM) (n ¼ 6, P < 0.05). There was no significant difference between apo-

ptosis rates in 23 mM and 30 mM glucose (n ¼ 6, P > 0.05) (Fig. 5C).




As this study showed, mRNAexpression of UCP1, UCP2 and

MnSOD increased as the glucose of media increased to 23 mM

from 5 mM. But when the glucose concentration increased to

30 mM, this compensatory mechanism disappeared. Only

mRNA expression of MnSOD of pericytes remained at a higher

level than low glucose. Echtay et al. (2002a,b) have proposed

a simple feedback cycle in which mitochondrial oxidative stress

acutely and chronically upregulates the proton translocation ac-

tivity of UCPs to lower theDj andthus decrease superoxide pro-

duction. When the compensatory mechanism cannot counteract

ROS overproduction, relevant pathology changes will occur.

This suggests that antioxidants targeted to mitochondria maybe potential therapies for diabetes (Echtay et al., 2002a,b). Ther-

apeutic strategies to counteract ROS through reinforcing com-

plementary action of UCPs and MnSOD should be explored.

Acknowledgements

We thank staff of the central laboratory of Shandong Pro-

vincial Hospital for cell culture assistance. This study was sup-

ported by National Nature Science Funding of China

(30471852), Project of Shanghai Science and Technology

Committee (054119561) and National Basic Research Pro-

gram of China (2005CB724302).

References

Adams, S.H., 2000. Uncoupling protein homologs: emerging views of physi-

ological function. J. Nutr. 130, 711e714.

Anderson, M.M., Requena, J.R., Crowley, J.R., Thorpe, S.R., Heinecke, J.W.,

1999. The myeloperoxidase system of human phagocytes generates N3-

(carboxymethyl) lysine on proteins. J. Clin. Invest. 104, 103e113.

Arsenijevic, D., Onuma, H., Pecqueur, C., Raimbault, S., Manning, B.,

Miroux, B., Goubern, M., Alves-Guerra, M.C., Couplan, E., Surwit, R.,

Bouillaud, F., Richard, D., Collins, S., Ricquier, D., 2000. Mice lacking

uncoupling protein-2 survive to Toxoplasma gondii infection: a link with

reactive oxygen species production and immunity. Nat. Genet. 26,

435e439.

Beltramo, E., Berrone, E., Buttiglieri, S., Porta, M., 2004. Thiamine and ben-fotiamine prevent increased apoptosis in endothelial cells and pericytes

cultured in high glucose. Diabetes Metab. Res. Rev. 20, 330e336.

Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic com-

plications. Nature 414, 813e820.

Capetandes, A., Gerritsen, M.E., 1990. Simplified methods for consistent and

selective culture of bovine retinal endothelial cells and pericytes. Invest.

Ophthalmol. Vis. Sci. 31, 1738e1744.

Du, X.L., Edelstein, D., Rossetti, L., Fantus, I.G., Goldberg, H., Ziyadeh, F.,

Wu, J., Brownlee, M., 2000. Hyperglycemia-induced mitochondrial super-

oxide overproduction activates the hexosamine pathway and induces plas-

minogen activator inhibitor-1 expression by increasing Sp1 glycosylation.

Proc. Natl. Acad. Sci. USA 97, 12222e12226.

Du, X.L., Edelstein, D., Dimmeler, S., Ju, Q., Sui, C., Brownlee, M., 2001. Hy-

perglycemia inhibits endothelial nitric oxide synthase activity by posttrans-

lational modification at the AKT site. J. Clin. Invest. 108, 1341e

1348.

Fig. 7. Expression of UCP1, UCP2 and MnSOD mRNA of bovine retinal endothelial cells and pericytes in different glucose concentrations (5 mM, 23 mM,

30 mM). Neither endothelial cells nor pericytes expressed UCP3 mRNA. The changing patterns of UCP1 and UCP2 mRNA expression of endothelial cells

and pericytes were similar. The expression level was greatest when glucose concentration was 23 mM. As glucose concentration increased to 30 mM, UCP1

and UCP2 mRNA expression level decreased to the level of low glucose. (A,B) UCPs and MnSOD mRNA expression of endothelial cells in 5 mM, 23 mM

and 30 mM glucose, respectively. MnSOD mRNA expression level of endothelial cells in 23 mM glucose was 2.22-fold the level in 5 mM glucose. In 30 mM

glucose, it decreased to the level in 5 mM glucose. (C,D) Pericytes. MnSOD mRNA expression level of pericytes in 23 mM glucose was 2.55-fold the level in

5 mM glucose. In 30 mM glucose, it decreased to 1.86-fold the level in 5 mM glucose.




Echtay, K.S., Roussel, D., St-Pierre, J., Jekabsons, M.B., Cadenas, S.,

Stuart, J.A., Harper, J.A., Roebuck, S.J., Morrison, A., Pickering, S.,

Clapham, J.C., Brand, M.D., 2002a. Superoxide activates mitochondrial

uncoupling proteins. Nature 415, 96e99.

Echtay, K.S., Murphy, M.P., Smith, R.A., Talbot, D.A., Brand, M.D., 2002b.

Superoxide activates mitochondrial uncoupling protein 2 from the matrix

side: studies using targeted antioxidants. J. Biol. Chem. 277, 47129e

47135.

Erlanson-Albertsson, C., 2002. Uncoupling proteins: a new family of proteins

with unknown function. Nutr. Neurosci. 5, 1e11.

Erlanson-Albertsson, C., 2003. The role of uncoupling proteins in the regula-

tion of metabolism. Acta Physiol. Scand. 178, 405e412.

Kim, J., Kim, K.S., Shinn, J.W., Oh, Y.S., Kim, H.T., Jo, I., Shinn, S.H., 2002.

The effect of antioxidants on glycated albumin-induced cytotoxicity in bo-

vine retinal pericytes. Biochem. Biophys. Res. Commun. 292, 1010e1016.

Koya, D., King, G.L., 1998. Protein kinase C activation and the development

of diabetic complications. Diabetes 47, 859e866.

Li, W., Liu, X., He, Z., Yanoff, M., Jian, B., Ye, X., 1998. Expression of ap-

optosis regulatory genes by retinal pericytes after rapid glucose reduction.

Invest. Ophthalmol. Vis. Sci. 39, 1535e1543.

Mattson, M.P., Kroemer, G., 2003a. Mitochondria in cell death: novel targets

for neuroprotection and cardioprotection. Trends Mol. Med. 9, 196e205.

Mattson, M.P., Liu, D., 2003b. Mitochondrial potassium channels and uncou-

pling proteins in synaptic plasticity and neuronal cell death. Biochem. Bio-

phys. Res. Commun. 304, 539e549.

Naruse, K., Nakamura, J., Hamada, Y., Nakayama, M., Chaya, S., Komori, T.,

Kato, K., Kasuya, Y., Miwa, K., Hotta, N., 2000. Aldose reductase

inhibition prevents glucose-induced apoptosis in cultured bovine retinal

microvascular pericytes. Exp. Eye Res. 71, 309e315.

Nishikawa, T., Edelstein, D., Du, X.L., Yamagishi, S., Matsumura, T.,

Kaneda, Y., Yorek, M.A., Beebe, D., Oates, P.J., Hammes, H.P.,

Giardino, I., Brownlee, M., 2000. Normalizing mitochondrial superoxide

production blocks three pathways of hyperglycemic damage. Nature 404,

787e790.

Porta, M., Molinatti, P.A., Dosso, A.A., Williams, F.M., Brooks, R.A.,

Kohner, E.M., 1994. Growth of bovine retinal pericytes and endothelial

cells in high hexose concentrations. Diabetes Metab. 20, 25e30.

Raha, S., Robinson, B.H., 2000. Mitochondria, oxygen free radicals, disease

and ageing. TIBS 25, 502e508.

Romeo, G., Liu, W.H., Asnaghi, V., Kern, T.S., Lorenzi, M., 2002. Activation

of nuclear factor-kB induced by diabetes and high glucose regulates a proa-

poptotic program in retinal pericytes. Diabetes 51, 2241e2248.

Skulachev, V.P., 1996. Role of uncoupled and non-coupled oxidations in main-

tenance of safely low levels of oxygen and its one-electron reductants. Q.

Rev. Biophys. 29, 169e202.

Skulachev, V.P., 1998. Uncoupling: new approaches to an old problem of bio-

energetics. Biochim. Biophys. Acta 1363, 100e124.

Wong, H.C., Boulton, M., Marshall, J., Clark, P., 1987. Growth of retinal cap-

illary endothelia using pericyte conditioned medium. Invest. Ophthalmol.

Vis. Sci. 28, 1767e1775.

Young, T.A., Cunningham, C.C., Bailey, S.M., 2002. Reactive oxygen species

production by the mitochondrial respiratory chain in isolated rat hepato-

cytes and liver mitochondria: studies using myxothiazol. Arch. of Bio-

chem. and Biophys. 405, 65e72.


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