Interaction between the Polyol Pathway and NonEnzymatic Glycation on Mesangial Cell Gene Expression

Life Sciences 76 (2004) 445–459www.elsevier.com/locate/lifescie

Interaction between the polyol pathway and non-enzymatic

glycation on aortic smooth muscle cell migration and

monocyte adhesion

Qinghong Dana, Rachel Wonga, Sookja K. Chungb, Stephen S.M. Chungb,

Karen S.L. Lama,*

aDepartment of Medicine, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong SAR, ChinabInstitute of Molecular Biology, The University of Hong Kong, Hong Kong, China

Received 30 April 2004; accepted 10 August 2004

Abstract

We investigated for the interaction between the polyol pathway and enhanced non-enzymatic glycation, both

implicated in the pathogenesis of diabetic atherosclerosis, in the activation of aortic smooth muscle cell (SMC)

function. Mouse aortas and primary cultures of SMCs from wildtype (WT) mice and transgenic (TG) mice

expressing human aldose reductase (AR) were studied regarding changes in AR activity, and SMC gene activation,

migration and monocyte adhesion, in response to advanced glycation end-product modified BSA (AGE-BSA).

Results showed that AGE-BSA increased AR activity in both WT and TG aortas, with greater increments (p b

0.05) in TG aortas which, basally, had elevated AR activity (2.8 fold of WT). These increments were attenuated by

zopolrestat, an AR inhibitor. Similar AGE-induced increments in AR activity were observed in primary cultures of

aortic SMCs from WT and TG mice (60% and 100%, respectively, P b 0.01). Such increments were accompanied

by increases in intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1)

mRNA levels (both P b 0.05), activation of membrane-associated PKC-h1 (P b 0.05) as well as increased SMC

migration and Tamm-Horsfall protein (THP)-1 monocyte adhesion to SMCs (both p b 0.01), with all changes

being significantly greater in TG SMCs (P b 0.05) and suppressible by either zopolrestat or transfection with an

AR antisense oligonucleotide. Our findings suggest that the effects of AGEs on SMC activation, migration and

monocyte adhesion are mediated partly through the polyol pathway and, possibly, PKC activation. The greater

0024-3205/$ -

doi:10.1016/j.l

* Correspo

E-mail add

see front matter D 2004 Elsevier Inc. All rights reserved.

fs.2004.09.010

nding author. Tel.: +86 852 2855 4769; fax: +86 852 2816 2187.

ress: [email protected] (K.S.L. Lam).

Q. Dan et al. / Life Sciences 76 (2004) 445–459446

AGE-induced changes in the TG SMCs have provided further support for the dependency of such changes on

polyol pathway hyperactivity.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Aldose reductase gene; Advanced glycation end-products (AGEs); Diabetic atherosclerosis; Transgenic mouse

Introduction

Diabetes mellitus is associated with accelerated atherosclerosis, leading to the increased incidence of

coronary artery disease and stroke (Pyorala et al., 1997). The mechanisms underlying the initiation and

progression of the atherosclerotic lesions are probably complex and involve multiple pathways. While

conventional cardiovascular risk factors, such as hypertension, hyperlipidemia and obesity are known to

be more prevalent in diabetic patients, recent interventional studies have provided compelling evidence

that hyperglycemia itself also plays an important contributory role in both type 1 and type 2 diabetes

(Nathan et al., 2003; Stratton et al., 2000). Various biochemical abnormalities induced by hyper-

glycemia, such as activation of the polyol pathway, enhanced non-enzymatic glycation with formation of

advanced glycation end-products (AGEs), and altered protein kinase C (PKC) activities have been

implicated in the pathogenesis of diabetic microangiopathic complications. Recent evidence suggests

that they are also involved in the pathogenesis of diabetic atherosclerosis (Massi-Benedetti and Federici,

1999; Kasuya et al., 1999; Ruef et al., 2000; Cerami et al., 1985; Bucala and Vlassara, 1995; Berg et al.,

1997; Ross, 1999; Higashi et al., 1997; Renier et al., 2003).

The aldose reductase (AR) gene, coding for the first and rate-limiting enzyme in the polyol pathway,

has been implicated in the etiology of diabetic atherosclerosis. Inhibition of AR prevents intimal

thickening in coronary arteries of galactose-fed beagle dogs (Kasuya et al., 1999), and mitogen-induced

DNA synthesis and cell proliferation in vascular smooth muscle cells (SMCs) (Ruef et al., 2000; Ramana

et al., 2002). Mitogen stimulation of cultured human vascular SMCs is associated with upregulation of

AR expression, which is also observed in the neointima of balloon-injured rat carotid arteries (Ruef et

al., 2000). These findings suggest that the activation of AR, known to be present in patients with

diabetes, can potentially contribute to the development of diabetic atherosclerosis.

Increased non-enzymatic glycation has also been proposed to contribute to the development of

vascular complications in diabetes (Cerami et al., 1985). AGE deposits are found in atherosclerotic

plaques and myocardium of patients with diabetes (Bucala and Vlassara, 1995), and increased

circulating plasma AGEs is associated with heart stiffness in patients with type 1 diabetes (Berg et al.,

1997). SMC migration from the media to the intima, and the adhesion of circulating monocytes to

endothelial cells and SMCs are thought to be early events in the development of atherosclerosis (Ross,

1993). Incubation of AGE-BSA results in increased rabbit aortic SMC migration (Higashi et al., 1997),

and increased monocyte adhesion to bovine endothelium (Renier et al., 2003), supporting a role of AGEs

in the early stages of diabetic atherosclerosis.

There is evidence that the various biochemical pathways, activated in the presence of hyperglycemia,

may interact in potentiating the hyperglycemia-induced tissue damage. The inhibition of AR has been

shown to prevent the activation of PKC induced by high glucose and growth factors (Keogh et al., 1997;

Ishii et al., 1998; Ramana et al., 2003a). In addition to its interaction with the PKC pathway, the polyol

pathway may also promote diabetic complications through the increased production of AGEs. In both

Q. Dan et al. / Life Sciences 76 (2004) 445–459 447

diabetic rats and humans, AR inhibitors have been reported to reduce the formation of AGEs (Suarez et al.,

1988; Hamada et al., 2000; Nakamura et al., 2003). On the other hand, AGE-BSA has been shown to

increase the expression of the AR gene in cultured humanmicrovascular endothelial cells (Nakamura et al.,

2000). More recently, it was reported that methylglyoxal, a precursor of AGEs, could induce AR mRNA,

protein and activity in rat aortic SMCs (Chang et al., 2002), suggesting another mechanism whereby the

polyol pathway and non-enzymatic glycation may interact in promoting diabetic atherosclerosis.

In the present study, we investigated whether AGE-BSA could also up-regulate the expression of AR

in aortic SMCs, using aortas and primary cultures of SMCs from wildtype and human AR (hAR)

transgenic mice. The effects of interaction of the two pathways on the gene expression of intercellular

adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), PKC-h1 activation,

as well as SMC migration and THP-1 monocyte adhesion, were also examined.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffer saline (PBS), penicillin/streptomycin

solution, trypin, fetal bovine serum (FBS), bovine serum albumin (BSA, fraction V, very low endotoxin),

Trizol Reagent, random primer labeling kit and Lipofectin AMINE Plus were purchased from GIBCO

BRL Life Technologies (Gaithersburg, MD, USA). Zopolrestat (Zop) was obtained from Pfizer (Groton,

CT, USA). Mouse monoclonal anti-PKC-h1 antibody, horseradish peroxidase-conjugated anti-rabbit andanti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA).

Tamm-Horsfall protein (THP)-1 cells, a human monocyte line, were purchased from the American Type

Culture Collection (ATCC, Rockville, MD, USA). Phosphorothioate AR antisense oligonucleotide (5V-CCTGGGCGCAGTCAATGTGG-3V) andmismatched control (Scrambled) oligonucleotide (5V-GGTGA-TAGCTGACGCGGTCC-3V) from Invitrogen (Gaithersburg, MD, USA). Other reagents used in Western

blot analysis and AR activity were obtained from Sigma Chemical (St. Louis, MO, USA).

TG mice expressing hAR gene in SMCs

TG mice were generated using modifications of a protocol previously used for generating TG mice

expressing hAR in the lens (Lee et al., 1995), another of our genetically manipulated mice for studying

the pathogenesis of diabetic complications. The entire 1.4 kb hAR cDNA was inserted into the pAL1

vector (Wu et al., 1994) which contains human scavenger receptor regulatory elements. A DNA

fragment containing the human scavenger receptor-hAR hybrid gene was microinjected into oocytes

from CBA egg donors fertilized by C57BL males. Mice carrying the hAR transgene were identified by

PCR amplification of tail DNA and further confirmed by Southern blot analyses. It was anticipated that

the transgene will be expressed in various cell types which normally express the scavenger receptor, such

macrophages, renal epithelial and mesangial cells, fibroblasts and SMCs, which are known to express

scavenger receptors (Pitas, 1990; Pitas et al., 1992). PCR was performed using primers specific for hAR

(Yamaoka et al., 1995). All procedures involving animals were carried out in compliance with the Guide

for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH

Publication No. 85-23, revised 1996).


Preparation of AGE-modified bovine serum albumin (BSA)

AGE-BSAwas prepared by incubating 20% BSA and 1.67 M glucose in 0.5 M phosphate buffer (PH:

7.4) for 12 weeks at 37 8C under sterile conditions. Unincorporated sugar was removed by dialysis

against PBS. Control non-glycated BSA was obtained by incubation of the same solution in the same

conditions without glucose. AGE formation was confirmed by measurement of fluorescence at 440nm of

wavelength. Contamination of BSA by endotoxin was checked using a commercial kit for endotoxin.

(Limulus J Single Test; Wako, Osaka, Japan). Both non-glycated BSA and AGE-BSA used in this study

contained b 0.2 ng/ml of endotoxin.

Isolation of mouse thoracic aortas and primary culture of SMCs

WT and TG mice thoracic aortas and aortic SMCs were isolated by an explant method as previously

described (Yasuda et al., 2000). SMCs were maintained in DMEM containing 10% FBS supplemented

with 100 U/ml penicillin and 100 Ag/ml streptomycin. SMCs were identified by their typical bhill-and-valleyQ growth pattern, and by positive fluorescence with antibodies against a-smooth muscle actin but

no fluorescence with antibodies against factor VIII antigen and cytokeratin.

Ex vivo study

Ex vivo study was conducted as previously described (Ramana et al., 2003b). The thoracic aorta

dissected from WT and TG mice was cut into six 5 mm strips. Aortic strips with 5 to 6 animals were

pooled and divided into groups with 6 random pieces in each group. The aortic strips were incubated in

DMEM supplemented with 10% FBS containing 100 Ag/ml of AGE-BSA or control BSA for 24 hours,

in the presence or absence of 10 AM of Zop. After treatment, the samples were washed with ice-cold

PBS and homogenized in 1 ml of 0.1 M phosphate (PH: 7.4) containing proteinase inhibitor cocktail. AR

activity was measured as described below.

AR activity measurement

Tissues or cells were homogenized in 1 ml of 0.1 M phosphate (PH: 7.4) containing protease cocktail.

AR activity was measured using glyceraldehyde as substrate as described previously (Nishinaka and

Yabe-Nishimura, 2001).

RNA extraction, Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR)

Total RNA was extracted from isolated aortas and aortic SMCs with Trizol Reagent according to the

manufacturer’s instructions. Heat-denatured RNAwas transferred onto Hybond-N nylon membrane. hAR

cDNA against hAR exon 10 was labelled with [a-32P]-dCTP using a random primer labeling kit. For RT-

PCR, 2 Ag of each total RNA sample was converted into cDNA with Superscript II RNase H reverse

transcriptase (RT) and random hexamer primers according to the manufacturer’s instructions. Equal

amounts of cDNAwere amplified by PCR. The sense and antisense primers for the amplification of each

fragment were as follows: mouse endogenous AR (mAR): sense: 5V-CCCAGGTGTACCAGAATGAGA-3V; antisense: TGGCTGCAATTGCTTTGATCC-3V; expected 580 bp; hAR: sense: 5V-GCCGTAT-


CCTGCTCAACAAC-3V; antisense: 5V-ACC ACAGCCTCAAAACTCTTC-3V; expected: 252 bp.

ICAM-1: sense: 5V-GGAGCAAGACTGTGAACACG-3V; antisense: 5V-GAGAACCACTGCTAGTC-CAC-3V; expected: 435 bp; MCP-1: sense:5V-ACTGAAGCCAGCTCTCTCTTCCTC-3V; antisense: 5V-TTCCTTCTTGGGGTCAGCACAGAC-3V; expected: 274 bp; GAPDH: sense:5V-TGATGACATCAA-GAAGGTGGTGAAG-3V, antisense:5V-CCTTGGAGGCCATGTAGGCCAT-3V, expected 239 bp. PCR

conditionswere as follows: 94 8C for 1min, 57 8C for 1min, and 72 8C for 1min. A final extension of 72 8Cfor 10 min also was included. 28 cycles were performed for ICAM-1, MCP-1 and GAPDH, and 35 cycles

for hAR and mAR. Primers for GAPDH were used as the internal standard.

Immunoblot for hAR

Western blot was performed as previously described (Nakamura et al., 2001). SMCs were lysed in lyses

buffer. Equal amount of proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis and

transferred onto polyvinylidene diffuoride (PVDF) membranes. Membranes were blocked overnight with

ovalbumin and reacted with anti-hAR polyclonal antibody diluted to 1:500 overnight at 4 8C and then

incubated with horseradish peroxidase-conjugated anti-rabbit antibodies diluted to 1:5000 for 2 h at room

temperature. Immunoreactive bands were detected using DAB peroxidase substrate developing reagent.

Cell fractionation and PKC-b1 immunoblots

PKC activation was determined by the method of Nakamura et al (Nakamura et al., 2001). Briefly,

SMCs were washed, resuspended in buffer, sonicated, and homogenates were ultra-centrifuged to isolate

the plasma membrane fractions. Equal amounts of membrane protein levels in each sample were loaded

and separated by SDS-PAGE under reducing conditions, and the membrane PKC isoform of PKC-h1were visualized by Western blot using specific monoclonal antibody against PKC-h1.

SMC migration and monocyte adhesion assays

The rate of migration of SMCs was determined using a scrape/wound assay as described previously

(Hamuro et al., 2002). Briefly, SMCs from WT and TG mice were grown in 35 mm dishes until

subconfluent. SMCs monolayer was rinsed with PBS and disrupted with a sterile rubber policeman to

create a cell-free zone. The cells were washed and treated with AGE-BSA (100 Ag/ml) in the presence or

absensc of Zop (10 AM) for 24 hours. In some experiments, instead of the addition of Zop, SMCs were

transfected with AR antisense or AR scrambled oligonucleotides (1.0 AM) followed by disruption. At the

end of the incubation, the cells were washed with PBS, fixed in methnol and stained with Giema stain

(Sigma). Stained cells were examined on an inverted microscope. SMC migration was quantified by

measuring the number of migrating cells that crossed the 2.0-mm length of wounded borderline into

denuded area. Measurements were performed at three different areas per dish.

Adhesion of THP-1 monocytic cell to SMCs was assessed as previously detailed (Lynn et al., 2000).

SMCs from WT and TG mice were cultured 24 hours in 35 mm dishes. After treatment, an aliquot of

THP-1 cells (2 � 105) was added to each dish, and cells were further incubated for 1 hour. After 3

washes with complete medium, cells were fixed with 1% glutaraldehyde. The number of attached THP-1

was determined by counting 10 random fields per dish under light microscopy (�100 magnification).

Co-cultured SMCs and THP-1 cells were identified by morphology (large, spindle shaped and small,


spherical, respectively), as well as by staining with hematoxylin. Hematoxylin-stained nuclei of SMCs

were light blue, while THP-1 nuclei were dark blue/black.

Transfection with antisense oligonucleotides (Ramana et al., 2003a)

WT and TG MCs grown to 60–70% confluency in DMEM were washed with serum-free DMEM

medium 3 times, 60 minutes before transfection. The cells were incubated with 1.0 AM AR antisense or

scrambled control oligonucleotides using Lipofect AMINE plus (15 Ag/ml) as the transfection reagent as

suggested by the supplier. After 12 hours, the medium was replaced with fresh DMEM (containing 10%

FBS) for another 12 hours followed by 24 hours of incubation in serum-free DMEM before stimulation

by AGE-BSA. The effect of AR ablation on AGE-BSA induced MCP-1 and ICAM-1 mRNA

expression, SMC migration and THP-1 monocyte adhesion to SMCs were assessed by incubating the

transfected cells with AGE-BSA (100 Ag/ml) for 24 hours.

Statistical analysis

Data are expressed as mean F SEM. Student’s t test or analysis of variance (ANOVA) in conjunction

with the Newman-Keuls test was employed as applicable. Differences between groups were considered

statistically significant when p b 0.05.

Results

AR expression in WT and TG aorta and aortic SMCs

Northern blot analysis employing a cDNA probe directed against exon 10 of hAR, specific for

the hAR gene, demonstrated the presence of hAR mRNA in the aortas and aortic SMCs of TG

mice, but not in that of WT mice (Fig. 1A). The protein expression of hAR gene products in

Fig. 1. A, B. hAR expression in aorta strips and aortic SMCs of transgenic mice. (A). Northern blot analysis showing a distinct

1.4 kb band representing the human AR transcript only in TG aortas and TG aortic SMCs. (B). Western blot analysis showing

the human AR gene product with an approximate molecular weight of 39 kDa in TG SMCs.


primary cultures of TG mice aortic SMCs was also confirmed using anti-hAR polyclonal antibody

(Fig. 1B). AR activity in the TG aorta was about 2.6 folds that in WT aorta (Fig. 2A), while the

TG SMCs had about twice the AR activity of WT SMCs (Fig. 2B).

Fig. 2. A, B. Effect of AGE-BSA on AR activity in mouse aortas and aortic SMCs. (A). After incubation, mouse aortas were

harvested and the AR activity in the supernatant fraction of aortas was measured. Results represent means F SEM of 3

experiments. * p b 0.05, ** p b 0.01 vs WT aortas treated with control BSA; y p b 0.05 vs WT aortas treated with AGE-BSA

plus Zop; # p b 0.05 vs TG aortas treated control BSA or TG aortas treated with AGE-BSA plus Zop. z P b 0.01 for TG aortas

treated with AGE-BSA vs WT aortas treated with AGE-BSA. (B). After incubation, SMCs were harvested and the AR activity

in the supernatant fraction of the cell hemogenate was measured. Results represent means F SEM of six experiments. AR SO:

AR scrambled oligonucleotide; AR AO: AR antisense oligonucleotide. * p b 0.01 vs cells treated with control BSA; y pb0.05 vscells treated with AGE-BSA+Zop; # p b 0.01 vs cells treated with AGE-BSA plus transfection with AR antisense

oligonucleotide, in each group. b p b 0.01 for TG SMC vs WT SMC in corresponding values.


Effect of AGE-BSA on AR activity in mice aortas and aortic SMCs

After exposure to 100 Ag/ml of AGE-BSA for 24 hours, significant increases in AR activity

were observed in the mice aortas (p b 0.05) and SMCs (p b 0.01). The increment was signi-

ficantly greater in TG aortas and SMCs compared to their WT counterparts (p b 0.01). To further

elucidate the role of AR, we examined the effect of an AR specific inhibitor, zopolrestat (Zop), on

AGE-stimulated AR activity. Our preliminary data showed that Zop at concentration of 1 AM, 10

AM and 100 AM inhibited AR activity by 37%, 76% and 88%, respectively in WT SMCs.

However, cell toxicity was observed when 100 AM Zop was used. The increment in AR activity in

response to AGE-BSA was largely abolished by 10 AM of Zop in both WT and TG aortas and

SMCs (Fig. 2).

Transgene hAR and endogenous mouse AR (mAR) expression in AGE-treated SMCs

To verify whether AGE-BSA caused the augmented AR activity via enhanced gene expression of

transgene hAR and endogenous mouse AR (mAR), SMCs from WT and TG mice were exposed to

AGE-BSA 100 Ag/ml for 24 hours. RT-PCR was performed using the primers specific for hAR and

mAR genes. As shown in Fig. 3, the addition of AGE-BSA resulted in an increase in mAR mRNA levels

Fig. 3. A, B, C. AGE-BSA increased mouse endogenous mAR and transgene-derived hAR expression in SMCs. After

treatment, SMCs were harvested and total RNA was extracted followed by RT-PCR analysis using mAR and hAR

specific primers. (A). A representative RT-PCR gel is shown. (B). Histogram represents means F SEM of the

densitomatric scans of the mAR mRNA band from five experiments, normalized by comparison with GAPDH mRNA.

*P b 0.05 vs cells treated with control BSA in each group. (C). Histogram represents means F SEM of the densitomatric

scans of the hAR mRNA band from 5 experiments, normalized by comparison with GAPDH mRNA. *p b 0.05 vs treatment

with control BSA.


in both WT and TG SMCs (p b 0.05). hAR mRNA levels were also significantly increased (by 2.2 fold)

in TG SMCs (p b 0.05; Fig. 3). As expected, no hAR mRNA could be demonstrated in WT SMCs,

before or after AGE-BSA treatment (Fig. 3A).

MCP-1 and ICAM-1 mRNA expression in AGE-treated SMCs

We next examined the effect of AGE-BSA on the mRNA levels of MCP-1 and ICAM-1 in SMCs.

Exposure of SMCs to 100 Ag/ml of AGE-BSA for 24 hours resulted in increased MCP-1 and ICAM-1

mRNA expression (both p b 0.05), with a greater increment being observed in TG SMCs (p b 0.05

versus WT SMCs; Fig. 4). The AR inhibitor, Zop (10 AM) attenuated the AGE-BSA induced MCP-1

and ICAM-1 mRNA increments in both WT and TG SMCs.

Fig. 4. A, B, C. Expression of MCP-1 and ICAM-1 mRNA in AGE-treated SMCs. After treatment, SMCs were harvested and

total RNA was extracted followed by RT-PCR analysis. AR SO: AR scrambled oligonucleotide; AR AO: AR antisense

oligonucleotide. (A). A representive gel is shown. Lanes 1–5, WT SMCs; lanes 6–10, TG SMCs. The results shown are the ratio

of the integrated absorbance of ICAM-1 (B) and MCP-1 (C) band and the corresponding GAPDH band in arbitrary units, and

are the means F SEM for 5 independent experiments. * p b 0.05 vs cells treated with control BSA; y p b 0.05 vs cells treated

with AGE-BSA+Zop; # p b 0.05 vs cells treated with AGE-BSA plus transfection with AR AO, in each group. b p b 0.05 for

TG SMC vs WT SMC in corresponding parameters.


SMC migration and monocyte adhesion in AGE-treated SMCs

To determine whether the enhanced MCP-1 and ICAM-1 mRNA levels were associated with

significant effects on SMC migration and monocyte adhesion in response to AGE-BSA, we tested the

effects of AGE-BSA on these two parameters in SMCs. As shown in Fig. 5, AGE-BSA treatment

significantly increased SMC migration and monocyte adhesion (both p b 0.01), with TG SMCs showing

significantly greater increments in SMC migration and monocyte adhesion (p b 0.01 versus WT SMCs

for both changes). AGE-BSA-induced SMC migration and monocyte adhesion to SMCs were also

attenuated by the addition of zopolrestat.

Fig. 5. A, B. Effect of AGE-BSA on SMC migration and monocyte adhesion to SMCs. After treatment, THP-1 monocyte

adhesion (A) and SMC migration (B) were measured. AR SO: AR scrambled oligonucleotide; AR AO: AR antisense

oligonucleotide. * p b 0.01 vs cells treated with control BSA; y p b 0.01 vs cells treated with AGE-BSA + Zop; # p b 0.01 vs

cells treated with AGE-BSA plus transfection with AR AO, in each group. b p b 0.01 for TG SMC vs WT SMC, in

corresponding values.


Effect of AGE-BSA on PKC-b1 isoform expression in SMCs

To assess whether PKC activation is involved in AGE-BSA-induced SMC migration and monocyte

adhesion, we examined the membrane-bound PKC-h1 activation in SMCs from WT and TG mice in

response to AGE-BSA. As shown in Fig. 6, the SMC expression of membrane-bound PKC-h1 isoform

protein was increased (p b 0.05) after treatment with 100 Ag/ml of AGE-BSA, but the change was more

pronounced in TG SMCs (p b 0.05 versus WT SMCs). The increment in the expression of membrane-

bound PKC-h1 protein was reduced by Zop (p b 0.05) in both WT and TG SMCs.

Antisense studies

As shown in Fig. 2, antisense oligonucleotide to AR at 1 AM significantly inhibited AGE-BSA

induced AR activity both in WT and TG SMCs. Antisense inhibition of AR was also accompanied by

significant reductions in MCP-1 and ICAM-1 mRNA levels (p b 0.05 for both), membrane-bound PKC-

h1 isoform protein expression (p b 0.05), and SMC migration (p b 0.01) as well as THP-1 monocyte

Fig. 6. A, B. Effect of AGE-BSA on membrane-bound PKC-h1 expression in SMCs. After treatment, SMCs were harvested

and membrane fractions were isolated. Western blot analysis was performed using monoclonal PKC-h1 antibody. AR SO: AR

scrambled oligonucleotide; AR AO: AR antisense oligonucleotide. (A) A respresentive Western blot analysis is shown. Lanes

1–5, WT SMCs; lane 6–10, TG SMCs. (B) Relative amounts of PKC-h1 protein in SMCs. Results represent the percentage of

WT SMC plus control BSA band, and are the means F SEM for 4 independent experiments. * p b 0.05 vs cells treated with

control BSA; y p b 0.05 vs cells treated with AGE-BSA+Zop; # p b 0.05 vs cells treated with AGE-BSA plus transfection with

AR AO, in each group. b p b 0.05 for TG SMC vs WT SMC in corresponding values.


adhesion (p b 0.01), in response to AGE-BSA (Figs. 4, 5 and 6). However, sense oligonucleotide to AR

(1 AM) had no effect on the above parameters.

Discussion

This study has demonstrated, for the first time that AGEs can upregulate the gene expression of AR in

SMCs, leading to increased AR activity in cultured SMCs as well as in incubated aortic strips. In the

cultured SMCs, this increase in AR expression was accompanied by increases in membrane PKC-h1protein expression, MCP-1 and ICAM-1 gene expression, as well as SMC migration and monocyte

adhesion. The attenuation of the above AGE-induced SMC activations by zopolrestat, an AR inhibitor,

or transfection with an AR antisense oligonucleotide, suggest that the stimulatory effects of AGEs on

SMC cytokine expression and migration are mediated, at least in part, through activation of the polyol

pathway. The much greater AGE-induced enhancement of SMC cytokine expression and migration

observed in the TG SMCs, in which the elevated basal AR activity showed a further two-fold rise in the

presence of AGEs, has provided additional evidence that these AGE-induced SMC changes, commonly

seen in the early stages of atherosclerosis, are dependent on the level of activity of the polyol pathway.

Previous studies have shown that AGE-BSA can induce oxidative stress in the vascular wall (Wautier

et al., 1994) and also activate various vascular cells through stimulating the activity of the extracellular

signal-regulated kinase (ERK) pathway (Treins et al., 2001; Lander et al., 1997). It has also been

reported that activation of the ERK pathway plays a major role in the upregulation of AR expression in

rat vascular SMCs under oxidative stress (Nishinaka and Yabe-Nishimura, 2001). Similar mechanisms,

mediated through AGE-induced oxidative stress, may be responsible for the enhancement of mAR gene

expression and activity in the mouse SMCs observed in this study. Indeed, the induction of AR activity

in human vascular endothelial cells by AGE-BSA was found to be associated with the enhancement of

specific DNA binding activity for AP-1 consensus sequence in these cells (Nakamura et al., 2000). The

authors suggested that the AGE-induced increase in AR activity may be mediated through AP-1, a

transcription factor implicated in the expression of various genes in response to oxidative stress, the

binding site of which is present in the AR gene promoter (Ko et al., 1997). In this study, AGE-BSA also

significantly increased the expression of hAR mRNA in the TG SMCs, suggesting that the AGE-induced

AR hyperactivity was also mediated through mechanisms independent of the endogenous mouse AR

gene promoter. In macrophages, AGE-BSA has been shown to increase type A scavenger receptor

mRNA expression (Iwashima et al., 2000). It is possible that, in this study, AGE-BSA also increased AR

activity in the TG SMCs indirectly via stimulation of the scavenger receptor promoter linked to the hAR

transgene. Nevertheless, our observations that AGE-BSA also increased AR activity and other responses

in the WT tissues (Figs. 2–6) suggest that the AR and AGE pathways interact even in non-TG mice in

which the AR gene is not coupled to the scavenger receptor promoter.

Atherosclerosis is now recognized as an inflammatory disorder (Ross, 1999) in which the adhesion of

monocytes to the vascular endothelium and SMCs and their subsequent migration into the vessel wall

are pivotal early events in pathogenesis (Ross, 1993). Experimental evidence strongly implicates

adhesion molecules including intercellular adhesion molecule ICAM-1 and inflammatory cytokines and

chemokines including monocyte chemoattractant peptide (MCP)-1, as mediators of the subintimal

monocyte accumulation in atherosclerosis (Ross, 1993, 1999). PKC activation has been reported to

induce ICAM-1 and MCP-1 expression in endothelial cells (Vielma et al., 2003) and macrophages (Nitti


et al., 2002) respectively. On the other hand, it has been shown that polyol pathway hyperactivity

contributes to glucose-induced SMC proliferation in cultured rat aortic SMCs through the activation of

PKC (Nakamura et al., 2001). In the present study, membrane-bound PKC-h1 activation was observed

in mouse SMCs in response to AGE-BSA, with greater activation being found in the TG SMCs. Thus

PKC activation, consequent to enhanced activity of the polyol pathway, could represent one plausible

mechanism for the changes in ICAM-1 and MCP-1 gene expression, and subsequent SMC migration and

monocyte adhesion to SMCs. This was supported by the finding of a complete normalization of the

PKC-h1 activation in the presence of the AR antisense oligonucleotide. On the other hand, the partial

inhibition of PKC-h1 activation by Zop was probably related to the incomplete suppression of AR

activity by this AR inhibitor at the dosage used.

In conclusion, there is evidence from these ex vivo and in vitro studies of an interaction between the

polyol pathway and non-enzymatic glycation, at the level of the aortic SMCs, in mediating the early

events of diabetic atherosclerosis. These events, which include enhancement of the polyol pathway by

AGEs, activation of membrane-bound PKC-h1, induction of inflammatory mediators such as ICAM-1

and MCP-1, and stimulation of SMC migration and monocyte adhesion to SMCs, may be more

pronounced in individuals with an underlying cause of increased AR gene activation, either

environmental or genetic.

Acknowledgments

This work was supported by a grant from the Hong Kong Research Grant council (HKU7270/98M).

We thank Dr Christopher K Glass for the generous gift of the pAL1 vector containing human scavenger

receptor regulatory elements.

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Interaction between the Polyol Pathway and NonEnzymatic Glycation on Mesangial Cell Gene Expression

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