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Diabetologia (2006) 49: 766–774
DOI 10.1007/s00125-005-0102-6
ARTICLE
A. C. Calkin .M. E. Cooper .
K. A. Jandeleit-Dahm .T. J. Allen
Gemfibrozil decreases atherosclerosis in experimental
diabetes in association with a reduction
in oxidative stress and inflammation
Received: 27 June 2005 / Accepted: 19 October 2005 / Published online: 4 February 2006
#Springer-Verlag 2006
Abstract Aims/hypothesis: It is postulated that peroxi-
some proliferator-activated receptor αagonists confer
cardiovascular benefits in diabetes, independently of their
effects on lipid metabolism. We investigated putative
mechanisms responsible for these anti-atherogenic effects
in an in vivo model of diabetes-associated atherosclerosis.
Materials and methods: Control and streptozotocin-in-
duced diabetic apolipoprotein-deficient mice received gem-
fibrozil (100 mg kg
−1
day
−1
) or no treatment for 20 weeks.
Aortic plaque deposition was assessed by Sudan IV staining
and subsequent en face quantification. Superoxide produc-
tion was measured using lucigenin-enhanced chemilumi-
nescence. Markers of pathways including inflammation and
oxidative stress were measured using real-time RT-PCR.
Results: No significant effect of gemfibrozil was observed
on glycated haemoglobin, cholesterol or insulin in diabetic
mice. Diabetes was associated with a three-fold increase in
plaque area and a significant increase in NADPH-dependent
superoxide compared with control mice. Gemfibrozil signif-
icantly attenuated plaque area and superoxide production in
diabetic mice. In addition, gemfibrozil reduced the expres-
sion of the genes encoding the NADPH oxidase subunits
p47phox, gp91phox and Rac-1. In addition, gemfibrozil
reduced the expression of the genes encoding nuclear factor
kappa B (NF-κB) subunit, p65, the NF-κB-dependent
chemokine monocyte chemoattractant protein-1, and tissue
factor. Conclusions/interpretations: This study demonstrates
that gemfibrozil exerts anti-atherogenic actions, indepen-
dently of changes in cholesterol and glucose metabolism.
Such findings emphasise the possible usefulness of fibrates
such as gemfibrozil in a setting of atherosclerosis even in
the absence of dyslipidaemia.
Keywords ApoE−/−mouse .Atherosclerosis .
Inflammation .Lipids .NAD(P)H oxidase
Abbreviations AGII: angiotensin II .ApoE−/−:
apolipoprotein-E-deficient .AGTR1: angiotensin II
receptor subtype 1 .GHb: glycated haemoglobin .MCP-1:
monocyte chemoattractant protein-1 .MMP: matrix
metalloproteinase .NF-κB: nuclear factor kappa B .
PPAR: peroxisome proliferator-activated receptor .AGER:
advanced glycation end product-specific receptor .
ROS: reactive oxygen species
Introduction
Fibrates are peroxisome proliferator-activated receptor
(PPAR) αagonists primarily used for the treatment of
dyslipidaemia. These agents regulate lipid metabolism by
reducing triglycerides and increasing HDL cholesterol as
well as by modulating LDL cholesterol, shifting the balance
towards large buoyant, less-atherogenic particles [1,2].
More recently it has been postulated that fibrates exert anti-
atherogenic actions independently of their effects on lipid
metabolism. This is supported by studies demonstrating
PPARαexpression in cells of the vessel wall, including
endothelial cells [3], smooth muscle cells [4]andmacro-
phages [5]. Furthermore, in vitro studies have demonstrated
that fibrates reduce a variety of mediators implicated in
atherosclerotic plaque development, including proteins in-
volved in cell recruitment [6], cell adhesion [7], cell
migration [8], foam cell formation [9] and plaque stability
Electronic Supplementary Material Supplementary material
is available for this article at http://dx.doi.org/10.1007/s00125-
005-0102-6
A. C. Calkin (*).M. E. Cooper .
K. A. Jandeleit-Dahm .T. J. Allen
Diabetes Complications Laboratory,
Baker Heart Research Institute,
P.O. Box 6492, St Kilda Rd Central,
Melbourne 8008, Australia
e-mail: anna.calkin@baker.edu.au
Tel.: +613-8532-1465
Fax: +613-8532-1288
A. C. Calkin
Department of Medicine, Monash University,
Alfred Hospital,
Melbourne, Australia
[10]. In addition, polymorphisms in the PPARαgene have
been linked to alterations in the risk of cardiovascular disease
in the absence of changes in lipid levels [11].
Large clinical trials investigating the effect of fibrates on
cardiovascular disease in both diabetic and non-diabetic
populations have demonstrated reductions in cardiovascu-
lar risk factors and events [2,12–14]. However, these
benefits were noted in the presence of changes in tri-
glyceride and cholesterol levels. Both low HDL and
increased LDL concentrations have independently been
associated with increased cardiovascular risk [15]. Further-
more, there is a vast array of evidence to support a pro-
atherogenic role for LDL, particularly oxidised LDL [16]
and an anti-atherogenic role for HDL [17,18]. Thus,
improvements in the lipid profile per se would be antic-
ipated to lead to a reduction in cardiovascular risk. There-
fore, it remains to be determined whether fibrates exert
direct anti-atherogenic effects, independently of their
effects on lipid metabolism in vivo.
People with diabetes commonly have a lipid profile high
in triglycerides and low in HDL cholesterol. In addition,
they often have high levels of modified LDL (oxidised or
glycated) [19]. Furthermore, diabetes is associated with an
upregulation of the renin–angiotensin system [20], ad-
vanced glycation endproducts [21] and oxidative stress
[22], all of which have been shown to contribute towards
the development and progression of atherosclerosis. Thus,
agents that not only improve the lipid profile but which
may also independently reduce atherosclerosis are of great
promise in diabetes. Indeed, such agents may be vasopro-
tective in a setting of atherosclerosis, even in the absence of
significant dyslipidaemia.
The apolipoprotein-E-deficient (apoE−/−) mouse is a
commonly used rodent model of atherosclerosis. The in-
duction of diabetes results in a model of hyperglycaemia and
hyperlipidaemia, metabolic changes often seen in human
diabetes. In this model, plaques exhibit foamy cells and a
lipid-rich necrotic core [20], a pathology similar to that seen
in humans. This model has been used to assess the role of the
renin–angiotensin system [20,23], the advanced glycation
pathway [24] and growth factors [25] on diabetes-associated
atherosclerosis. In these studies minimal effects on the lipid
profile were observed, making this an ideal model to assess
the role of the fibrate, gemfibrozil, on atherosclerosis in the
absence of large alterations in lipid profile.
Thus, the aim of this study was to examine the potential
anti-atherosclerotic effects of gemfibrozil in the strepto-
zotocin-diabetic apoE−/−mouse, an in vivo model of dia-
betes-associated atherosclerosis where the effects of these
agents on lipids are minimised.
Materials and methods
Animals
Six-week-old apoE−/−mice (backcrossed 20 times to a
C57BL/6 background; Animal Resource Centre, Canning-
vale, WA, Australia) were housed at the Precinct Animal
Centre at AMREP, Melbourne, Australia and studied in
accordance with the National Health and Medical Research
Council and Alfred Hospital/Baker Heart Research Institute
Animal Ethics guidelines. Mice were randomised to the
following groups: control, receiving citrate buffer alone, and
rendered diabetic by i.p. injection of streptozotocin (55 mg
kg
−1
day
−1
)for5days[23]. Control and diabetic mice were
then randomised to treatment with gemfibrozil (100 mg kg
−1
day
−1
) by gavage or notreatment for 20weeks (n=22/group).
Mice had free access to standard chow and water. Mice were
killed by i.p. injection of Euthal (10 mg/kg) (Delvet, Seven
Hills, Australia) and subsequent exsanguination by cardiac
puncture. Excised aortae were cleaned of excess fat and
placed in neutral buffered formalin for analysis of the en face
plaque area, then embedded in paraffin (n=8), snap frozen in
liquid nitrogen and stored at −80°C for subsequent RNA
extraction (n=8) or used fresh to quantify vascular superox-
ide production (n=6).
Metabolic parameters
Fasting glucose, cholesterol and triglycerides were measured
using an automated system (Abbott Architecture ci8200;
Abbott Laboratories, Abbott Park, IL, USA). Fasting insulin
was measured by an RIA kit (Linco Research, St Charles,
MI, USA; intra- and intervariability [CV] were 4.8 and 7.4%,
respectively). Glycated haemoglobin (GHb) was measured
by HPLC [26].
Plaque area quantification
Plaque area was quantified as described previously [20].
Briefly, excised aortae were cleaned of adventitial fat and
then stained with Sudan IV (0.5% w/v) (Gurr, BDH, Poole,
UK). The aortae were cut longitudinally and then divided
into arch, thoracic and abdominal aorta. After pinning onto
wax, aortae were photographed using an Axiocam camera
(Zeiss, Heidelberg, Germany). Total and segmental plaque
areas were quantified as percentage area visualised red as
stained by Sudan IV. Tissue was then embedded in paraffin
blocks and 4-μm sections cut for subsequent immunohis-
tochemical analysis.
Vascular superoxide production
Vascular superoxide production was measured by lucigen-
in-enhanced chemiluminescence as established by Omar et
al. [27]. Freshly excised aortae were cleaned of adventitial
fat, cut into 2-mm sections and placed in a 96-well plate.
The origin of the aorta (arch, thoracic or abdominal) was
randomised. NADPH (125 μmol/l) was added to wells in
the presence of rotenone (100 μmol/l), a mitochondrial
complex I inhibitor (Sigma-Aldrich, St Louis, MO, USA).
Lucigenin (Sigma-Aldrich) was added to a final concen-
767
tration of 3.75 μmol/l. Luminescence readings were taken
every 6 min for 1 h and results averaged per well. Data
were expressed as relative light units per 10 mg tissue.
Real-time RT-PCR
As described previously [20], RNA was extracted from
whole aortae by homogenisation using the Trizol method
(Life Technologies, Rockville, MD, USA). RNA was
subsequently treated with DNase (DNA Removal Kit;
Ambion, Austin, TX, USA) and then the cDNA was reverse
transcribed (Pierce, Rockford, IL, USA). Quantitative real-
time RT-PCR was carried out using the Taqman system as
previously described [20] on an ABI Prism 7700 Sequence
Detector (Applied Biosystems, Foster City, CA, USA). Gene
expression was normalised to 18S mRNA. Probes and
primers were designed in Primer Express (v1.3) (see Table
S1).
Immunostaining
Sections were stained with haematoxylin and eosin to
assess plaque complexity using a standard protocol. Serial
sections were stained with the macrophage marker F4/80
(Serotec, Oxford, UK) as previously described [28].
Statistical analysis
Data were analysed by ANOVA using Statview (v5.0).
Fisher’s test of least significant difference was used for post
hoc analysis. Significance was defined as p<0.05. Results
are represented as means±SEM unless otherwise specified.
Table 1 Metabolic parameters at conclusion of the study
Parameter Control C+G Diabetic D+G
Body weight (g) 30.8±0.4 28.3±0.4* 22.8±0.5* 23.1±0.5
Glucose (mmol/l) 12.6±1.7 10.1±0.9 32.6±1.2* 35.5±1.7
GHb (%) 4.8±0.1 3.4±0.1* 16.7±0.4* 15.7±0.7
Insulin (ng/ml) 0.47±0.07 0.19±0.05* 0.20±0.05* 0.14±0.03
Triglycerides (mmol/l) 1.4±0.3 1.0±0.1 2.8±0.2* 3.4±0.7
Total cholesterol (mmol/l) 11.8±1.0 7.6±1.2 19.4±1.9* 19.8±1.8
LDL cholesterol (mmol/l) 7.9±0.7 4.6±1.1* 16.1±0.9* 14.8±1.5
HDL cholesterol (mmol/l) 2.7±0.2 2.6±0.2 3.8±0.3 3.5±0.5
Data are expressed as means±SEM. n=21–22/group for body weight; n=8/group for all other parameters
*p<0.05 compared with control mice
C+G control+gemfibrozil; D+G diabetic+gemfibrozil
Fig. 1 Plaque area as quantified
by the en face approach. aTotal
plaque area; baortic arch;
cthoracic aorta; dabdominal
aorta. Ccontrol; Ddiabetic;
C+G control+gemfibrozil; D+G
diabetic+gemfibrozil. *p<0.05
**p<0.01 ***p<0.001 vs C;
†p<0.01 ††p<0.001 vs D
768
Results
Metabolic parameters
Diabetic mice had increased plasma glucose and GHb and
decreased plasma insulin concentrations compared with
control mice. Gemfibrozil did not affect these parameters in
the streptozotocin-diabetic mice (Table 1). Diabetes was also
associated with significant increases in triglycerides, total
cholesterol and LDL cholesterol (Table 1). Gemfibrozil did
not significantly alter lipid levels in diabetic mice. In control
mice gemfibrozil significantly reduced GHb and insulin
levels and LDL cholesterol. There was no difference in HDL
cholesterol among all four groups (Table 1).
Plaque area quantification
Diabetes was associated with an approximately three-fold
increase in plaque area compared with control mice
(p<0.001; Fig. 1a). Subsequent analysis of plaque localisa-
tion revealed a significant increase in arch, thoracic and
abdominal plaque deposition in diabetic mice (Fig. 1b–d).
Gemfibrozil treatment was associated with an attenuation of
plaque deposition at all three sites within the aortae in
diabetic mice. By contrast, gemfibrozil only attenuated
plaque deposition significantly in the aortic arch of control
mice. Figure 2a–d shows representative pictures of plaque
complexity in the absence and presence of gemfibrozil
treatment. Cross-sectional analysis revealed that diabetic
Fig. 2 Representative sections
stained with haematoxylin
and eosin demonstrating plaque
complexity for control (a), con-
trol+gemfibrozil (b), diabetic
(c) and diabetic+gemfibrozil
(d) mice. Magnification ×850
Fig. 3 a Superoxide production
(RLU relative light units) as
assessed by lucigenin-enhanced
chemiluminescence. Expression
of bNAD(P)H oxidase
subunits gp91phox, cp47phox
and dRac-1 as quantified by
real-time RT-PCR. Ccontrol;
Ddiabetic; C+G control+
gemfibrozil; D+G diabetic+
gemfibrozil. *p<0.05, **p<0.01,
***p<0.001 vs C; †p<0.05 vs D
769
mice had more complex, advanced lesions with a thickened
intima and fibrous cap covering macrophages, smooth
muscle cells and cholesterol clefts, as has been previously
described by our group [20]. Gemfibrozil attenuated the
presence and complexity of plaques in both control and
diabetic mice, as seen in Fig. 2b,d.
Oxidative stress
NADPH-dependent superoxide production was increased in
the aortae of diabetic mice (Fig. 3a; p<0.01). Gemfibrozil
significantly abrogated superoxide production in both
control and diabetic mice. To further investigate the mech-
anism underlying the observed reduction in vascular super-
oxide production, we assessed the gene regulation of the
various NAD(P)H oxidase subunits. We demonstrated that
diabetes was associated with an upregulation of Cybb
(control vs diabetic, p<0.001), Ncf1 (control vs diabetic,
p<0.01) and Rac1 (p<0.05), the genes encoding gp91phox,
p47phox and Rac-1, respectively. Gemfibrozil attenuated
gene expression of each of these subunits in diabetic mice
(p<0.05; Fig. 3b–d).
Inflammation
We measured the regulation of nuclear factor kappa B
(NF-κB), a mediator of inflammation, via gene expression of
the NF-κB subunit, Rela. Diabetes was associated with an
upregulation of the expression of Rela and this was atten-
uated by gemfibrozil treatment (Table 2). We subsequently
investigated the expression of the gene (Ccl2) encoding the
chemokine monocyte chemoattractant protein-1 (MCP-1),
an NF-κB-dependent protein which has been shown to play
an important role in the initial stages of plaque development
[29]. We demonstrated an upregulation of Ccl2 gene expres-
sion in the aortae of diabetic mice which was attenuated by
gemfibrozil (p<0.001). An increase in Ccl2 gene expression
may indicate increased macrophage infiltration, and thus
immunostaining for the macrophage marker F4/80 was
undertaken [28]. Representative pictures of macrophage
staining in the vessel wall (Fig. 4a–d) demonstrate localisa-
tion of macrophages to the intima and media, which appear
to be more pronounced in the diabetic mice compared with
all other groups, consistent with enhanced Ccl2 expression.
Tissue factor (F3) has also been demonstrated to be
under the regulation of NF-κB[30]. Thus, we investigated
Table 2 Gene expression (arbitrary units) of markers and mediators
of atherosclerosis at conclusion of the study
Parameter Control C+G Diabetic D+G
Agtr1 1.2±0.2 0.4±0.1* 1.4±0.2 0.6±0.1†
Ccl2 1.2±0.2 1.2±0.3 6.0±0.8* 3.2±0.4†
Mmp2 1.1±0.1 0.5±0.1 1.8±0.2* 1.2±0.2†
Mmp9 1.1±0.2 1.2±0.4 1.4±0.2 0.6±0.1†
Rela 1.1±0.2 0.8±0.3 3.3±1.0* 0.6±0.2†
Ager 0.9±0.2 0.8±0.3 2.9±0.7* 1.2±0.3†
F3 1.0±0.1 0.6±0.2 4.1±1.1* 1.4±0.4†
Vcam1 1.4±0.4 0.9±0.3 3.0±0.7* 3.3±0.9
Data are expressed as means±SEM. n=7–8/group
*p<0.05 compared with control mice
†p<0.05 compared with diabetic mice
C+G control+gemfibrozil; D+G diabetic+gemfibrozil; VCAM1
vascular cell adhesion molecule-1
Fig. 4 Representative sections
stained with the macrophage
marker, F4/80 for control
(a), control+gemfibrozil
(b), diabetic (c) and diabetic+
gemfibrozil (d) mice.
Magnification ×850
770
the effect of gemfibrozil on F3 gene expression. Diabetes
was indeed associated with a four-fold increase in F3
expression and this was abrogated by gemfibrozil treat-
ment. Finally, we investigated the effect of fibrate therapy
on expression of the adhesion molecule, vascular cell
adhesion molecule. Whilst we observed a marked increase
in Vcam1 gene expression in diabetic mice, gemfibrozil did
not affect this parameter.
Renin–angiotensin system
Angiotensin II (AGII) has been shown to exert effects on
both inflammation and oxidative stress via the AGII receptor
subtype 1 [31,32]. In the current study we demonstrated a
reduction in Agtr1 gene expression in the aortae of both
control (p<0.05) and diabetic mice (p<0.05) with gemfibro-
zil treatment (Table 2).
Regulation of advanced glycation end-product-
specific receptor
In diabetic mice, expression of Ager, the gene encoding the
advanced glycation end product-specific receptor (AGER),
was upregulated (p<0.01) and this was abrogated by
gemfibrozil treatment (p<0.05) (Table 2).
Markers of plaque stability
Matrix metalloproteinases (MMPs) are involved in degra-
dation of extracellular matrix and both MMP2 and MMP9
have been associated with plaque stability [33]. We demon-
strated that diabetic mice had an upregulation in aortic gene
expression of Mmp2 and this was attenuated with gemfi-
brozil treatment (Table 2). Furthermore, whilst diabetes was
not associated with an alteration in Mmp9 gene expression,
gemfibrozil significantly downregulated gene expression of
this enzyme in diabetic mice (p<0.05; Table 2).
Discussion
This study clearly demonstrated that gemfibrozil attenuates
diabetes-associated atherosclerosis in the absence of readily
detectable changes in lipid or glucose metabolism. By
contrast, a range of other pathways that have been implicated
in the development of atherosclerosis, in the presence or
absence of diabetes, were influenced by gemfibrozil treat-
ment. Indeed, in aortae from our diabetic mice, gemfibrozil
treatment was associated with changes in: (1) various
markers and mediators of inflammation; (2) oxidative stress;
(3) AGER; (4) the renin–angiotensin system; and (5) MMPs
implicated in plaque stability.
While a small number of studies have assessed the role of
fibrates and PPARαin rodents, albeit in a non-diabetic
context, these studies reported conflicting findings. A study
of high-fat-fed PPARα−/−apoE−/−mice demonstrated
lower en face atherosclerotic lesions compared with PPA
Rα+/+ apoE−/−mice [34]. Whether these effects were lipid
independent could not be determined, since the genetic
deletion of PPARαwas associated with higher VLDL levels.
Fu et al. have reported that fibrate therapy in apoE−/−
mice increases fatty acid oxidation as expected. Some-
what surprisingly, they also observed an increase in plas-
ma cholesterol level and subsequent plaque deposition
[35]. In contrast, Duez et al. observed no increase in ath-
erosclerosis in the aortic sinus of younger apoE−/−mice in
the presence of increased plasma cholesterol and trigly-
ceride levels with fenofibrate treatment [36]. Further-
more, in older apoE−/−mice, in the absence of changes in
plasma lipid levels, these authors demonstrated a reduc-
tion in aortic cholesterol content. The latter study, whilst
difficult to interpret, does suggest direct effects of this
fibrate on the vessel wall. Indeed, our findings clearly
support a direct anti-atherogenic role of fibrates in the
setting of diabetes. We also observed an attenuation of
plaque deposition with gemfibrozil in control mice;
however, this was in the presence of a reduction in LDL
cholesterol and therefore cannot be attributed purely to a
direct vascular effect.
The apparent independent effects of gemfibrozil demon-
strated in the current study are supported by a number of
small clinical studies examining the role of fibrates on
endothelial function and inflammation. Playford et al. dem-
onstrated an improvement in endothelial function, as
assessed by flow-mediated dilation, which was negatively
correlated with changes in lipid profile in patients with type 2
diabetes [37]. Okapcova and Gabor demonstrated reductions
in soluble vascular cell and intercellular adhesion molecule-1
and soluble E-selectin, which were not correlated with lipid
profile in the presence or absence of diabetes [38].
Diabetes is recognised as a proinflammatory state, as
shown by increased levels of adhesion molecules, cytokines
and chemokines which accelerate the development of athero-
sclerotic plaques [38]. NF-κB is a proinflammatory tran-
scription factor which has been demonstrated to regulate
various pro-atherogenic cytokines and growth factors. PPAR
agonists are now thought to mediate many of their anti-
inflammatory actions via the transrepression pathway, inter-
fering with the activation of NF-κB and activator protein-1
pathways [39]. We have confirmed that gemfibrozil treat-
ment is associated with a reduction in Rela gene expression
in vivo. This may be a direct effect or may be secondary to an
attenuation in the aortic gene expression of Agtr1,asAGII
has been reported to be associated with increased expression
of the Rela subunit of NF-κB[32]. This effect on Rela
expression appears to be biologically relevant since altered
expression of the chemokine Ccl2, known to be under the
regulation of NF-κB[40], was also observed. MCP-1 is a
key atherogenic molecule responsible for migration of
monocytes to the intima [29], and indeed we observed
fewer macrophages in the vessel wall of gemfibrozil-
treated mice. Our findings confirm previous studies in
endothelial cells which demonstrated a role for fibrates in
771
the attenuation of MCP-1 expression [6] and suggest that
this drug may also downregulate other NF-κB-dependent
pro-atherogenic molecules.
Diabetic mice demonstrated an upregulation of AGER, as
has been shown previously [41]. We have demonstrated a
novel finding, that gemfibrozil treatment was associated with
an attenuation of Ager expression in diabetic mice. The
effect of interference with AGER has clearly been demon-
strated by Bucciarelli et al., who observed a marked re-
duction in atherosclerosis with soluble AGER treatment [24]
in association with reductions in inflammation and mono-
nuclear and smooth muscle cell activation. This finding is,
however, not totally surprising since Ager expression is
regulated by NF-κBwithanNF-κB-binding site in its
promoter region [42]. Interestingly, the converse is also true
with AGER inducing NF-κB expression [43]. In the current
study, we have demonstrated a reduction in expression of the
genes encoding AGER and NF-κB and cannot at this stage
determine which of these pathways is upstream and most
closelylinkedtoPPARαagonism.
Tissue factor has also been shown to be under the
regulation of NF-κB[30] and be upregulated in diabetes, a
state of increased thrombosis associated with plaque
rupture [24]. In the current study we observed an increase
in F3 expression in the vessels of diabetic mice and have
confirmed previous findings demonstrating the attenuation
of F3 with fibrate treatment in endothelial cells [30],
monocytes and macrophages [44].
MMPs play a role in extracellular matrix degradation. In
the setting of atherosclerosis this can lead to degradation of
the fibrous cap, promoting plaque rupture [45]. Both
MMP2 and MMP9 are produced in atherosclerotic plaques
and appear to localise around the shoulder region of the
plaque [33]. In the current study diabetes was associated
with an upregulation of Mmp2, but no change in Mmp9
expression. Gemfibrozil abrogated both Mmp2 and Mmp9
gene expression, implicating a further role for this drug in
improving plaque stability.
Oxidative stress is well recognised to promote plaque
formation via effects on cell adhesion, migration, prolifer-
ation, endothelial dysfunction and inflammation [46]. Super-
oxide, generated from NADPH oxidase, appears to be the
most abundant form of reactive oxygen species (ROS) in the
vessel wall, although other ROS formed include hydrogen
peroxide, peroxynitrite and nitrotyrosine [46]. Superoxide
production has been correlated with endothelial function and
clinical risk factors of atherosclerosis [47]. Previous lit-
erature demonstrates conflicting effects on fibrates in the in
vitro setting. Studies in macrophages demonstrate that
PPARαactivation leads to increased superoxide and hydro-
gen peroxide production via an upregulation of NADPH
oxidase subunits, p47phox, p67phox and gp91phox [48].
Conversely, studies in endothelial cells demonstrated a
decrease in p22phox and p47phox and increased antioxidant
expression with fibrate therapy [49]. In the current study we
observed an upregulation of superoxide production in the
aortae of diabetic mice. Gemfibrozil significantly attenuated
superoxide production in both control and diabetic mice
Furthermore, gemfibrozil abrogated the diabetes-induced
increase in gene expression of various NADPH oxidase
subunits including p47phox, gp91phox and Rac-1. Our
findings are consistent with those of Evans et al., who
demonstrated a reduction in oxidative stress after 3 months
of treatment withciprofibrate in subjects with type 2 diabetes
[50].
Gemfibrozil treatment also had effects on the renin–
angiotensin system with a reduction in Agtr1 gene expres-
sion in diabetic mice. AGII influences a variety of pathways
to induce pro-atherogenic effects. In the model of AGII
infusion, albeit in a non-diabetic context, fibrates have been
associated with reductions in inflammation and oxidative
stress [51].
AGII is widely recognised to increase the production of
ROS, and in particular, to enhance NADPH oxidase activity
[52]. Furthermore, ROS can activate MMPs, in particular by
activation of NADPH oxidase, and more specifically, the
p47phox subunit of NADPH oxidase [53]. Interestingly, a
recent study has demonstrated that AGII reduces MMP2 in a
p47phox-dependent manner, although these studies were
performed in cultured smooth muscle cells [31]. In the
current study we observed a reduction in Agtr1,Ncf1 and
Mmp2 gene expression. We postulate that the observed
reduction in AGTR1 expression in diabetic mice resulted
in a downregulation of AGII-mediated effects, reducing
NAD(P)H oxidase subunit expression, and thus ROS, and
subsequently MMP expression.
In diabetic mice, gemfibrozil had no effect on glucose,
GHb or insulin levels. In control mice, however, gemfi-
brozil treatment resulted in a reduction in GHb and insulin
levels. Both animal [54,55] and clinical studies [56] have
reported an insulin-sensitising effect of fibrate therapy,
which they attribute to a reduction in lipids. Moreover, Ide
and co-workers demonstrated that fibrates can regulate the
insulin receptor signalling pathway [57]. It is possible that
in contrast to the control mice, gemfibrozil had no effect on
glycaemic control in diabetic mice, since this is a state of
insulin deficiency with less opportunity for the potential
insulin-sensitising effects of the fibrate to be operative.
Thus, this study demonstrates clearly, in an in vivo model
of diabetes-associated atherosclerosis, that gemfibrozil
abrogates plaque development in the absence of significant
changes in lipid and glucose metabolism. Moreover, this
drug acts on a number of pathways to reduce inflammation
and oxidative stress and potentially to improve plaque
stability via effects on MMPs and F3. Whilst these findings
expand our understanding of the independent vascular
effects of fibrates, further studies are required to fully
elucidate specific inflammatory and oxidative stress path-
ways by which gemfibrozil confers its anti-atherogenic
effects. These might include clinical studies assessing the
effect of fibrates on markers of inflammation and endothelial
function after a short duration in which effects on lipids are
not yet apparent. Furthermore, delayed interventional studies
such as those by Bucciarelli et al. [24], which more closely
resemble the clinical setting, will assist in determining the
role of fibrates in the treatment of atherosclerosis. Finally, the
increasingly recognised pleiotropic actions of fibrates may
be highly relevant to their potential wider use in the clinic
772
and in particular to the high-risk diabetic population with or
without dyslipidaemia.
Acknowledgements A. C. Calkin is supported by a postgraduate
scholarship from the National Heart Foundation. K. A. Jandeleit-
Dahm is a recipient of a National Heart Foundation Fellowship. T. J.
Allen is an R. D. Wright Fellow of the National Health and Medical
Research Council and Diabetes Australia. The authors would like to
thank J. Pete, C. Tikellis, M. Arnstein, V. Thallas-Bonke, C. Smith
and M. Lassila for their excellent technical assistance. We would also
like to thank S. Miljavec, G. Langmaid and P. Aldersea for their
expertise in animal handling. We acknowledge the gift of gemfibrozil
compound from Pfizer Pty. Limited.
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