Pan-PPAR Agonist, Bezafibrate, Restores Angiogenesis in Hindlimb Ischemia in Normal and Diabetic Rats.

Abstract

Introduction. The aim of this study was to investigate the effect of bezafibrate as a pan-PPAR agonist on angiogenesis and serum nitrite, the main metabolite of nitric oxide (NO), vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) concentrations in hindlimb ischemia model of normal and type I diabetic rats. Methods. 28 male Wistar rats were divided into control and diabetic groups. Then, all rats underwent unilateral hindlimb ischemia. After recovery, they were randomly assigned to one of the following experimental groups: (1) control; (2) control + bezafibrate (400 mg/kg/day); (3) diabetic; (4) diabetic + beztafibrate. After three weeks, blood samples were taken and capillary density was evaluated in the gasterocnemius muscle of ischemic limb. Results. Bezafibrate increased capillary density and capillary/fiber ratio in ischemic leg of diabetic and control rats (P < 0.05). Serum VEGF and VEGFR-2 concentrations did not alter after bezafibrate administration, however, serum nitrite concentration was significantly higher in bezafibrate-treated groups than non-treated groups (P < 0.05). Discussion. It seems that bezafibrate, as a pan PPAR agonist, restores angiogenesis in hindlimb ischemic diabetic animals and is useful for prevention and/or treatment of peripheral artery disease in diabetic subjects.

Full text

Page 1

Hindawi Publishing Corporation
International Journal of Peptides
Volume 2012, Article ID 637212, 5 pages
doi:10.1155/2012/637212
Research Article
Pan-PPAR Agonist, Bezafibrate, Restores Angiogenesisin
HindlimbIschemiainNormalandDiabetic Rats
M. Khazaei,1E.Salehi,1andB. Rashidi2
1Department of Physiology, Isfahan University of Medical Sciences, Hezar Jarib Ave, Isfahan, Iran
2Department of Anatomy, Isfahan University of Medical Sciences, Isfahan, Iran
Correspondence should be addressed to M. Khazaei, khazaei@med.mui.ac.ir
Received 4 February 2012; Accepted 16 April 2012
Academic Editor: Ayman El-Faham
Copyright © 2012 M. Khazaei et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction. The aim of this study was to investigate the effect of bezafibrate as a pan-PPAR agonist on angiogenesis and serum
nitrite, the main metabolite of nitric oxide (NO), vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-
2) concentrations in hindlimb ischemia model of normal and type I diabetic rats. Methods. 28 male Wistar rats were divided
into control and diabetic groups. Then, all rats underwent unilateral hindlimb ischemia. After recovery, they were randomly
assigned to one of the following experimental groups: (1) control; (2) control+bezafibrate (400mg/kg/day); (3) diabetic; (4)
diabetic+beztafibrate. After three weeks, blood samples were taken and capillary density was evaluated in the gasterocnemius
muscle of ischemic limb. Results. Bezafibrate increased capillary density and capillary/fiber ratio in ischemic leg of diabetic and
control rats (P < 0.05). Serum VEGF and VEGFR-2 concentrations did not alter after bezafibrate administration, however, serum
nitriteconcentrationwassignificantlyhigherinbezafibrate-treatedgroupsthannon-treatedgroups(P < 0.05).Discussion.Itseems
that bezafibrate, as a pan PPAR agonist, restores angiogenesis in hindlimb ischemic diabetic animals and is useful for prevention
and/or treatment of peripheral artery disease in diabetic subjects.
1.Introduction
Peroxisome proliferator-activated receptors (PPARs) are
ligand-inducible transcription factors that regulate expres-
sion of genes involved in some biological effects including
lipid metabolism, inflammatory responses, and glucose
homeostasis [1]. There are three isotypes of PPAR super-
family: PPARα, PPARβ, and PPARγ. Today, PPAR agonists
have clinical importance in management of dyslipidemia
(such as clofibrate, a PPARα agonist) and reducing insulin
resistance and antidiabetic activity (such as rosiglitazone,
a PPARγ agonist). Bezafibrate, a PPARα agonist, is high-
affinity ligand of PPARγ and PPARβ and is considered as
a pan-PPAR agonist [2, 3]. Bezafibrate is more efficient in
reducing body weight and blood glucose than fenofibrate
in overweight mice fed with high-fat diet [4]. It also raises
HDL, reduces triglyceride, and improves insulin sensitivity
in diabetic subjects [5, 6].
Diabetes is associated with several cardiovascular abnor-
malities including abnormal angiogenesis [7]. Enhanced or
insufficient angiogenesis plays a role in some complications
of diabetes including diabetic retinopathy or impaired
wound healing, respectively [8]. In recent years, it is sug-
gestedthatPPARsmaybeinvolvedinregulatingangiogenesis
[9]; however, the role of these receptors in angiogenesis
process still remained unclear [9].
Angiogenesis is a growth of new blood vessels from
preexisting vessels and stimulated by tissue hypoxia via
activation of hypoxia-inducible factor [10]. Then, activation
oftranscriptionofseveralangiogenicfactorsoccurs.Vascular
endothelial growth factor (VEGF) is a 45kD glycopro-
tein which is involved in physiological and pathological
angiogenesis [11]. VEGF has two tyrosine kinase receptors:
VEGFreceptor-1(VEGFR-1)andVEGFreceptor-2(VEGFR-
2). VEGFR-2 is the predominant effector of proangiogenic
signaling in angiogenesis process [12]. Nitric oxide (NO) is
anotherangiogenicfactorwhichplaysakeyroleinangiogen-
esis [13] especially in postischemic revascularization [14].
Since peripheral artery disease is one of the most important
complications of diabetes, this study aimed to investigate the

Page 2

2International Journal of Peptides
0
2
4
6
8
10
12
14
Serum nitrite concentration (µmol/L)
Pre
Post
P = 0.08
P < 0.05
ControlDiabetic
Control +
bezafibrate
Diabetic +
bezafibrate
Figure 1: Serum nitrite concentration (μmol/lit) before (pre) and
after (post) experiment in the study groups. n = 7 each group.
effect of bezafibrate as a pan-PPAR agonist on angiogenesis
in hindlimb ischemia model of normal and type I diabetic
rats.
2.MaterialsandMethods
2.1. Animals. In total, 28 male Wistar rats (Pasteur Institute
of Iran) at 8–10 weeks old were studied. The animals were
kept two per cage in animal room with 12h light/dark cycle
and room temperature between 20 and 25◦C and had free
accesstowaterandfoodadlibitum.Theethicalcommitteeof
the authors’ institution approved all experimental protocol.
2.2. Hindlimb Ischemia Model and Treatment Groups. Dia-
betes was induced by single dose of intraperitoneal injec-
tion of streptozotocin (55mg/kg). Control groups received
normal saline injection with the same volume. After 48h,
blood glucose levels were measured and the animals with
blood glucose level higher than 16.7mmol/lit were con-
sidered as diabetic [15]. Then, all rats were anaesthetized
by intraperitoneal injection of ketamine (75mg/kg) and
xylazine (7.5mg/kg) and underwent unilateral hindlimb
ischemia as previously described [16, 17]. For this purpose,
femoral artery and all major branches were closed and
excised. Then, the skin was closed with 3–0 surgical suture.
Diabetic and control groups were randomly divided into
two groups: treated with bezafibrate and nontreated groups.
Bezafibrate was dissolved in corn oil and administered
400mg/kg/day by gavage on day after operation [18]. Non-
treated groups received the vehicle. The rats were assigned
to one of the following experimental groups: (1) control; (2)
control + bezafibrate; (3) diabetic; (4) diabetic + bezafibrate
(n = 7 each). Before experiment (after randomization)
and after three weeks of treatment, blood samples were
taken and centrifuged at 3000c/s for 20 minutes. Serum
sampleswerepoured in Eppendorf tubesand saved at −70◦C
for further analysis of serum nitrite, VEGF, and VEGFR-2
concentrations.
2.3. Serum Nitrite, VEGF, and VEGFR-2 Measurements. The
serum concentration of nitrite, the main metabolite of NO,
was assayed by griess reagent method (Promega Corp, USA)
according to the manufacture’s instruction. The serum levels
of VEGF and VEGR-2 were measured using a sandwich
enzyme immunoassay kit (R&D systems, USA). Serum lipids
and blood glucose concentrations were measured using
commercially available kits.
2.4. Measurement of Capillary Density. The animals were
sacrified by cervical dislocation at 21 days after treatment,
and capillary density was evaluated in the gasterocnemius
muscle of ischemic limb. The samples were embedded in
paraffin. Sections with 5μm thickness were stained with
rat monoclonal antibody against murine CD31 (Abcam,
Cambridge, UK). Capillary endothelial cells were identified
by CD31-positive cells and counted by a light microscope.
Ten microscopic fields (×400) from three different sections
in each tissue block were randomly selected, and the
number of capillaries was counted by two blinded observers.
Capillary density was expressed as the number of capillaries
per square millimeter. Capillary/muscle fiber ratio was also
expressed, because muscle atrophy or interstitial edema may
overestimate or underestimate capillary density.
2.5. Statistical Analysis. All data are expressed as mean ±
SE. The Kolmogorov-Smirnov test for evaluation of normal
distribution of data. One-way ANOVA using Tukey’s post
hoc test was used to compare data between groups. Bivariate
correlations were calculated using Pearson’s correlation
coefficient. Paired data was analyzed by paired t-test. P value
less than 0.05 was considered statistically significant.
3.Results
3.1. Serum Lipid Profile. All of the animals were survived
after induction of hindlimb ischemia. Serum lipid profile
of experimental groups is shown in Table 1. Our results
showed that bezafibrate did not change the plasma levels
of triglyceride (TG), high-density lipoprotein cholesterol
(HDL-C), low-density lipoprotein cholesterol (LDL-C), and
total cholesterol (TC) in control and diabetic animals (P >
0.05).
3.2. Serum Nitrite Concentration. Figure 1 illustrates serum
nitrite concentration in all experimental groups. Results
showed that diabetic animals had lower serum nitrite
concentration than control group (P = 0.08). Also, the
diabetic animals who received bezafibrate had higher serum
nitrite concentration than non-treated group (P < 0.05).
3.3. Serum VEGF and VEGFR-2 Concentrations. Results
showed that there were no significant differences in serum
VEGF and VEGFR-2 concentrations between diabetic and
control groups (P > 0.05). Bezafibrate administration did

Page 3

International Journal of Peptides3
Serum VEGF concentration (pg/mL)
0
20
40
60
80
100
ControlDiabeticControl +
bezafibrate
Diabetic +
bezafibrate
(a)
Serum VEGFR-2 concentration (ng/mL)
0
20
40
60
80
100
120
ControlDiabeticControl +
bezafibrate
Diabetic +
bezafibrate
(b)
Figure 2: Serum VEGF (a) and VEGFR-2 (b) concentrations in experimental groups. n = 7 each group.
Table 1: Serum lipid profile before and after experiments. There were no significant differences between bezafibrate-treated and nontreated
groups (P > 0.05).
GroupsTotal cholesterol (mg/dL)
Before
66.00 ±6.79 84.40 ±8.74
Triglyceride (mg/dL)
Before
93.16 ±6.72
63.40 ±5.88
HDL-C (mg/dL)
Before
28.33 ±4.19 44.66 ±5.01 25.56 ±1.94 28.52 ± 3.66
44.66 ±5.01 33.60 ±4.24 19.63 ±2.44 20.76 ± 2.56
LDL-C (mg/dL)
Before After AfterAfter After
Control
Control + bezafibrate 84.40 ±8.74 79.80 ±9.35
Diabetic 70.33 ±5.44 72.25 ±7.06 81.80 ± 15.02 71.25 ±12.37 26.00 ±3.55 33.40 ±2.76 19.63 ±2.44 20.76 ± 2.56
Diabetic + bezafibrate 72.25 ±7.06 85.42 ±9.18 71.25 ± 12.37 93.75 ±10.43 33.40 ±2.76 37.14 ±4.74 21.16 ±2.78 24.20 ± 5.67
Data are expressed as mean ± SE.
73.40 ±5.88
87.00 ±6.77
ControlDiabeticControl +
bezafibrate
Diabetic +
bezafibrate
P < 0.05
P < 0.05
0
0.3
0.6
0.9
1.2
1.5
1.8
Capillary : fiber ratio
Figure 3: Effect of pan-PPAR agonist, bezafibrate, on capillary
density/fiber ratio of hindlimb ischemia. n = 7 each group.
not significantly change serum VEGF and VEGFR-2 values
in control and diabetic animals (P > 0.05) (Figure 2).
3.4. Capillary Density. Figure 3 illustrates that the capillary
density/muscle fiber ratio in ischemic gastrocnemius muscle
of diabetic animals was significantly lower than control
(P < 0.05). Capillary/fiber ratio was also significantly lower
in diabetic than control group (P < 0.05). Bezafibrate
significantly restored capillary density and capillary/fiber
ratio in both diabetic and control groups (P < 0.05).
Samples of immunohistochemical stained with anti-CD31
monoclonal antibody are shown in Figure 4.
3.5. Correlation Analysis. In the correlation analysis, we
found that capillary density in skeletal muscle tissue was
positively correlated with the serum nitrite level (r = 0.55).
4.Discussion
Diabetes is one of the most important risk factors for
development of peripheral artery disease [19], and thera-
peutic angiogenesis has been considered to improve tissue
perfusion in these subjects. In this study, we found that
neovascularization in hindlimb ischemic diabetic animals
was lower than control and bezafibrate significantly restored
capillary density and serum nitrite level in diabetic hindlimb
ischemic rats.
Among many genes induced after hypoxia to increase
new blood vessel growth, the VEGF family genes and
NO synthase are most important [9]. VEGF is one the
most important growth factors which affects all aspects of
angiogenesis including matrix degradation and endothe-
lial cell migration and proliferation [8, 11, 20]. VEGFR-
2 is expressed primarily on endothelial cells and is the
predominant effector of angiogenesis signaling [12]. The
presentstudyshowedthatbezafibrateadministrationdidnot
change serum VEGF and VEGFR-2 concentrations, however,
significantly increased serum nitrite, the main metabolite of
NO,level.NOhasakeyroleinphysiologicalandpathological
neovascularization process [13, 21]. NO regulates endothe-
lial cell proliferation, migration, and apoptosis [22, 23]. It

Page 4

4International Journal of Peptides
Control
(a)
Diabetic
(b)
Control +
bezafibrate
(c)
Diabetic +
bezafibrate
(d)
Figure 4: Representative photographs of immunohistochemical staining (×400) with anti-CD31 monoclonal antibody in experimental
groups. Arrows indicates CD31-positive cells.
also mediates the angiogenic response to VEGF or other
angiogenic factors [24].
In the recent years there has been increasing evidences
that PPARs are involved in regulation of angiogenesis;
however, the exact role of these receptors still remained
controversial [9, 25, 26]. Bezafibrate is a high-affinity ligand
for all three isoforms of PPARs and considered as a pan-
PPAR agonist [2, 3]. In clinical studies, antidiabetic and
antihyperlipidemic effects of bezafibrate have been docu-
mented [5, 6]. It also prevents ischemic heart injury and
reduces coronary artery disease [27, 28]. In the present
study, we found that bezafibrate restored angiogenesis in
hindlimb ischemic diabetic rats. According to our knowl-
edge, this is the first study in this model of angiogenesis in
diabetic animals. Balance between pro- and antiangiogenic
factors regulates angiogenesis process. PPARs may modulate
angiogenesis through the action of some growth factors
and cytokines [9]. Biscetti et al. suggested that the effect of
PPARs on angiogenic process is dependent on VEGF activity
[29]. In the present study, we found that bezafibrate did
not significantly alter serum VEGF and VEGFR-2 concen-
trations in diabetic animals. Instead, it increased serum NO
level in diabetic group. A clinical study in patients with
metabolic syndrome demonstrated that bezafibrate reduced
incidence of myocardial infarction [3]. The importance
of NO in angiogenesis in hindlimb ischemia model has
been documented in previous studies [14, 17]. Thus, with
considering the positive correlation between serum nitrite
level and capillary density, our results suggest the possible
involvement of NO pathway in increased neovascularization
after bezafibrate administration.
In conclusion, while bezafibrate, as pan-PPAR agonist,
restores angiogenesis in hindlimb ischemic diabetic animals
and may be beneficial for prevention or treatment of periph-
eral artery disease in diabetic subjects, further studies for
evaluation of its impact on other angiogenic, antiangiogenic,
and endothelial markers, especially in long-term use in
human, are required.

Page 5

International Journal of Peptides5
Acknowledgment
This study was supported by a grant from the Isfahan
University of Medical Sciences (number: 188138).
References
[1] B. Desvergne and W. Wahli, “Peroxisome proliferator-acti-
vated receptors: nuclear control of metabolism,” Endocrine
Reviews, vol. 20, no. 5, pp. 649–688, 1999.
[2] T. M. Willson, P. J. Brown, D. D. Sternbach, and B. R. Henke,
“The PPARs: from orphan receptors to drug discovery,”
Journal of Medicinal Chemistry, vol. 43, no. 4, pp. 527–550,
2000.
[3] A. Tenenbaum, M. Motro, and E. Z. Fisman, “Dual and
pan-peroxisome proliferator-activated receptors (PPAR) co-
agonism: the bezafibrate lessons,” Cardiovascular Diabetology,
vol. 4, p. 14, 2005.
[4] C. Fernandes-Santos, R. E. Carneiro, L. de Souza Mendonca,
M. B. Aguila, and C. A. Mandarim-de-Lacerda, “Pan-PPAR
agonist beneficial effects in overweight mice fed a high-fat
high-sucrose diet,” Nutrition, vol. 25, no. 7-8, pp. 818–827,
2009.
[5] A. Tenenbaum, M. Motro, E. Z. Fisman et al., “Effect of
bezafibrate on incidence of type 2 diabetes mellitus in obese
patients,” European Heart Journal, vol. 26, no. 19, pp. 2032–
2038, 2005.
[6] A. Tenenbaum, E. Z. Fisman, V. Boyko et al., “Attenuation
of progression of insulin resistance in patients with coronary
artery disease by bezafibrate,” Archives of Internal Medicine,
vol. 166, no. 7, pp. 737–741, 2006.
[7] M. Simons, “Angiogenesis, arteriogenesis, and diabetes: para-
digm reassessed?” Journal of the American College of Cardiol-
ogy, vol. 46, no. 5, pp. 835–837, 2005.
[8] M. Simons, “Angiogenesis: where do we stand now?” Circula-
tion, vol. 111, no. 12, pp. 1556–1566, 2005.
[9] F. Biscetti, G. Straface, D. Pitocco, F. Zaccardi, G. Ghirlanda,
and A. Flex, “Peroxisome proliferator-activated receptors
and angiogenesis,” Nutrition, Metabolism and Cardiovascular
Diseases, vol. 19, no. 11, pp. 751–759, 2009.
[10] P. Carmeliet, “Angiogenesis in health and disease,” Nature
Medicine, vol. 9, no. 6, pp. 653–660, 2003.
[11] N. Ferrara, “Vascular endothelial growth factor,” Arteriosclero-
sis, Thrombosis, and Vascular Biology, vol. 29, no. 6, pp. 789–
791, 2009.
[12] R. S. Kerbel, “Tumor angiogenesis,” New England Journal of
Medicine, vol. 358, no. 19, pp. 2039–2049, 2008.
[13] J. P. Cook, “NO and angiogenesis,” Atherosclerosis Supple-
ments, vol. 4, no. 4, pp. 53–60, 2003.
[14] C. D. Luque, H. Vargas Robles, E. Romo, A. Rios, and B.
Escalante, “The role of nitric oxide in the post-ischemic
revascularization process,” Pharmacology and Therapeutics,
vol. 112, no. 2, pp. 553–563, 2006.
[15] Y. Taniyama, R. Morishita, M. Aoki et al., “Therapeutic
angiogenesis induced by human hepatocyte growth factor
gene in rat and rabbit hindlimb ischemia models: preclinical
study for treatment of peripheral arterial disease,” Gene
Therapy, vol. 8, no. 3, pp. 181–189, 2001.
[16] T.Murohara,T.Asahara,M.Silveretal.,“Nitricoxidesynthase
modulates angiogenesis in response to tissue ischemia,” Jour-
nal of Clinical Investigation, vol. 101, no. 11, pp. 2567–2578,
1998.
[17] A. Katayama, Y. Yamamoto, K. Tanaka et al., “Fenofibrate
enhances neovascularization in a murine ischemic hindlimb
model,” Journal of Cardiovascular Pharmacology, vol. 54, no. 5,
pp. 399–404, 2009.
[18] D. Panigrahy, A. Kaipainen, S. Huang et al., “PPARα agonist
fenofibrate suppresses tumor growth through direct and
indirect angiogenesis inhibition,” Proceedings of the National
Academy of Sciences of the United States of America, vol. 105,
no. 3, pp. 985–990, 2008.
[19] J. A. Beckman, M. A. Creager, and P. Libby, “Diabetes and
atherosclerosis epidemiology, pathophysiology, and manage-
ment,” Journal of the American Medical Association, vol. 287,
no. 19, pp. 2570–2581, 2002.
[20] N. Ferrara, “Vascular endothelial growth factor: molecular
and biological aspects,” Current Topics in Microbiology and
Immunology, vol. 237, pp. 1–30, 1999.
[21] P. Carmeliet, “Mechanisms of angiogenesis and arteriogene-
sis,” Nature Medicine, vol. 6, no. 4, pp. 389–395, 2000.
[22] K. Kawasaki, R. S. Smith Jr, C. M. Hsieh, J. Sun, J. Chao,
and J. K. Liao, “Activation of the phosphatidylinositol 3-
kinase/protein kinase Akt pathway mediates nitric oxide-
induced endothelial cell migration and angiogenesis,” Molec-
ular and Cellular Biology, vol. 23, no. 16, pp. 5726–5737, 2003.
[23] T. Murohara, B. Witzenbichler, I. Spyridopoulos et al., “Role
of endothelial nitric oxide synthase in endothelial cell migra-
tion,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol.
19, no. 5, pp. 1156–1161, 1999.
[24] D. Fukumura, T. Gohongi, A. Kadambi et al., “Predominant
roleofendothelialnitricoxidesynthaseinvascularendothelial
growth factor-induced angiogenesis and vascular permeabil-
ity,” Proceedings of the National Academy of Sciences of the
United States of America, vol. 98, no. 5, pp. 2604–2609, 2001.
[25] A. Pozzi and J. V. Capdevila, “PPARalpha ligands as anti-
tumorigenic and antiangiogenic agents,” PPAR Research, vol.
2008, Article ID 906542, 8 pages, 2008.
[26] D. Bishop-Bailey, “Peroxisome proliferator-activated receptor
beta/delta goes vascular,” Circulation Research, vol. 102, no. 2,
pp. 146–147, 2008.
[27] R. S. Elkeles, J. R. Diamond, C. Poulter et al., “Cardiovascular
outcomes in type 2 diabetes: a double-blind placebo- con-
trolled study of bezafibrate: the St. Mary’s, Ealing, Northwick
Park diabetes cardiovascular disease prevention (SENDCAP)
study,” Diabetes Care, vol. 21, no. 4, pp. 641–648, 1998.
[28] C. G. Ericsson, J. Nilsson, L. Grip, B. Svane, and A. Ham-
sten, “Effect of bezafibrate treatment over five years on
coronary plaques causing 20% to 50% diameter narrowing
(The Bezafibrate Coronary Atherosclerosis Intervention Trial
[BECAIT]),” American Journal of Cardiology, vol. 80, no. 9, pp.
1125–1129, 1997.
[29] F. Biscetti, E. Gaetani, A. Flex et al., “Selective activation
of peroxisome proliferator-activated receptor (PPAR)alpha
and PPARgamma induces neoangiogenesis through a vascular
endothelial growth factor-dependent mechanism,” Diabetes,
vol. 57, no. 5, pp. 1394–1404, 2008.