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Hypercholesterolemic diet induces vascular smooth muscle cells apoptosis insympathectomized rats via intrinsic pathway

Rafik Hachani, Houcine Dab, Anouar Feriani, Sami Saber, Mohsen Sakly,Eric Vicaut, Jacques Callebert, Richard Sercombe, Kamel Kacem

PII: S1566-0702(14)00037-XDOI: doi: 10.1016/j.autneu.2014.02.007Reference: AUTNEU 1636

To appear in: Autonomic Neuroscience: Basic and Clinical

Received date: 26 September 2013Revised date: 19 February 2014Accepted date: 26 February 2014

Please cite this article as: Hachani, Rafik, Dab, Houcine, Feriani, Anouar, Saber, Sami,Sakly, Mohsen, Vicaut, Eric, Callebert, Jacques, Sercombe, Richard, Kacem, Kamel,Hypercholesterolemic diet induces vascular smooth muscle cells apoptosis in sympathec-tomized rats via intrinsic pathway, Autonomic Neuroscience: Basic and Clinical (2014), doi:10.1016/j.autneu.2014.02.007

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Hypercholesterolemic diet induces vascular smooth muscle cells apoptosis in

sympathectomized rats via intrinsic pathway

Hachani Rafik1,2,3

, Dab Houcine1, Feriani Anouar

4, Saber Sami

5 Sakly Mohsen

1, Vicaut Eric

2,

Callebert Jacques3, Sercombe Richard

2 and Kacem Kamel

1

1Laboratoire d’Étude de Pathologies Vasculaires, Unité de Physiologie Intégrée, Faculté des

Sciences de Bizerte. 7021 Jarzouna, TUNISIA.

2Laboratoire d’Étude de la Microcirculation (EA 3509), Université Paris 7, FRANCE.

3Laboratoire de Biochimie, Hôpital Lariboisière, Paris, FRANCE.

4Laboratoire d’Ecophysiologie Animale, Faculté des Sciences de Sfax, 3000, TUNISIA.

5Faculté de Médecine de Sfax, 3000, TUNISIA.

Corresponding author

Dr Rafik Hachani

Laboratoire d’Étude de Pathologies Vasculaires, Unité de Physiologie Intégrée, Faculté des

Sciences de Bizerte. 7021 Jarzouna, TUNISIA.

E-mail: rafik.hachani@fsb.rnu.tn,

Tel: +216 52677208;

Fax: +216 76211026

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Abstract

In this study, we intend to investigate the role of hypercholesterolemic diet, a high risk factor

for atherosclerosis, on vascular cell apoptosis in rats that have been previously

sympathectomized. Thus, newborn male Wistar rats received injections of guanethidine for

sympathectomy. Sham received injections of vehicle. The two groups were fed 1%

cholesterol diet for 3 months. Sympathectomy alone group was also exploited. Apoptosis in

abdominal aortic tissue was identified by TUNEL method and conventional agarose gel

electrophoresis to detect specific DNA fragmentation. Caspases 3 and 9, Bcl-2, Bax and

cytochrome c were examined by immunoblotting. Oil Red O staining was used to reveal lipid

in the arterial wall. Vascular smooth muscle cells (VSMCs) and macrophages were identified

by immunostaining for α-smooth muscle actin and rat macrophage marker (ED1),

respectively. The efficacy of sympathectomy was evaluated by analysis of perivascular

sympathetic fibers. Our study showed that hypercholesterolemic diet, when performed in rats

with neonatal sympathectomy, 1) increased aortic TUNEL-positive cells compared to sham

and sympathectomy alone groups, 2) illustrated a typical apoptotic DNA ladder on agarose

gel electrophoresis, 3) induced Bax translocation from cytosol to mitochondria, 4) enhanced

cytochrome c release from mitochondria to cytosol, 5) increased expression of active caspases

3 and 9, and 6) decreased Bcl-2 expression. VSMCs are identified as the major cell type

exhibiting apoptosis in this model. Taken together, it can be concluded that

hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, induces

vascular cell apoptosis in an intrinsic pathway.

Keywords: sympathectomy, aorta, hypercholesterolemia, rat, apoptosis, intrinsic pathway.

Introduction

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There is increasing evidence that apoptosis in atherosclerotic lesions occurred in both early

and advanced stages. In early stages, apoptosis may delay atherosclerotic process. However,

once the plaque is formed, apoptosis may lead to plaque rupture and thrombosis (Karaflou,

2008).

Apoptosis is triggered by a number of upstream signaling pathways. The best studied of these

upstream pathways include those generated through disruption of mitochondrial membrane

potential that leads to the release of cytochrome c (Jia et al., 2001), referred to as intrinsic

pathway.

This pathway is controlled by a multigene family of Bcl-2-like proteins. Some of these

proteins such as Bcl-2 itself inhibit apoptosis (Allsopp et al., 1993) and others such as Bax

promote it (Davies, 1997). In response to apoptotic signals, Bax is redistributed from the

cytosol to the mitochondria, where it decreases membrane potential leading to

cytochrome c release and caspase activation (Jia et al., 2001). Once released from the

mitochondria, cytochrome c binds and activates procaspase 9. The clustering of procaspase 9

in this manner leads to caspase 9 activation (Elmore, 2007).

The intrinsic pathway ends at the point of the execution phase, considered the final pathway

of apoptosis. Caspase 3 appears to be the most important of the executioner caspases, cleaving

various substrates that ultimately cause the morphological and biochemical changes seen in

apoptotic cells (Slee et al., 2001).

In our previous studies, we showed that hypercholesterolemic diet when combined with

sympathectomy induces neointimal formation containing poorly differentiated VSMCs and

abnormal extracellular matrix components (Hachani et al., 2010 and 2011). However, we do

not know if hypercholesterolemic diet combined with sympathectomy triggers apoptosis of

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vascular cells, reported to be associated with a dedifferentiated VSMCs phenotype and intimal

thickening (Slomp et al., 1997). To our knowledge, this is the first study to investigate the role

of hypercholesterolemic diet on vascular cell apoptosis in rats that have been previously

sympathectomized.

To shed some light on this issue, chemical sympathectomy was conducted with guanethidine

in newborn male Wistar rats before giving them a hypercholesterolemic diet. Apoptosis in

aortic tissue was identified by TUNEL method and conventional agarose gel electrophoresis

to detect specific DNA fragmentation. Caspases 3 and 9, Bcl-2, Bax and cytochrome c were

examined by immunoblotting. SMCs and macrophages were identified by immunostaining for

α-smooth muscle actin and macrophage-specific marker (ED1), respectively. Red-Oil-O

staining was used to reveal lipid in the arterial wall. The efficacy of sympathectomy was

evaluated by analysis of perivascular sympathetic fibers.

Materials and methods

Animals

The animal protocols used for this study were approved by the University Animal Care and

Use Committee of University of Paris VII (France), the Faculty of Sciences of Bizerte

(Tunisia), and were in accordance with the United States National Institutes of Health

Guidelines for the Care and Use of Laboratory Animals.

During treatment, all animals had access to diet and water ad libitum. They were housed in

clean, dry polypropylene cages and maintained in a well ventilated animal house. Light was

controlled in a 12-h light-12-h dark cycle. The room temperature was set at 20 °C.

Every possible step was taken to reduce the number of animals used and their distress.

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Neonatal male Wistar rats received subcutaneous injections of guanethidine (Sigma, St.

Louis, MO, USA) for sympathectomy (Gua+HC group), as previously described (Hachani et

al., 2010, 2011, 2012). Sham rats received equal volumes of saline. After weaning, sham and

sympathectomized animals were fed standard rat pellets incorporating 1% cholesterol (Sigma,

St. Louis, MO, USA) for 3 months. Sympathectomy alone group (Gua) was also exploited.

Intact rats fed standard rat pellets without cholesterol were used too to verify

hypercholesterolemia in sham and Gua+HC groups.

Sampling and fluorescence labelling of catecholamine containing fibers

At the end of the experiment, rats were weighed and blood was collected for serum

cholesterol assay before sacrifice by an overdose of pentobarbital. The abdominal aorta was

rapidly removed between the renal artery level and the bifurcation of the iliac arteries. After

cleaning to eliminate blood and connective tissue, thirty six artery segments (n=12/each

group, sham, Gua and Gua+HC) were used either immediately for the visualization of

sympathetic fibers (n=6/each group) by the glyoxylic acid method as previously described

(Hachani et al., 2010 and 2011) or quickly frozen in liquid nitrogen and stored at −80 °C until

use for DNA fragmentation assay on agarose gel electrophoresis (n=6/each group).

Eighteen others (n=6/each group) were divided into two parts, one (10 mm length) was used

for immunoblotting, and the second (5 mm length) was used for Oil Red O staining, TUNEL

method, immunohistochemistry and ethidium bromide staining for nuclei. Segment destined

for immunoblotting was quickly frozen in liquid nitrogen; the other was embedded in O.C.T.

compound (Tissue Tek II, Lab-Tek Products) and stored at −80 °C until use.

Serum cholesterol measure

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To verify the hypercholesterolemia, total cholesterol concentration was enzymatically

determined in serum from intact normocholesterolemic rats, sham, Gua and Gua+HC groups

(n=6/group), as previously described (Hachani et al., 2011).

Oil Red O staining

Lipid revelation in the arterial wall was assessed by Oil Red O (ORO) staining of cross-

sections (16 μm thick) from sham and sympathectomized groups (n=6/group), as previously

described (Hachani et al., 2010).

TUNEL assay

Visualization of apoptotic DNA fragmentation was performed on abdominal aortic cross-

sections (16 μm thick) of sympathectomized and sham animals (n=6/group) by TdT-mediated

dUTP-biotin nick-end labelling (TUNEL) method, using the TUNEL Apoptosis Detection Kit

(GenScript USA Inc.) and according to the manufacturer's procedure. Sections were then

counterstained with hematoxylin for 5 min for nuclear tissue.

Cells with a brown-red nuclear labelling were defined as TUNEL positive. Positive controls

were provided by sections pretreated with DNAse I Buffer (100 U/ml) for 10 minutes at 15-

25°C to induce DNA strand degradation. In negative control experiments, TdT was omitted

from the labelling mixture, and no staining was detected.

The labelled nuclei by TUNEL and Hematoxylin staining were counted in a fixed box in three

different regions of medial and neointimal areas (Kockx et al., 1996). The percentage of

TUNEL-positive cells (TUNEL index) in each area was estimated after averaging values of

the three different regions. It was calculated by the following formula:

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TUNEL index in each region = (100%×[number of TUNEL-positive nuclei/total number of

nuclei])

Ethidium bromide staining of nuclei

To determine the cellularity of the vessel wall, aortic cross-sections (16 μm thick) from sham,

Gua and Gua+HC groups (n=6/group) were incubated with ethidium bromide (10 µg/ml;

Sigma, St. Louis, MO, USA), a nuclear fluorescent dye which labels nuclei bright red and

readily countable.

The labelled nuclei were counted in an arbitrary box of 6500 µm2

in three different regions of

medial and neointimal areas. The number of nuclei/box/area was estimated after averaging

values of the three different regions.

DNA Extraction and agarose gel electrophoresis

50mg of abdominal aorta from sham and sympathectomized groups (n=6/group) was

homogenized in liquid nitrogen using a mortar and pestle. Total tissue DNA was extracted by

the phenol and chloroform procedure, following tissue digestion steps with proteinase K and

RNase A in the presence of EDTA, as previously described (Teiger et al., 1996). DNA

concentration was determined by spectrophotometry. To quantify the degree of

oligonucleosomal DNA fragmentation in the aorta, 1μg of extracted DNA was subjected to

2% agarose gel electrophoresis, stained with ethidium bromide (0.5 μg/ml), and visualized

under UV light.

Smooth muscle α-actin and macrophage immunostaining

Abdominal aorta from sympathectomized and sham animals (n=6/group) were used to reveal

VSMCs and macrophages on serial sections (16 μm thick).

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Smooth muscle α-actin immunostaining was performed as previously described (Hachani et

al., 2010). As for macrophage staining, we used rat macrophage marker ED1 (dilution 1/200,

mouse monoclonal, Serotec) and we proceeded exactly as previously described (Law et al.,

2000). Slides were then counterstained with Mayer’s acid hematoxylin.

Microscopy

The fluorescence of catecholamines induced by the glyoxylic acid was examined under a

confocal laser-scanning microscope (Zeiss LSM 510 Meta) equipped with UV laser. The

immunofluorescence labelling of smooth muscle α-actin and the fluorescence labelling of

nuclei were examined under a confocal laser-scanning microscope (CLSM, Bio-Rad MRC

600, Microscience Division) associated with a Nikon optiphot microscope. The

immunofluorescence labelling of macrophages and the staining of Oil Red O and in situ

TUNEL procedures were examined by light microscopy. Then, images were acquired with a

color digital camera (OLYMPUS BX 50).

Preparation of Cytosolic and Mitochondrial Fractions

Preparation of cytosolic and mitochondrial fractions from sham and sympathectomized

animals (n=6/group) was performed as previously described (Penchalaneni et al., 2004).

Protein concentration was determined by the method of Lowry et al. (1951). Cytosolic and

mitochondrial fractions were used for the quantification of apoptotic proteins by western blot

analysis.

Western Blot Analysis

Western blot analysis was performed as previously described (Penchalaneni et al., 2004).

Equal amounts of proteins (30 μg) were separated by appropriate SDS-PAGE: 12% for Bcl-2,

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Bax and caspase-9; and 15% for cytochrome c and caspase-3. For the detection of proteins on

nitrocellulose membrane, we have used antibodies either from Santa Cruz Biotechnology,

Santa Cruz CA, to cytochrome c (1:1000, catalog no. SC-13156), Bcl-2 (1:1000, catalog no.

SC-492), Bax (1:1000, catalog no. SC-426), or from Abcam to caspase 3 (1:500, catalog no.

ab2302) and caspase 9 (1:500, catalog no. ab32539). Polyclonal β-tubulin antibody (catalog

no. SC-9104) and monoclonal heat shock protein 60 (HSP60) antibodies (catalog no. SC-

13115; Santa Cruz Biotechnology) were used at a dilution of 1:1000. Blots were exposed for

1 h to horseradish peroxidase-conjugated anti-rabbit IgG (caspase 3, caspase 9, Bcl-2 and β-

tubulin), or anti-mouse IgG (cytochrome c, Bax and HSP60) secondary antibodies (diluted

2000- to 5000-fold, Santa Cruz Biotechnology). The blots were rinsed, and the enhanced

chemiluminescence reagent (ECL Kit; Amersham Life Science, Piscataway, NJ) was added

and incubated for 1 min and then exposed for 1 min to X-ray film (Kodak BioMax). The

intensity of specific immunoreactive bands was quantified by a densitometric scanning

program (Image J, NIH). All replicates from each group were run in one gel, and the proteins

are expressed as a ratio of protein signal to the β-tubulin signal (for cytosolic fractions) or to

HSP60 signal (for mitochondrial fractions). Pre-stained molecular markers were used to

assess molecular weight.

Statistical analysis

Values are expressed as mean±Standard Error Mean (SEM). The data were analyzed by

analysis of variance (ANOVA). Differences were considered statistically significant at

p<0.05.

Results

Body weight and serum total cholesterol

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There was no difference in the body weight of sham, Gua and Gua+HC groups (Table 1).

Total cholesterol measured in serum at the end of the experiment was significantly increased

by 70% (p<0.01) and 32% (P<0.05) in sham (hypercholesterolemic) and sympathectomy

alone animals, respectively, compared to normocholesterolemic rats (NC). The effect of 1%

cholesterol diet on serum total cholesterol was increased further, by about 38% (P<0.05), by

combination with sympathectomy (Gua+HC) (Table 1).

Catecholamines, Oil Red O and ethidium bromide staining

The adventitia of abdominal aortae from sham group showed a well developed network of

fluorescent catecholamine containing fibers, whereas guanethidine treatment induced entire

disappearance of fluorescent catecholamine containing fibers (Fig. 1A, B, C).

Compared with sham and Gua groups, a thickened intima (NI) is developed in the form of a

streak in the Gua+HC group which was strictly labelled with ORO (Fig. 1D, E, F). Lipids

stained with ORO are limited to the thickened intima and bordering regions (Fig. 1F).

As attested by ethidium bromide staining of nuclei, the cellularity of the arterial wall is

decreased in Gua+HC group by 18% and 19% (p<0.05) in the media and neointima,

respectively, compared with sham media (Table 2). Nuclei in the medial layers typically

appeared spindle-shaped in sham, Gua and Gua+HC groups, whereas those of the neointima

(Gua+HC group) were predominantly discoid, probably because of differences in cell

orientation (Fig. 1G, H, I). No thickened intima was revealed in the sympathectomized only

rats (Gua group) where the cellularity of the arterial wall remained unchanged, compared with

sham group.

In situ apoptosis detection using TUNEL method

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In sham and Gua groups, only particularly rare TUNEL-positive cells are identified in the

media. However, in Gua+HC group, there are significantly higher numbers of TUNEL-

positive cells, compared with sham (Fig. 2A, B, C). These cells are randomly dispersed

throughout the whole media, whereas other TUNEL-positive cells are also localized at the

plaque shoulders and fibrous cap of neointima. Consequently, apoptotic index is increased

and attained approximately 16% (p<0.001) in both intimal and medial areas (Fig. 2D).

DNA fragmentation on agarose gel electrophoresis

Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal fragments

of 180-200 base pairs (bp) that are readily detected as a DNA ladder by agarose gel

electrophoresis.

In sham group, our results reveal tow scarcely perceptible apoptotic DNA fragments of 800-

and 1000bp. However, hypercholesterolemic diet, when combined with sympathectomy,

exhibits a spectacular typical DNA ladder with clearly increased intensity of DNA fragments

at ~200bp intervals (Fig. 2E). As in sham animals, sympathectomy alone group revealed a

few apoptotic DNA fragments.

Immunolabelling of VSMCs and Macrophages

In sham and sympathectomy alone groups, immunohistochemical analysis shows that α-SM

actin appears uniformly and strongly labelled in all cells of the media (Fig. 3A, B).

Consequently, the rare TUNEL-positive cells identified in the media are recognized as SMCs

in origin. Conversely, macrophages are not identified in any of the three tunics (intima, media

and adventitia) (Fig. 3D, E).

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After a hypercholesterolemic diet in rats with neonatal sympathectomy (Gua+HC), cells are

less labelled for α-SM actin (Fig. 3C). All TUNEL-positive cells are stained intensely for α-

SM actin in both medial and neointimal areas, indicating that VSMCs are the major cell type

undergoing apoptosis after a hypercholesterolemic diet in rats with neonatal sympathectomy

(Fig. 3G). TUNEL-positive cells are detected mainly at the plaque shoulders and fibrous cap

of neointima. Conversely, we failed to detect macrophage staining in the media of the

GUA+HC group (Fig. 3F). Only a few cells are identified as macrophages in the neointima;

they are revealed mainly around the lipid core and did not exhibit TUNEL positivity.

Bax, Bcl-2, cytochrome c, caspases 3 and 9 analysis

Figure 4 shows the changes in cytochrome c, Bax, Bcl-2 and caspase-3 and -9 proteins in

sham and sympathectomized (Gua and Gua+HC groups) rats.

Our results showed that Bax levels decreased in cytosol by 32% (p<0.05) and increased in

mitochondria by 34% (p<0.05), in Gua+HC group compared with sham. However, Bcl-2

protein was significantly decreased by 24% (p<0.05) in animals with neonatal sympathectomy

fed a hypercholesterolemic diet. Neither Bax nor Bcl-2 was significantly affected after

sympathectomy alone (Gua group).

Cytochrome c release from mitochondria is a critical component in the apoptotic process.

Thus, we measured cytochrome c content in mitochondrial and cytosolic fractions in sham

and sympathectomized rats. Our results revealed that cytochrome c levels decreased by 29%

(p<0.05) in mitochondria and increased in cytosol by 31% (p<0.05), in Gua+HC group

compared with sham.

Consistent with cytochrome c efflux from mitochondria, the proteolytically cleaved, active

caspases 3 (17 kDa) and 9 (35 kDa) increased by 37% (p<0.01) and 21% (p<0.05),

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respectively, in the Gua+HC group compared with sham. Our data showed also that

cytochrome c levels and active caspases 3 and 9 were unaffected after sympathectomy alone

(Gua group).

Discussion

We have previously reported that chemical sympathectomy by guanethidine, combined with

hypercholesterolemic diet, induced atherosclerosis in abdominal aorta of rats (Hachani et al,

2010 and 2011). The present study, interpreted again this background, indicates that a

hypercholesterolemic diet in rats that have been previously sympathectomized causes a

decrease in aortic cell number in both medial and neointimal areas. Our results suggest that

the reduction in cell number observed under our experimental conditions is achieved to a

great extent through apoptosis. This was evidenced by TUNEL and agarose gel

electrophoresis methods, as well as by identifying apoptotic proteins by means of western

immunoblot.

In the present study, the hypercholesterolemia was checked by dosage of serum total

cholesterol. Our results showed that diet enriched with 1% of cholesterol supplied during

three months increased the level of serum total cholesterol by 70% in sham. Similar results

were obtained with a 2% cholesterol diet given for one month in the same species (Yan et al.,

2006). We showed here that sympathectomy alone increased significantly serum total

cholesterol. These data are compatible with the study of Fronek and Turner (1980), which

demonstrated that sympathectomy induced an abnormal accumulation of lipid, a major risk

factor for atherosclerosis. Moreover, it has been reported that sympathectomy induced by 6-

OHDA increases plasmatic total cholesterol in the rat (Lelorier et al., 1976). Additionally, we

presently showed that 1% cholesterol diet is also able to potentiate the effect sympathectomy,

induced by guanethidine, on serum total cholesterol. Along with this concept, sympathectomy

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aggravates the process of atherosclerosis induced by high cholesterol diet in rabbits (Murphy

et al., 1957; Kacem et al., 2006).

The fact that sympathectomy alone or in combination with a hypercholesterolemic diet

increased serum total cholesterol incites us to know why the level of serum total cholesterol is

higher in the sympathectomized (Gua and Gua+HC) rats?

The answer comes from our previous study demonstrating that sympathectomy, when

combined with a hypercholesterolemic diet, increased both LDL-cholesterol and native LDL

particles in plasma (Hachani et al. 2012). This might indicate a reduction in LDL clearance

by receptor-mediated pathways. This hypothesis is strengthened by our previous findings that

hepatic LDL receptors, which account for 60–80% of LDL clearance (Stucchi et al., 1995),

decreased dramatically after a hypercholesterolemic diet in rats with neonatal sympathectomy,

a phenomenon which could reduce the removal and uptake of the cholesterol-enriched LDL

particles from the circulation (Hachani et al. 2012).

These results suggest that hypercholesterolemic diet in conjunction with sympathectomy

allows the blood accumulation of more lipid and cholesterol, and this might be a contributing

factor to the effects we describe here.

Our results showed that cellularity of the arterial wall remained unchanged after

sympathectomy alone. However, it was decreased after a hypercholesterolemic diet combined

with sympathectomy, in both neointimal and medial areas. This could reflect an imbalance

between cell survival and death which may reduce the arterial wall cellularity (Kockx et al.,

1996). It seems that this imbalance is related to an increased apoptotic cell death. In

accordance with this idea, it has been found that the long-lasting process of atherogenesis

involves dramatic alterations in cellularity of the arterial wall which is related to abundant

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apoptotic cell death (Liu et al., 2005). Furthermore, Bochaton-Piallat et al. (1995) have shown

that apoptosis contributes to the regulation of cellularity in experimental intimal thickening in

the rat.

To begin to verify and understand the possible implication of apoptotic cell death in the

cellularity alteration seen herein, we performed TUNEL assay. Our results showed

significantly higher numbers of TUNEL-positive cells in the aorta of rats fed a

hypercholesterolemic diet with neonatal sympathectomy, compared with sham and

sympathectomy alone groups. Consequently, TUNEL index is increased and attained

approximately 16% in both intimal and medial areas. This is in good agreement with the

percentage of cellularity loss detected after sympathectomy (18% and 19% in neointimal and

medial areas, respectively). These data suggest that increased apoptosis is responsible, at least

in part, for the large reductions in vessel wall cellularity observed under our experimental

conditions.

Different studies have used TUNEL to demonstrate that cells can die in atherosclerotic

plaques through apoptosis. However, a large variation in the percentage of TUNEL positive

nuclei has been found, ranging from less than 2% (Isner et al., 1995; Hegyi et al., 1996;

Kockx et al., 1996a and 1996b) up to 30% (Han et al., 1995; Mallat et al., 1997). The level of

apoptotic cell death is strongly related to the stage of development of the atherosclerotic

plaque (Zou et al., 1997). Therefore, a large variability can be expected when atherosclerotic

plaques of different stages are compared.

We next attempted to determine the cell types that are undergoing TUNEL positivity. We

have focused on VSMCs and macrophages, since these two cell types were identified as the

major cellular components of atherosclerotic lesions (Fuster et al., 2010).

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Notably, we find that all TUNEL-positive cells are stained intensely for α-SM actin either in

media or neointima of Gua+HC group, indicating that VSMCs are the major cell type

undergoing apoptosis after a hypercholesterolemic diet combined with sympathectomy.

Increased levels of TUNEL-positive VSMCs in the Gua+HC group suggest a defect in

clearance within the plaque micro-environment, as has been suggested in humans (Schrijvers

et al., 2005).

Concomitantly, neointima displayed faint staining for the macrophage marker ED1. Staining

is revealed mainly around the lipid core of atherosclerotic lesion, where TUNEL positivity

was absent. These results indicate that macrophages, compared to VSMCs, are implied with a

lesser extend in the composition and progression of the neointima induced by

hypercholesterolemic diet in rats that have been previously sympathectomized. Similarly,

Clarke et al. (2006) have found that VSMCs are highly effective phagocytes in the vessel

wall. Thus, unlike VSMCs, they cannot find evidence of significant sequelae of macrophage

apoptosis in established plaques. It was even shown that VSMCs have significant phagocytic

capacity, and that clearance of apoptotic bodies does not require recruitment of professional

phagocytes like macrophages (Clarke and Bennett, 2006).

The lack of intact and TUNEL-positive macrophages in the media of the three groups (sham,

Gua and Gua+HC) may not be surprising given that an infiltrating macrophage would have to

degrade and migrate through multiple layers of VSMCs surrounding the internal elastic

lamina. However, the deficiency of TUNEL-positive macrophages in the neointima is

unexpected. For example, macrophage death in established lesions would be predicted to

enlarge the necrotic core and to produce inflammation (Tabas, 2005). Thus, it is possible that

TUNEL-positive macrophages decreased after a hypercholesterolemic diet in rats with

neonatal sympathectomy to prevent secondary necrosis and inflammation.

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Although TUNEL method is continued to be used in many studies to detect apoptotic cells,

this method does not, by itself, fully discriminate between apoptosis and necrosis.

Furthermore, it has been suggested that the best way to differentiate apoptosis from necrosis is

through a combination of biochemical, identifying apoptotic proteins, and anatomical, which

recognizes DNA fragmentation, methodologies (Stadelmann and Lassmann, 2000).

Oligonucleosomal DNA fragmentation into 180- to 200-bp integer fragments is a hallmark of

apoptosis (Bortner 1995). This specific pattern of DNA fragmentation appears as a ladder of

DNA fragments after conventional agarose gel electrophoresis in various cell types

undergoing apoptosis (Bortner 1995), including VSMCs (Bennett et al., 1995).

Our results showed that a hypercholesterolemic diet in rats that have been previously

sympathectomized increased the number of DNA fragments on agarose gel electrophoresis,

compared with sham. The pattern of DNA fragmentation illustrates a typical apoptotic DNA

ladder of ~200bp intervals. These results confirm those obtained by TUNEL method, showing

that apoptotic cell death increased in the Gua+HC group compared to sham animals.

We presently showed that a hypercholesterolemic diet in rats that have been previously

sympathectomized induced Bax translocation from cytosol to mitochondria; however, it

decreased Bcl-2 concentration. This effect resulted in a significantly elevated ratio of

cytosolic Bax to Bcl-2 in this group. An elevated ratio of cell death effector Bax to the cell

death inhibitor Bcl-2 may also be indicative of apoptosis (Wu et al., 2000).

Our data demonstrate that, in Gua+HC group, there is an increase in cytochrome c release

from mitochondria to cytosol. Consequently, aortic active caspases 3 and 9 increased,

indicating that this treatment induces apoptosis in rat vascular cells through an intrinsic

signaling pathway. Our data are in accordance with previous studies reporting that vascular

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cells derived from atherosclerotic plaques are intrinsically sensitive to apoptosis, compared

with cells from normal vessels (Bennett et al., 1995).

Our data showed that sympathectomy alone (Gua group) was unable to increase apoptotic

events in rats. However, when combined with a hypercholesterolemic diet, sympathetic

denervation produced neointimal formation containing apoptotic vascular cells. These results

proved that the differences observed between animals with neonatal sympathectomy fed a

hypercholesterolemic diet (Gua+HC group) and control rats fed a hypercholesterolemic diet

(sham group) are not due to the effects of sympathectomy alone, but the contribution of the

tow treatments (sympathectomy and hypercholesterolemic diet). However, it is tempting to

speculate that some of previous studies reported that norepinephrine (sympathetic mediator)

induces apoptosis in rat (communal et al, 1998; Zaugg et al., 2000; Harrell et al., 2005). Thus,

it seems that sympathetic-dependant apoptosis is tissue- and/or context-dependant, since all

these previous studies were carried out either in vitro (we in vivo), or in non aortic tissues

(cardiomyocytes and brain), we in aorta. Moreover, it is very likely that sympathetic-

dependant apoptosis may have different intracellular transduction signal pathways, an aspect

that warrants further investigation.

Another explanation, sympathetic nervous system may play a double role in vascular cell

apoptosis (1) by mediating a direct stimulation of the programmed cell death on cells, (2) and

possibly by an indirect inhibition through an unknown factor (either locally and/or

systemically) which is altered by sympathectomy. In this context, we previously showed that

sympathectomy, when combined with a hypercholesterolemic diet, increased the oxidized

LDL concentrations in both plasma and aorta (Hachani et al., 2012). Interestingly, it was

demonstrated that low density lipoprotein can induce apoptosis in VSMCs, particularly

oxidized LDL (Nishio et al., 1996; Diez et al., 1997). The effect of oxidized LDL has been

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shown to occur particularly via ketocholesterol, possibly through the downregulation of Bcl-2

(Nishio et al., 1996).

Additionally, the role of scavenger receptors in inducing apoptosis in plaques after a

hypercholesterolemic diet in rats that have been previously sympathectomized should not be

underestimated. In this context, we have recently showed that the same treatment increased

SR-AI expression (a scavenger receptor) at the mRNA and protein levels (Hachani et al.,

2012). Intriguingly, it has been reported that engagement of SR-A pathways by modified

lipoproteins triggers apoptotic cell death in the atherosclerotic lesions (Devries-Seimon et al.,

2005).

Apoptosis within the atherosclerotic plaque may also be regulated by cell–matrix interactions.

The presence of the extracellular matrix (ECM) prevents apoptosis in many cell types, via

specific integrin-mediated signaling (McGill et al., 1997). We showed previously (Hachani et

al. 2011) that combination of a hypercholesterolemic diet with sympathectomy decreases the

amounts of collagen IV, elastin and laminin, which are involved together in the binding of

VSMCs to the ECM. Clearly, this treatment, by degrading extracellular matrix, may disrupt

the cell–matrix interaction, and therefore promotes apoptosis. This idea is strengthen by

Newby (2006) who reviewed the evidence that matrix degradation regulates migration,

proliferation and apoptosis of SMCs.

As the animals will have been almost completely sympathectomized, one could imagine that

the changes observed in the aorta could potentially result from loss of sympathetic control of

other organs, rather than a direct consequence of denervation of the aorta. Previous studies, of

us and other authors, militate against this hypothesis since local surgical sympathectomy,

which causes specific denervation, exerted the same type of intimal thickening effects on ear

arteries (Kacem et al., 1997) and aggravation of atherosclerosis on aorta (Murphy et al., 1957)

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of hyperlipidemic rabbits. Moreover, a study in monkeys (Lichtor et al., 1987) fed a

hypercholesterolemic diet found that 12 months after surgical thoracic sympathectomy there

were identical plaques (as we presently show) in the aorta. Additionally, in their review,

Azevedo and Osswald (1986) reported ultrastructural evidence of SMC dedifferentiation

towards a more secretory state, which is associated with apoptotic death, in both dog

saphenous vein and rabbit ear artery after unilateral surgical sympathectomy, and a

comparable result was obtained in dog mesenteric arteries after sympathectomy by 6-OHDA.

Intriguingly, it has been reported also that guanethidine treatment irreversibly inhibits the

development of innervation to the vasculature, without affecting the adrenal glands and the

brain (Johnson et al. 1976).

In our present study, chemical sympathectomy with guanethidine was preferred to other

chemical methods such as 6-hydroxydopamine (6-OHDA) because it induces a dramatic loss

of catecholamines in the circulation (Johnson et al., 1976). Its destructive effect is more

efficient in rats (Johnson et al., 1976). We selected chemical sympathectomy since surgical

periarterial sympathectomy may directly induce vessel wall injury, which may affect cell

survival/death cycle in the arterial wall. However, limited (surgical) as opposed to generalized

(chemical) disruption of sympathetic impulse may offer a better perception of the mechanism

involved in VSMC apoptosis, differentiating to some extent between systematic

(hemodynamic) and regional (direct) effects of sympathectomy.

By contrast to other animal species like mouse ApoE-/- (Daugherty, 2002) or humans,

hypercholesterolemia was unable to induce intimal thickening and atherosclerosis

development in rats, even though they were fed cholesterol at high concentration and for a

long period (Clowes et al., 1977; Cole et al., 1984; Sasaki et al., 1994). Moreover, the

hypercholesterolemia did not aggravate atherosclerotic lesions induced by endothelium injury

(Clowes et al., 1977). Thus, our study was carried out on rats to verify if a

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hypercholesterolemic diet, combined with sympathectomy, is able to break this protection and

provoke atherosclerotic lesions with vascular cells apoptosis.

We have examined the distribution of sympathetic fibers in the aortic arch, thoracic and

abdominal aortae (data not shown). We have found that sympathetic innervation in the aortic

arch and thoracic aorta appeared very sparsely distributed compared with the abdominal aorta

where it was very dense. Thus, the abdominal aorta was selected as the site of focus rather

than the other aortic sites.

In conclusion, the present study demonstrates that a hypercholesterolemic diet induces, in rats

that have been previously sympathectomized, aortic apoptotic death through down-regulation

of Bcl-2 and activation of caspases 3 and 9, cytochrome c and Bax pathways. VSMCs are

identified as the major cell type exhibiting apoptosis in this model. However, additional

studies will be necessary to identify the intracellular transduction signal pathway underlying

the activation of this intrinsic pathway after this treatment. Cell surface death receptor

pathway needs to be investigated too.

Acknowledgements

Rafik Hachani received a grant from the Ministry of Higher Education, Scientific Research

and Technology (Tunisia) to work on this study in the Laboratoire d'Etude de la

Microcirculation (EA 3509), Faculté de Médecine Lariboisière St-Louis, Université Paris VII,

Paris.

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Figure legends

Figure 1. Catecholamines (A, B and C), Oil Red O (D, E and F) and ethidium bromide (G, H

and I) staining in the abdominal aorta of sham (A, D and G), Gua (B, E and H) and Gua+HC

(C, E and I) groups.

Catecholamines were revealed by glyoxylic acid method. Note that fluorescent catecholamine

containing fibers are present in sham aorta (arrows) but totally absent in the

sympathectomized animals (Gua and Gua+HC groups). Lipids stained with ORO (Fig. F) are

limited to the neointima (NI) and bordering regions in the Gua+HC group. As attested by

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ethidium bromide staining of nuclei, the cellularity of the arterial wall is decreased in animals

with neonatal sympathectomy fed a hypercholesterolemic diet (Gua+HC group) (Fig. I). Nuclei in

the medial layers appeared spindle-shaped in all groups, whereas those of the neointima

(Gua+HC group) were predominantly discoid. Elastic laminae coursing in the media are

autofluorescent (solid arrows). Sham: hypercholesterolemic intact rats. Gua: rats treated with

guanethidine for sympathectomy; Gua+HC: rats treated with guanethidine for sympathectomy

and fed 1% cholesterol for three months. n=6/group. A: adventitia, M: media, L: lumen.

Bar=10μm in A, B and C; Bar=25μm in D, E, F, G, H and I.

Figure 2. TUNEL assay (A, B and C), TUNEL index (D) and agarose gel electrophoresis

analysis (E) of abdominal aorta from sham (A), Gua (B) and Gua+HC (C) groups.

In sham and Gua groups, none or rare TUNEL-positive cells are identified in the media

(outlined arrows). In Gua+HC group, there are significantly higher numbers of TUNEL-

positive cells. Consequently, TUNEL index is increased in both neointimal and medial areas.

Note that the hypercholesterolemic diet combined with sympathectomy (Gua+HC group)

exhibits, on agarose gel electrophoresis, a typical apoptotic DNA ladder of ~200bp intervals.

Sham: hypercholesterolemic intact rats. Gua: rats treated with guanethidine for

sympathectomy; Gua+HC: rats treated with guanethidine for sympathectomy and fed 1%

cholesterol for three months. Data are shown as mean values±SEM. ***<0.001 versus sham.

n=6/group. A: adventitia, M: media, L: lumen, NI: neointima, MW: molecular weight

markers. Bar=25μm.

Figure 3. Cross-sections of abdominal aorta from sham (A), Gua (B) and Gua+HC (C)

animals. Immunolabelling for α-SM actin (A, B and C) and macrophages (D, E and F). G:

colocalization of α-SM actin (figure 3C) with TUNEL staining (figure 2C), taken exactly at

the same region of Gua+HC aorta.

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Note that α-SM actin appears uniformly and strongly labelled in all cells of sham and Gua

media. In the Gua+HC group, cells are less labelled for α-SM actin. Macrophage staining was

absent in sham, Gua and Gua+HC media. Only a few cells are identified as macrophages in

the neointima of Gua+HC group, particularly around the lipid core where TUNEL positivity

was absent (outlined arrows). Note that all TUNEL-positive nuclei are colocalized with α-SM

actin staining either in media or neointima of the Gua+HC group (solid arrows). Sham:

hypercholesterolemic intact group; Gua: rats treated with guanethidine for sympathectomy;

Gua+HC: rats treated with guanethidine for sympathectomy and fed 1% cholesterol for three

months.n=6/group. A: adventitia, M: media, L: lumen, NI: neointima, Bar=25μm.

Figure 4. Western blot analysis and densitometric quantification of Bax (A), cytochrome c

(B), Bcl-2 (C), and caspases 3 (D) and 9 (E) from sham, Gua and Gua+HC rats.

Values are presented as ratio of protein signal to β-tubulin signal (for cytosolic fractions) or to

HSP60 signal (for mitochondrial fractions).

Data are shown as mean values±SEM, n=6 in each group. *<0.05, **<0.01 versus sham.

Sham: hypercholesterolemic intact group; Gua: rats treated with guanethidine for

sympathectomy; Gua+HC: rats treated with guanethidine for sympathectomy and fed 1%

cholesterol for three months.

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Table 1. Body weight and serum total cholesterol recorded at the end of experiments.

NC Sham Gua Gua+HC

Body weight (g) 195.75±9.56 202.83±11.64 199.77±10.58 199.84±10.27

Serum total

cholesterol (mmol/L) 1.48±0.26 2.53±0.31** 2.02±0.30* 3.49±0.41*

NC: normocholesterolemic intact rats. Sham: intact rats fed 1% cholesterol. Gua: rats treated

with guanethidine for sympathectomy; Gua+HC: rats treated with guanethidine for

sympathectomy and fed 1% cholesterol for three months. **<0.01 versus

normocholesterolemic; *<0.05 versus sham; n=6 for NC; Animals of sham, Gua and Gua+HC

group (n=6/group) are the same as those used for DNA fragmentation assay on agarose gel

electrophoresis.

Table 2. Number of nuclei per arbitrary box in sham, Gua and Gua+HC groups.

Sham media Gua media

Gua+HC

Media Neointima

Number of nuclei 44±4 46±5 36±3* 35±3 *

Aortic cross-sections from sham, Gua and Gua+HC groups (n=6/group) were incubated with

ethidium bromide. The labelled nuclei were counted in an arbitrary box of 6500 µm2

in three

different regions of medial and neointimal areas. The number of nuclei/box/area was

estimated after averaging values of the three different regions. *<0.05 versus sham. Sham:

intact rats fed 1% cholesterol. Gua: rats treated with guanethidine for sympathectomy.

Gua+HC: rats treated with guanethidine for sympathectomy and fed 1% cholesterol.

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Figure 1

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Figure 2

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Figure 3

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Figure 4