Hypercholesterolemic diet induces vascular smooth muscle cell

Autonomic Neuroscience: Basic and Clinical 183 (2014) 49–57

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Hypercholesterolemic diet induces vascular smooth muscle cell apoptosis in sympathectomized rats via intrinsic pathway Rafik Hachani a,b,c,⁎, Houcine Dab a, Anouar Feriani d, Sami Saber e, Mohsen Sakly a, Eric Vicaut b, Jacques Callebert c, Richard Sercombe b, Kamel Kacem a a

Laboratoire d Étude de Pathologies Vasculaires, Unité de Physiologie Intégrée, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisia Laboratoire d Étude de la Microcirculation (EA 3509), Université Paris 7, France Laboratoire de Biochimie, Hôpital Lariboisière, Paris, France d Laboratoire d Ecophysiologie Animale, Faculté des Sciences de Sfax, 3000, Tunisia e Faculté de Médecine de Sfax, 3000, Tunisia b c

a r t i c l e

i n f o

Article history: Received 26 September 2013 Received in revised form 19 February 2014 Accepted 26 February 2014 Keywords: Sympathectomy Aorta Hypercholesterolemia Rat Apoptosis Intrinsic pathway

a b s t r a c t 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. © 2014 Elsevier B.V. All rights reserved.

1. Introduction 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 et al., 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.

⁎ Corresponding author at: Laboratoire d Étude de Pathologies Vasculaires, Unité de Physiologie Intégrée, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisia. Tel.: +216 52677208; fax: +216 76211026. E-mail address: rafi[email protected] (R. Hachani).

http://dx.doi.org/10.1016/j.autneu.2014.02.007 1566-0702/© 2014 Elsevier B.V. All rights reserved.

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, 2011). However, we do not

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R. Hachani et al. / Autonomic Neuroscience: Basic and Clinical 183 (2014) 49–57

know if hypercholesterolemic diet combined with sympathectomy triggers apoptosis of vascular cells, reported to be associated with a dedifferentiated VSMC 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. Oil Red O staining was used to reveal lipid in the arterial wall. The efficacy of sympathectomy was evaluated by analysis of perivascular sympathetic fibers.

2. Materials and methods 2.1. Animals The animal protocols used for this study were approved by the University Animal Care and Use Committee of University of Paris VII (France) and 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. 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.

2.2. Sampling and fluorescence labeling 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, 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.

2.3. Serum cholesterol measure 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). 2.4. 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). 2.5. 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 labeling (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 labeling were defined as TUNEL positive. Positive controls were provided by sections pretreated with DNAse I Buffer (100 U/ml) for 10 min at 15–25 °C to induce DNA strand degradation. In negative control experiments, TdT was omitted from the labeling mixture, and no staining was detected. The labeled nuclei by TUNEL and hematoxylin staining were counted in a fixed box in three different regions of medial and neointimal areas (Kockx et al., 1996a,b). 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:

TUNEL index in each region ¼ ð100% ½number of TUNEL−positive nuclei=total number of nucleiÞ:

2.6. 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 labeled 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. 2.7. DNA extraction and agarose gel electrophoresis 50 mg of the 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, 1 μg of extracted DNA was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide (0.5 μg/ml), and visualized under UV light.


2.8. Smooth muscle α-actin and macrophage immunostaining The abdominal aorta from sympathectomized and sham animals (n = 6/group) were used to reveal VSMCs and macrophages on serial sections (16 μm thick). 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.

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for 1 min to X-ray film (Kodak BioMax). The intensity of specific immunoreactive bands was quantified by a densitometric scanning program (ImageJ, 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. 2.12. 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 b 0.05.

2.9. 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 labeling of smooth muscle α-actin and the fluorescence labeling 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 labeling 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). 2.10. 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. 2.11. 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, 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

3. Results 3.1. Body weight and serum total cholesterol 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 b 0.01) and 32% (p b 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 b 0.05), by combination with sympathectomy (Gua + HC) (Table 1). 3.2. Catecholamines, Oil Red O staining and ethidium bromide staining The adventitia of the 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 labeled 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 b 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. 3.3. In situ apoptosis detection using TUNEL method 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

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

Body weight (g) Serum total cholesterol (mmol/l)

NC

Sham

Gua

Gua + HC

195.75 ± 9.56 1.48 ± 0.26

202.83 ± 11.64 2.53 ± 0.31⁎⁎

199.77 ± 10.58 2.02 ± 0.30⁎

199.84 ± 10.27 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. 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. ⁎⁎ p b0.01 versus normocholesterolemic. ⁎ p b0.05 versus sham.

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Fig. 1. Catecholamines (A, B and C), Oil Red O staining (D, E and F) and ethidium bromide staining (G, H and I) 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 (panel F) are limited to the neointima (NI) and bordering regions in the Gua + HC group. As attested by 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) (in this figure). 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 = 10 μm in A, B and C; Bar = 25 μm in D, E, F, G, H and I.

(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 b 0.001) in both intimal and medial areas (Fig. 2D).

In sham group, our results reveal two scarcely perceptible apoptotic DNA fragments of 800- and 1000 bp. However, hypercholesterolemic diet, when combined with sympathectomy, exhibits a spectacular typical DNA ladder with clearly increased intensity of DNA fragments at ~ 200 bp intervals (Fig. 2E). As in sham animals, sympathectomy alone group revealed a few apoptotic DNA fragments.

3.4. DNA fragmentation on agarose gel electrophoresis 3.5. Immunolabeling of VSMCs and macrophages 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. Table 2 Number of nuclei per arbitrary box in sham, Gua and Gua + HC groups. Sham media

Number of nuclei

44 ± 4

Gua media

46 ± 5

Gua + HC Media

Neointima

36 ± 3⁎

35 ± 3⁎

Aortic cross-sections from sham, Gua and Gua + HC groups (n = 6/group) were incubated with ethidium bromide. The labeled 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. 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. ⁎ p b0.05 versus sham.

In sham and sympathectomy alone groups, immunohistochemical analysis shows that α-SM actin appears uniformly and strongly labeled in all cells of the media (Fig. 3A, B). Consequently, the rare TUNELpositive 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). After a hypercholesterolemic diet in rats with neonatal sympathectomy (Gua + HC), cells are less labeled 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


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Fig. 2. TUNEL assay (A, B and C), TUNEL index (D) and agarose gel electrophoresis analysis (E) of the 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 ~200 bp 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. ***pb0.001 versus sham. n = 6/group. A: adventitia, M: media, L: lumen, NI: neointima, MW: molecular weight markers. Bar = 25 μm.

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

3.6. Bax, Bcl-2, cytochrome c, caspases 3 and 9 analysis Fig. 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 b 0.05) and increased in mitochondria by 34% (p b 0.05), in Gua + HC group compared with sham. However, Bcl-2 protein was significantly decreased by 24% (p b 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 b 0.05) in mitochondria and increased in cytosol by 31% (p b 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 b 0.01) and 21% (p b 0.05), 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).

4. Discussion We have previously reported that chemical sympathectomy by guanethidine, combined with hypercholesterolemic diet, induced atherosclerosis in the abdominal aorta of rats (Hachani et al., 2010, 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

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Fig. 3. Cross-sections of the abdominal aorta from sham (A), Gua (B) and Gua + HC (C) animals. Immunolabeling for α-SM actin (A, B and C) and macrophages (D, E and F). G: colocalization of α-SM actin (panel C in this figure) with TUNEL staining (Fig. 2C), taken exactly at the same region of Gua + HC aorta. Note that α-SM actin appears uniformly and strongly labeled in all cells of sham and Gua media. In the Gua + HC group, cells are less labeled 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 the 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 = 25 μm.

with this concept, sympathectomy aggravates the process of atherosclerosis induced by high cholesterol diet in rabbits (Kacem et al., 2006; Murphy et al., 1957). 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., 1996a,b). 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 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


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Fig. 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. *pb0.05, **pb0.01 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.

in the percentage of TUNEL positive nuclei has been found, ranging from less than 2% (Hegyi et al., 1996; Isner et al., 1995; Kockx et al., 1996a, 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). Notably, we find that all TUNEL-positive cells are stained intensely for α-SM actin either in the 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, the 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. 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 et al., 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 et al., 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 ~ 200 bp 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

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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 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 two 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; Harrell et al., 2005; Zaugg et al., 2000). Thus, it seems that sympathetic-dependent apoptosis is tissue- and/or context-dependent, since all these previous studies were carried out either in vitro (we in vivo), or in non-aortic tissues (cardiomyocytes and brain), we in the aorta. Moreover, it is very likely that sympatheticdependent apoptosis may have different intracellular transduction signal pathways, an aspect that warrants further investigation. Another explanation, the 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 the plasma and aorta (Hachani et al., 2012). Interestingly, it was demonstrated that low density lipoprotein can induce apoptosis in VSMCs, particularly oxidized LDL (Diez et al., 1997; Nishio et al., 1996). The effect of oxidized LDL has been 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 integrinmediated 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 the 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 the ear arteries (Kacem et al., 1997) and aggravation of atherosclerosis on the aorta (Murphy et al., 1957) 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 hypercholesterolemic diet, combined with sympathectomy, is able to break this protection and provoke atherosclerotic lesions with vascular cell 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. Acknowledgement Rafik Hachani received a grant from the Ministry of Higher Education,Scientific Research and Technology (Tunisia)(264/2006)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. References Allsopp, T.E., Wyatt, S., Paterson, H.F., Davies, A.M., 1993. The protooncogene bcl-2 can selectively rescue neurotrophic factor dependent neurons from apoptosis. Cell 73, 295–307.

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