Effect of policosanol on intimal thickening in rabbit cuffed carotid artery

International Journal of Cardiology 67 (1998) 125–132

Effect of policosanol on intimal thickening in rabbit cuffed carotid artery ´ Rosario Mesa Miriam Noa*, Rosa Mas, Laboratory of Histology, Center of Natural Products, National Center for Scientific Research, 25 Ave. and 158 St, P.O. Box 6990, Havana, Cuba Received 3 June 1998; accepted 10 September 1998

Abstract We studied the effect of policosanol on smooth muscle cell proliferation in the cuffed carotid artery of the rabbit. Policosanol is a mixture of higher aliphatic primary alcohols isolated from sugar cane wax, with cholesterol lowering effects proved in experimental models and patients with type II hypercholesterolemia. It acts by inhibiting cholesterol biosynthesis. The positioning of a nonocclusive silicone collar around the rabbit carotid artery results in the formation of a neointima. We wished to determine whether policosanol orally administered prevented intimal thickening. Collars were placed around the left carotid for 15 days. The contralateral artery was sham operated. We included three experimental groups: a control received vehicle and two others policosanol at 5 and 25 mg Kg until sacrificed. Samples of arteries were examined by light and electron microscopy. To evaluate intimal thickening the cross-sectional area of intima and media were measured. Neointima was significantly reduced in policosanol-treated animals compared with controls. The smooth muscle cell proliferation was studied by the immunohistochemical detection of proliferating cell nuclear antigen and a significant reduction was observed in policosanol treated rabbits. It is concluded that policosanol has a protective effect on the neointima formation in this experimental model.  1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Policosanol; Smooth muscle cell proliferation

1. Introduction Neointimal formation related to smooth muscle cell proliferation is a crucial and perhaps the primary lesion in the progression of the atherosclerotic plaque [1]. Accelerated proliferation of smooth muscle cell also appears to be a cause of early coronary occlusion in patients undergoing heart transplantation, coronary artery bypass graft and percutaneous transluminal coronary angioplasty [2,3]. Factors controlling vascular smooth muscle cell proliferation are thought to be important in the development of atherosclerotic disease [4]. It has been reported that some cholesterol-lowering *Corresponding author. Fax: 1537 33 0497 / 33 3837.

drugs acting by inhibiting cholesterol biosynthesis, such as, the inhibitors of hydroxy methylglutaryl CoA reductase, can prevent smooth muscle cell proliferation both in vivo and in vitro [5]. Policosanol is a natural mixture of higher aliphatic primary alcohols isolated from sugar cane (Saccharum officinarum, L) wax with cholesterol-lowering effects demonstrated in different experimental models [6,7], healthy volunteers [8] and in patients with type II hypercholesterolemia [9–18]. Policosanol acts by inhibiting cholesterol biosynthesis in one of the steps located between acetate consumption and mevalonate production [19]. In addition, in vitro experiments shows that policosanol does not inhibit directly the hydroxy methylglutaryl CoA reductase activity.

0167-5273 / 98 / $ – see front matter  1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 98 )00305-2

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Also, policosanol, orally administered produces a significant reduction of thromboxane B 2 (a stable metabolite of thromboxane A 2 ) [20] and increases prostacyclin levels in rodents [21]. On the other hand, it has been demonstrated that policosanol reduces lipofundin-induced atherosclerotic lesions in rabbits and rats, including foam cell formation [22,23], and also prevents the development of foam cells in carrageenan-induced granulomas in rats [24]. Furthermore, long-term administration of policosanol significantly prevented the development of atherosclerotic lesions in Macaca arctoides monkeys [25]. Taking into account these data, the aim of the present study was to investigate if policosanol also showed inhibitory effects on smooth muscle cell proliferation in the cuffed carotid artery of the rabbit.

2. Materials and methods

2.1. Animals Twenty seven male New Zealand rabbits (2.5–3 kg) from National Center for Laboratory Animals production (CENPALAB) were adapted to laboratory conditions for 1 week with free access to food and water. Animals care and handle were in accordance with the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care) [26]. Study protocols are in accordance with our institution’s guidelines. They were randomly distributed in four groups. (eight rabbits / group).

2.2. Administration and dosage Policosanol was suspended in a 2% Tween 20 / H2O vehicle and orally administered by gastric gavage (1 ml kg) for 15 days. The experimental groups were: Group 1. Rabbits treated with Tween 20 / H2O (1 ml kg during 15 days). Group 2. Animals treated with policosanol at 5 mg kg. Group 3. Rabbits treated with policosanol at 25 mg kg. Group 4. Sham-operated rabbits. A satellite group, consisting of three rabbits with a sham-operated

carotid artery without the placement of cuff in the contralateral carotid.

2.3. Experimental induction of neointimal formation The day before the beginning of the treatment, rabbits were anesthetised with sodium pentobarbital (30 mg kg body wt i.v.) and both carotid arteries were exposed and dissected from the surrounding tissues. A nonocclusive, biologically inert, soft and flexible silicone cuff (20 mm long, 4.7 mm inner diameter) was placed around the left carotid artery as described [27]. The right carotid artery was sham operated, i.e, it was separated from the surrounding connective tissue and the vagus nerve and received a similar stretch as the contralateral cuffed artery. Animals were killed 15 days after cuff placement.

2.4. Microscopic studies The cuffed segment and the control artery were cut into transverse sections for light microscopy. The specimens were fixed in a 10% buffer formaldehyde. Subsequently, they were embedded in paraffin, sectioned and stained with haematoxylin and eosin.

2.5. Quantification of neointimal formation The neointimal formation was assessed by measuring with light microscopy the cross-sectional thickness of intimal (I) and medial (M) tissue of fixed arteries. The intima-to-media ratio was calculated. An ocular graticule and an Olympus micrometric slide with the same magnification of 1003 was used. Five arterial segments per animal were always evaluated. Measurement of the intima was always performed from the elastic and its values were expressed in mm. Mean values and standard deviations for the intima and media of each animal were obtained and afterwards, the average value for each group.

2.6. Immunohistochemistry For the immunohistochemical studies of cell proliferation sections of the four experimental groups were assessed, and three others groups of rabbits (eight / group) were studied in order to examine the evolu-

M. Noa et al. / International Journal of Cardiology 67 (1998) 125 – 132

tion of the intimal and medial SMC proliferation. They were: a– rabbits killed after 7 days of cuff implantation, in this group the contralateral arteries were studied as sham; b and c– rabbits sacrificed at 7 days of the positioning of the cuff and treated with policosanol at 5 and 25 mg kg, respectively. To determine proliferation in the intima and media the monoclonal antibody anti-proliferating cell nuclear antigen (PCNA. clone PC10, 1 / 10 dilution) from DAKO, Glostrup, Denmark, was used. The method was performed as previously described by Immunocytochemistry protocols of Amersham and as instructions of the specification sheet of DAKO. In brief: Sections were de-waxed and rehydrated, treated with 1% hydrogen peroxide in methanol for 20 min to block endogenous cellular peroxidase activity. The incubation time with monoclonal antibody was 30 min at appropriate dilution. With intervening PBS washes, sections were incubated in 1:500 dilutions in PBS of biotynilated rabbit antimouse Ig G for 30 min. After PBS washes, Streptavidin biotynilated peroxidase complex (Amersham) was used as detection system, during 30 min and then were incubated in 0.1% 3.3, diaminobenzidine (DAB, Sigma Chemical Company) in 0.05 M Tris buffer (pH 7.6) to which 0.75 ml of 3% hydrogen peroxide has been added. Sections were counterstained with methyl green, mounted and examined under a light microscope Carl Zeiss. Control preparations to ensure that streptavidin biotin staining was specific for PCNA included substitution of PBS for the PCNA monoclonal antibody.

2.7. Quantification of PCNA activity The immunoreactive SMC nuclei were counted in the media and the neointima (if present). After measuring the total medial area per section, the number of SMC nuclei was counted under a X400 microscope (equipped with a X10 eyepiece and a X40 objective) for entire area of all specimens (more than 50 high-power fields per specimen) [28]. In this way, the total number of SMC nuclei in the intima and media per section could be calculated. The PCNA-immunoreactive SMC nuclei were counted in the total medial and neointimal areas. Five arterial segments per animal were always evaluated, mean

127

values and standard deviations were obtained and afterwards, the average value for each group. The PCNA activity was expressed as the percentage of PCNA-positive SMC nuclei per total number of medial or neointimal SMC nuclei [27].

2.8. Transmission electron microscopy ( TEM) Segments of the cuffed arterial region and the sham-operated artery were cut into 1 to 2 mm thick slices (maximum) and kept for 1 h in 3.2% glutaraldehyde, 0.1 M phosphate buffer and then for another hour in 1% osmium tetroxide. After dehydration the tissues were included in Epon 812, and ultrathin sections were performed using an LKB ultramicrotome (Ultratome III) and stained with uranyl acetate and lead citrate. The study was conducted in a JEOL JEM 100S electron microscope in a double blind fashion. Synthetic smooth muscle cells were identified when showed a prominent rough endoplasmic reticulum, occasionally dilated cisternae, free ribosomes, with few and sometimes no evident myofilaments. One hundred cells of each group were examined. Apoptosis was measured by counting apoptotic cells in one hundred cells (in the intima and the media) of each group by transmission electron microscope. It was considered when cells rounds up, detaches from its neighbours, its cytoplasm condenses and were accompanied by chromatin condensation and aggregation at the periphery of the nuclear membrane. The three groups assessed for PCNA in addition to the others, were used too for the study of apoptosis.

2.9. Statistical analysis Comparisons between groups of thickness of intima (I) and media (M) and Intima / Media ratio and the percentage of PCNA-positive SMC of the neointima and media of arteries were performed using the non-parametric Mann-Whitney U9 test. Comparison among groups of rabbits presenting synthetic smooth muscle cells in the neointima and apoptotic cells were performed using the Fisher Exact probability test.

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3. Results

percentage of replicating medial SMC nuclei was 0.38260.1%, and the neointima contained 0.60460.007% PCNA-positive SMC nuclei. At 15 days values were of 0.6460.09% in the neointima and 0.38260.0.2% in the media. In 25 mg kg policosanol treated animals at 7 days the percentage was 0.38360.02% in the medial cells and 0.6260.15% in the intimal cells and at the end of the experiment values were of 0.6760.15% in the intimal cells and 0.38260.02% in the media (Table 2).

3.1. Light microscopy In all groups, the contralateral sham-operated arteries were lined by a normal endothelium. The media consisted of regular circularly orientated smooth muscle cells. There was a fibroblastic granulation tissue in the adventitial layer. The sham-operated rabbits of the satellite group showed similar results as the contralateral sham-operated rabbits. The cuffed arteries in the control group showed a great cellular proliferation (neointimal formation), some elastic fibers were present in the neointima. The endothelial cells were well preserved. The medial smooth muscle cells appeared to be normal. In the adventitial tissue there was a circumferential sheet of cellular fibrous tissue. In the cuffed arteries of policosanol-treated rabbits, the endothelial cells were well preserved, but there was a significant reduction of the neointimal formation (Table 1). The media was normal and the adventitial tissue presented cellular fibrous tissue.

3.2. Electron microscopy In the sham-operated arteries, the endothelial cells were lying immediately on the internal elastic lamina. Beneath the endothelium, collagen fibers with cross banding were identified and smooth muscle cells oriented in a circular direction. They displayed the contractile phenotype (filled with myofilaments and dense bodies but contain a relatively poorly developed Golgi apparatus and rough endoplasmic reticulum). In the cuffed arteries the endothelial cells were well preserved and demonstrated intact junctions. The neointima contained elastic fibers, cross-banded collagen fibers and the smooth muscle cells in a synthetic phenotype. The cells showed a prominent rough endoplasmic reticulum, occasionally dilated cisternac with few and sometime no evident myofilaments. The smooth muscle cells of the neointima were surrounded by basal lamina (Fig. 1). The media consisted of smooth muscle cells in a contractile phenotype, elastic laminae and collagen fibers. Many activated platelets were seen near the endothelium and in the adventitial layer in these cuffed arteries. In the cuffed arteries of rabbits treated with policosanol at 5 and 25 mg kg, the endothelial cells were well

3.1.1. PCNA activity In the cuffed carotid arteries at 7 days, the percentage of replicating medial SMC nuclei was 2.1960% and 0.3860.1% in the sham operated arteries. In the cuffed arteries 12.760.41% of the neointimal cell nuclei were PCNA-positive, and in the sham-operated rabbits 0.01860.01%. At the end of the experiment (15 days), the media of the cuffed carotid arteries contained (0.50360.1%) and 0.38760.1% in the sham-operated rabbits. The neointima contained 2.19460% PCNA-positive SMC nuclei, and 0.01860.01% in the sham-operated rabbits. In 5 mg kg policosanol treated animals at 7 days, the

Table 1 Values of intimal and medial carotid thickening in cuffed rabbits treated with policosanol for 15 days Treatment

Sham Cuff1tween Cuff1policosanol Cuff1policosanol

Doses

Thickness (mm)

mg / kg

Intima

Media ab

5 25

0.00760.0 40763.8 ab 0.09460.0 ab 0.09460.3 ab

Values are means1standard deviations. a , P,0.0005 (Comparisons with sham) (Mann Whitney U Test). b , P,0.0005 (Comparisons with positive control) (Mann-Whitney U Test).

Intima / Media ratio

419.1160.92 419.2560.70 418.7560.70 419.3760.74

0.01 ab 0.97 ab 0.02 ab 0.02 ab

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Table 2 Percentage of PCNA positive neointirnal and medial SMC nuclei and apoptotic cells in cuffed rabbits treated with policosanol Groups

Intima

Media

%PCNA1 intima

%PCNA1 media

AC-I

AC-M

Sham (7d) n56 Sham (15d) n56 Cuff (7d) n53 Cuff (15d) n55 Policosanol 5 mg kg 21 (7d) n56 Policosanol 5 mg kg 21 (15d) n56 Policosanol 25 mg kg 21 (7d) n56 Policosanol 25 mg kg 21 (15 d) n56

1861.09 1.864.30 111.466.65 a 219.467.56 a 13.261.48 ab

1272649.8 1292617.0 1286.4634.3 1259.2650.2 1276.2634.2

0.01860.01 0.01860.01 12.760.41 a 2.19460.07 a 0.60460.07 ab

0.38160.1 0.38760.1 2.19460 a 0.50360.1 0.38260.1

0 0 6c 8d 1

0 0 0 1 0

12.860.83 ab

1275.8626.1

0.6460.09 ab

0.38260.02

0

0

12.861.30 ab

1278626.2

0.6260.15 ab

0.38360.02

0

0

12.861.30 ab

1275.4625.1

0.6760.15 ab

0.38260.02

0

0

a

, , c , d , a

P,0.009 P,0.009 P,0.003 P,0.004

sham vs cuff and policosanol (Mann Whitney U9 Test). cuff vs. Policosanol (Mann Whitney U9 Test). (sham 7 d vs cuff 7 d and policosanol) Fisher Exact Test. (sham 15 d vs cuff 15 d and policosanol) Fisher Exact Test.

preserved. The neointima were in all cases smaller than in the cuffed arteries only. The majority of the smooth muscle cells showed the features of smooth muscle cells in a contractile state (Fig. 2), only a few one were in a synthetic state (4%) in the group of 5 mg kg, and in the group of 25 mg kg this phenotype was not observed. Comparison between groups presenting modified smooth muscle cells in the neointima showed significantly differences (positive control vs policosanol-treated groups). In these arteries the neointima contained lesser elastic laminae and

Fig. 1. Electron micrograph of a synthetic smooth muscle cell from the neointima of a cuffed carotid from a control group rabbit showing a cytoplasm rich in rough endoplasmic reticulum (er), ribosomes and vacuoles surrounded by ribosomes (v), containing a fine granular material. The intercellular space of the intima is wide and electron lucent and contains granulofibrillar material (gf). 37500.

collagen fibers than in the cuffed arteries without policosanol treatment. The media consisted of smooth muscle cells in a contractile phenotype, elastic laminae and collagen fibers. In cuffed arteries of policosanol treated animals, platelets were never seen. Apoptotic cells (AC) were never seen in arteries of control animals (sham), while in cuffed rabbits at 7 days, AC were seen in the neointima in 6%.In the group of cuffed rabbits at 15 days, AC were seen in 8%. In policosanol treated rabbits at 5 and 25 mg kg AC were seen at 1% and 0% respectively (Table 2).

Fig. 2. Electron micrograph of a contractile phenotype of the reduced neointima from a cuffed carotid rabbit treated with policosanol at 25 mg kg, showing a cytoplasm filled with myolilaments that often condensed into the characteristic elongated densities (ed) and only a few cell organelles. 37800

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4. Discussion The cuff system is a rapid method to study neointinial formation [27,29], which has been used for different pharmacological studies [30–33]. In the present work, characteristics of neointimal formation in the cuffed carotid artery of the rabbit were similar to those reported by Kockx et al. [27]. In a more recent work, it has been demonstrated that although the endothelial cell layer was morphologically intact after the cuff placement, the endothelial cells were metabolically altered as demonstrated by immunohistochemistry [33]. Policosanol administered at 5 and 25 mg kg for 15 days to rabbits with cuffed arteries significantly reduced the neointimal formation in these cuffed carotid arteries, thus indicating a decrease in the smooth muscle cell proliferation. The SMC replication was studied by the immunohistochemical detection of PCNA, which is an auxiliary protein of DNA d polymerase [34]. This protein appears during the G1, S, and G2 phases of the cell cycle and is a reliable marker for cell proliferation [35,36]. Values obtained with PCNA are similar as it compares with other well known S-phase markers such as BrdU or Tdr [37]. Our study shows that SMC replication in the media precedes the accumulation of SMCs in the neointima, which suggests that the cells divide first and migrate later. In policosanol treated groups the PCNA-positive cells in the intima were significantly reduced which agrees with the reduction of the neointimal formation in these arteries, it is likely that policosanol acts inhibiting SMC proliferation, values of PCNA-positive SMC are reduced both in the medial and intimal cells indicating some interference with myocyte proliferation and not by preventing chemotaxis. An interesting aspect to consider could be the of higher rates of apoptosis in positive control group than in policosanol treated rabbits. It has been demonstrated the presence of apoptosis in human atherosclerosis and in experimental model [38,39]. Most features of apoptotic cell death can be detected and are still best observed by electron microscopy [40]. The SMC lost could be attributable to apoptosis, which may modulate the cellularity of the lesion. Nevertheless, these results are not so reliable because features of apoptosis limit its detection. apoptotic

cells have a very short half-life and may undergo a rapid lysis or phagociytosis by neighbours [41]. Another work should be needed to a better understanding of apoptosis in the mechanism of the reduction of SMC proliferation in the policosanol treated rabbits. It has been showed a significant reduction of the neointimal formation induced by insertion of a flexible collar around one carotid artery of normocholesterolemic rabbits treated with lovastatin, simvastatin and fluvastatin. Intima / Media tissue ratios were 0.24, 0.20 and 0.17 respectively, vs. 0.36 in control animals (cuffed only) [28]. The present results show that the Intima / Media tissue ratio in policosanoltreated rabbits was significantly lower than in control animals (cuff1tween) (0.97 vs. 0.02, 0.02, respectively). In addition, indirect comparison indicate that the inhibition of intimal hyperplasia in rabbits treated with policosanol look to be greater than in animals treated with statins. A direct relationship between cholesterol synthesis and cell growth has been suggested, [5] hence the inhibition of the cholesterol biosynthesis at the level of the hydroxy methylglutaryl CoA reductase induced by the statins can explain their inhibitory effects on smooth muscle cell proliferation. A number of mevalonate-derived molecules, others than cholesterol, are potential adjuvant as cell cycle and cell survival products. For example, the activity of many signal transduction protein (i.e., Ras protein) is dependent on their localisation in the plasma membrane through a post-translational modification which covalently bind a lipid moiety (farnesyl or geranylgeranyl pyrophosphate derived from mevalonate [42,43]. The local delivery of mevalonate at the lesion site prevented the inhibitory effect of neointima formation induced by systemic fluvastatin administration [44]. Nevertheless, a review of the data show that cholesterol-lowering efficacy of policosanol and the strength of its inhibitory effect on cholesterol biosynthesis is not higher than the effect induced by statins [19]. So. other effect could support a higher effect on smooth muscle cell proliferation. A plausible explanation could be related with the increase of prostacyclin levels induced by policosanol Thus, eicosanoids, in particular prustacyclin play a role in

M. Noa et al. / International Journal of Cardiology 67 (1998) 125 – 132

mediating smooth muscle cell phenotype [45]. Experimental data demonstrated that eicosanoids derived from the arterial wall maintain smooth muscle cells in a contractile, quiescent phenotypic state, in which they are responsive to vasoactive stimuli but not to mitogens. Direct comparison amongst policosanol and statins on this experimental model can be developed in further experiments to coroborate the magnitude of their relative effectiveness. The treatment and prevention of atherosclerosis may be obtained not only through a reduction of risk factors, but also with a direct action on the arterial wall. Thus, policosanol is a drug with cholesterollowering and antiplatelet effects, which could explain its preventive effects on the development of atherosclerotic lesions. The policosanol-induced interference with myocite proliferation observed in this study are consistent with this product profile.

Acknowledgements The authors wish to thank M.E. Ramos, C. Agui˜ and A. Oquendo for techni´ lar, R. Gamez, F. Brinis cal assistance.

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