Journal of Bioscience and Bioengineering

VOL. 119 No. 5, 558e563, 2015

www.elsevier.com/locate/jbiosc

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Effects of oral administration of tripeptides derived from type I collagen

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(collagen tripeptide) on atherosclerosis development in

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hypercholesterolemic rabbits
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Lihua Tang,1, x Yasuo Sakai,2, * Yoshimichi Ueda,1 and Shogo Katsuda1
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Department of Pathophysiological and Experimental Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Ishikawa 920-0293, Japan1 and
Central Research Institute, Jellice Co., Ltd., 4-4-1 Sakae, Tagajo-shi, Miyagi 985-0833, Japan2

Received 23 April 2014; accepted 17 October 2014

Available online 14 November 2014
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Digestion of type I collagen with a collagenase-type protease yields a collagen tripeptide (Ctp) fraction comprising Gly-

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X-Y sequences that exhibit diverse biological activities. We previously demonstrated that Ctp inhibits the proliferation
and migration of cultured aortic smooth muscle cells (SMCs) in vitro. These cells contribute to the pathogenesis of
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atherosclerosis and other cardiovascular diseases. In order to evaluate the effects of Ctp on atherosclerosis development

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in vivo, here we used the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit model of familial hypercholes-
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terolemia to determine the effects of oral administration of Ctp for three months. Ctp induced a significant decrease in
the area occupied by atherosclerotic plaques in the aorta and in the level of total serum cholesterol. The components of

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atherosclerotic plaques underwent distinct changes, including reduction in the populations of macrophages and SMCs
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and a significant decrease in the proportion of macrophages to SMCs. Ctp administration decreased the number of cells
in plaques that expressed proliferating cell nuclear antigen and the number of cells with oxidative damage to DNA as

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indicated by 8-hydroxy-20 -deoxyguanine detection. These findings are the first to define the mechanism underlying the
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inhibitory effects of Ctp on atherosclerosis development in hypercholesterolemic rabbits, and suggest that Ctp provides
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an effective therapy for treating atherosclerosis.
Ó 2014, The Society for Biotechnology, Japan. All rights reserved.
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[Key words: Collagen tripeptide; Atherosclerosis; Plaque; Kurosawa and Kusanagi-hypercholesterolemic rabbit; Smooth muscle cells; Total
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cholesterol]

Atherosclerosis is the principal cause of most diseases related to matrix metalloproteinases (MMPs); and growth factors such as
cardiovascular disease-related deaths in both the developed and platelet-derived growth factor and insulin-like growth factor I is
developing countries (1). Long considered to arise from the pro- critical to their role, along with SMCs, in the damage and repair that
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gressive accumulation of lipids within the arterial wall, athero- ensue as atherosclerotic lesions progress (4). SMCs in the lesions
sclerosis is now recognized as a chronic inflammatory disease synthesize the bulk of the connective tissue matrix, including
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(2e4). Although effective measures to prevent and treat athero- collagen, elastic fibers, and proteoglycans (9,10). Thus, SMCs play
sclerosis are available, it remains refractory. Smooth muscle cells the principal role in the fibro-proliferative component of this dis-
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(SMCs) and macrophages derived from tissues and blood, respec- ease. The proliferation of macrophages and SMCs is therefore an
tively, are the most important cells in atherosclerotic lesions important feature of atherosclerosis.
because they play crucial roles in determining disease development Using a protease that specifically hydrolyzes the peptide bonds
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and prognosis (5,6). Moreover, any disturbance of their homeo- of type I collagen at the amino terminus of glycine residues, we
stasis may directly decrease the stability of the atherosclerotic obtained a highly purified, nonantigenic, and low-allergenic tri-
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plaque and cause thrombotic complications (7). peptide fraction containing Gly-X-Y sequences (where X and Y
Macrophages internalize atherogenic lipoproteins such as represent any amino acid residue, but most frequently proline and
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oxidized low-density lipoproteins (LDLs) via scavenger receptors,
and the generation of lipid-loaded macrophages containing large
amounts of cholesterol esters (foam cells) is a hallmark of early and
late atherosclerosis (4,8). The ability of macrophages to produce
cytokines such as tumor necrosis factor-a, interleukin-1, and
transforming growth factor-b; proteolytic enzymes, particularly
* Corresponding author. Tel.: þ81 22 361 6710; fax: þ81 22 361 6713.
E-mail address: sakai@jellice.com (Y. Sakai).
x
Present address: Department of Pathology, Yichang Central People’s Hospital,
Yichang, Hubei, China.
hydroxyproline) called collagen tripeptide (Ctp) (11). After oral
administration, evidence indicates that intact Ctp is transported to
the blood from the small intestine, for example, the transport of
tritium-labeled Ctp (Gly-Pro-Hypro) was rapid in comparison with
the absorption of tritium-labeled proline (12).
Ctp exerts multiple effects on cultured cells as well as on
experimental animals and affects the function of many organs and
tissues, including skin, bone, and cartilage (12e14). For example,
Ctp apparently accelerates the healing of bone fractures (12),
stimulates the calcification of human osteoblastic cells (13), and
increases the production of type I collagen by human osteoblastic

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2014.10.011
 VOL. 119, 2015 EFFECTS OF COLLAGEN TRIPEPTIDE ON ATHEROSCLEROSIS 559

cells (13). DNA microarray and quantitative RT-PCR analyses
demonstrate that Ctp up-regulates osterix, which is a bone-specific
transcription factor (13). Moreover, Ctp enhances the production of
hyaluronic acid and collagen by human dermal fibroblasts in vitro
and in murine skin in vivo (15). Oral administration of Ctp in a
mouse model of dry skin significantly reportedly decreased the
Immunohistochemistry The aortas were cut in cross-section into small
segments (5-mm each), embedded in paraffin, and stained with hematoxylineeosin
and Elastica van Gieson. Representative lesioned segments were selected, and
immunohistochemical analysis was performed using specific antibodies and the
streptavidin-biotin immunoperoxidase procedure. For the double-labeling proce-
dure, sequential avidin-biotin immunoalkaline phosphatase procedures were
employed using Fast Red as the chromogen to generate a red reaction product. The
primary mouse monoclonal antibodies were as follows: RAM11 (specific for mac-

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transepidermal loss of water, suppressed scratching behavior, and
dramatically inhibited the growth of intraepidermal nerves.
Quantitative PCR and immunohistochemical analyses reveal that
rophages, diluted 1:50), HHF35 (specific for smooth muscle actin, diluted 1:50), and
PC10 [specific for proliferating cell nuclear antigen (PCNA), diluted 1:200; all three
purchased from DakoCytomation, Glostrup, Denmark] and N45.1 [specific for 8-

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the increased levels of nerve growth factor and Sema3A returned to hydroxy-20 -deoxyguanosine (8-OHdG), diluted 1:10; purchased from JaICA, Shi-
normal in a mouse model of dry skin and rehydrated the epidermis zuoka, Japan]. The aortas were prepared as described in the preceding section. After

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hematoxylineeosin staining, we chose six lesioned segments of each aorta. Image-
(15). Pro Plus 5.1 was used to measure the total aortic area and lesion areas (1 pixel/unit).
In our previous study on atherosclerosis, we found that Ctp in- For analysis using the antibodies against RAM11, HHF35, and 8-OHdG, the number of

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hibits the proliferation and migration of cultured human aortic pixels in the positive area was divided by that in the lesioned area, and the result is

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SMCs and increases the synthesis of type I and IV collagen in vitro expressed as a percentage. For PCNA, we determined the number of stained cells in
the lesioned area, and the number in each 104 pixels was calculated for statistical
(16). The obtained results provided a clue for understanding the
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mechanism underlying the modulatory effects of Ctp on the path-
Data analysis and statistical analysis Data were analyzed using the Statis-
ogenesis of atherosclerosis. tical Package for the Social Sciences 10.0 (IBM Corp., NY, USA). One-way ANOVA
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The Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit followed by least significant difference post hoc tests were used to determine the
is a useful model for studying familial hypercholesterolemia statistical significance of differences between the means.
(17e19) caused by the deficiency of the LDL-receptor, which leads
to persistent hypercholesterolemia and the gradual development of RESULTS
atherosclerosis (20). Here we determined the effects of Ctp on
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atherosclerosis development using this rabbit model. Effects of Ctp on serum lipid levels Rabbits were orally

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administered 400 mg Ctp in distilled water or only water for three
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months, and the levels of serum lipids (TC, HDL-C, and TG) were
MATERIALS AND METHODS

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measured and compared with those before Ctp administration. In
comparison with the control group, which did not show any sig-
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Animal model Ten male KHC rabbits aged 3 months and weighing
nificant differences, TC levels in the Ctp group markedly decreased.

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2.275  0.167 kg mean  standard deviation (SD) were purchased from Japan
Laboratory Animals (Tokyo, Japan) and were randomly separated into a control and There were no significant differences between the levels of HDL-C
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an experimental group (hereinafter, Ctp group), comprising five rabbits each. The or TG between the control and Ctp groups during treatment (Fig. 1).

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rabbits in the Ctp group were continuously administered Ctp (400 mg in 5 ml of
distilled water) daily via a gastrointestinal feeding tube for three continuous
Macroscopic analyses of the areas in the aorta harboring
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months. The rabbits in the control group received 5 ml of distilled water using the atherosclerotic plaques Atherosclerotic plaques were present
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same route. All rabbits were housed individually in a pathogen-free animal facility on the inner surfaces of the aortas in the control and Ctp groups
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under a 12 h-dark/12 h-light cycle with free access to food and water. All protocols in
this study were performed in accordance with the guidelines of the Animal Research
Committee of Kanazawa Medical University.
Ctp preparation Ctp was prepared as previously described (9). Briefly, Ctp
was prepared from porcine skin collagen digested using a collagenase-type
protease (Protease N, Nagase Chemtex Corporation, Osaka, Japan). The digest
was deionized using an ion exchange resin (Diaion type SK, Mitsubishi
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(Fig. 2). The areas of atherosclerotic plaques in figure were
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indicated in red color by the image analysis software although
the actual color was white. The plaques were white with irregular
areas protruding into the aortic lumen. The distribution of
atherosclerotic lesions was not uniform in the three areas, and
severe involvement of the arch portion was observed, in contrast

Chemical, Tokyo, Japan), passed through a 0.2-mm filter, and was then to only slight involvement of the thoracic and abdominal areas
subjected to ion exchange chromatography using Toyopearl DEAE-650 (Tosoh
Corp., Japan). The tripeptide fraction was isolated using reverse-phase high-
around branch sites and bifurcations. Neither superimposed
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pressure liquid chromatography (HPLC). The mean molecular mass of Ctp is thrombus nor ruptured plaque was observed on the vessel
approximately 1970 Da. The CTP contains more than 25% of the tripeptide surface of either group.
component having the sequence (Gly-XY). The remaining 75% is composed of Using the first and last intercostal artery as the boundaries, we
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dimers/trimers of (Gly-X-Y) which means the precursor of tripeptides (10). Its

subdivided the aorta into the arch, thoracic, and abdominal regions
major components of tripeptide include peptides such as Gly-Pro-Hypro (9.7%),
Gly-Pro-Ala (4.7%), Gly-Ala-Hypro (1.7%), and Gly-Pro-Pro (1.1%). The purity of (Fig. 3A). The percentages of atherosclerotic plaques and the dif-
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Ctp was analyzed using HPLC with a Superdex Peptide gel filtration column ferences between the control and Ctp groups in each are shown in
(GE Healthcare UK Ltd., UK) and quantitated by integrating the absorbance at
214 nm of the major peak. The composition of tripeptide components is
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expressed as the area ratio (%).

Before administrarion After administration
Analysis of serum lipids Blood samples were collected from an aural artery
Relative TC, HDL-C, TG

1.5
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and added to tubes containing an anticoagulant. Total serum cholesterol (TC), HDL-
cholesterol (HDL-C), and total triglyceride (TG) contents were determined using a
cholesterol (Determiner-L TC II), triglyceride (Determiner-L TG II) (Kyowa Medex,
Tokyo, Japan), and cholesterol Qualigent HDL (Sekisui Medical, Tokyo, Japan) kit, 1.0
respectively. *
Quantitative analysis of atherosclerotic plaque area in the aorta Rabbits
were sacrificed using an injection of an overdose of sodium pentobarbital. A tho- 0.5
racotomy and laparotomy were performed to expose the heart and the entire aortal
tree. The aorta was separated and removed intact from its origin to the origin of the
iliac arteries. After the aorta was cut along its length and fixed with 4% para-
0.0
formaldehyde, photographs were taken at the same magnification. The aortic lumen Control Ctp Control Ctp Control Ctp
involved in the lesion was selected manually according to the different color by the
computer-assisted image analysis software Image-Pro Plus 5.1. Briefly, by using the TC HDL TG
menu of “select color” and “color cube based”, the parts of lesion were chosen and
shown in red color by the software automatically (shown in Fig. 2). Then the FIG. 1. Effects of Ctp on serum levels of TC, HDL-C, and TG. Each value represents the
percentage of lesion was calculated by the software and the statistic analysis was relative mean  SD values of TC/HDL-C/TG levels of five animals. * P < 0.05 compared
performed. with the Ctp group before Ctp administration.
560 TANG ET AL. J. BIOSCI. BIOENG.,

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FIG. 2. Macroscopic observations of the inner surfaces of the aortas with atherosclerotic plaques. (A, A1eA5) Aortas from the control group. (B, B1eB5) Aortas from the Ctp group.
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A30 and B50 are enlargements of the white rectangular areas of A3 and B5, respectively. The white protruding areas on the inner surface are atherosclerotic plaques. A300 and B500 are

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shown in red color of the white parts of lesion of A30 and B50 by the software automatically, respectively. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.).
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Fig. 3B. In the control group, atherosclerotic lesions covered positive SMCs (Fig. 4C). The positive area in the Ctp group was
16.4%  5.9% of the total aortic area. However, in the Ctp group, the

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approximately equivalent to that of SMCs and macrophages.
extent of the lesions decreased significantly to 4.5%  1.6%. The
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Effects of Ctp on PCNA expression PCNA-positive cells were

percentage of the area of atherosclerotic plaques in the Ctp group observed in the lesions in each group and were more numerous in

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decreased markedly as well as in the total aorta compared with that the upper area (Fig. 5A and B). In comparison with the control
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in the control group. group, the number of PCNA-positive cells in the lesions
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significantly decreased after Ctp administration (i.e., in the Ctp
Immunohistochemical analysis of cell type HHF35-positive
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group; Fig. 5C).

SMCs and RAM11-positive macrophages were dispersed in the le-
sions without any distinct predilection for location (Fig. 4A).
Compared with the control group, the areas positive for HHF35
and RAM11 were significantly decreased in the Ctp group
(Fig. 4B), and the ratio of the positive areas of the two cell types
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Effects of Ctp on oxidative damage to DNA We analyzed the
expression of 8-OHdG to evaluate oxidative damage to DNA. Cells
that reacted with the antibody against 8-OHdG were observed in
the lesions in the control and Ctp groups (Fig. 6A and B) and were
scattered throughout the lesions, although at a relatively higher

(RAM1-and HHF35-positive) in the Ctp group (1.12  0.17) was

significantly lower than that in the control group (2.28  0.91) density in the upper area. The detection of 8-OHdG in the lesions
(Fig. 4C). The area occupied by RAM11-positive macrophages in was significantly lower in the Ctp group (Fig. 6C).
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the control group was approximately twice that of HHF35-

DISCUSSION
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In the present study, serum TC levels were significantly

decreased in the Ctp group than in the control group. Macroscopic
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analysis of atherosclerotic lesions demonstrated that 16.4%  5.9%

and only 4.5%  1.6% of the intimal surface of the aortic area was
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covered with atherosclerotic lesions in the control and in the Ctp

groups, respectively. Thus, Ctp inhibited the development of the
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atherosclerotic lesions in KHC rabbits.

FIG. 3. Regions of the aorta. (A) Quantitative analysis of the lesioned area of the entire
aorta and different portions (mean  SD, n ¼ 5). (B) The lesioned area (%) is expressed
as the percentage of the atherosclerotic area/total area of the aorta in each region.
* P < 0.05, ** P < 0.01 compared with the control group.
Epidemiologic analyses such as the famous Framingham study
have demonstrated a significant correlation between atheroscle-
rosis severity and TC or LDL levels (21). Lowering serum cholesterol
level via diet or drugs slows the rate of progression of atheroscle-
rosis, causes regression of some plagues, and reduces the risk of
cardiovascular events (22). Hypercholesterolemia is thus a major
risk factor underlying atherosclerosis development. Therefore, the
decrease in TC levels induced by Ctp was considered an important
factor that inhibited the development and progression of athero-
sclerosis in KHC rabbits. Although there may be no direct rela-
tionship between collagen peptide and serum cholesterol, a
preventive effect of collagen peptide on lipid levels in blood has
been previously reported (23,24). However, the detailed
 VOL. 119, 2015 EFFECTS OF COLLAGEN TRIPEPTIDE ON ATHEROSCLEROSIS 561

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FIG. 4. Immunohistochemical analysis of RAM11 and HHF35 expression. (A) Immunohistochemical detection of RAM11 and HHF35. The cytoplasm stains brown. Original

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magnifications: 200. Scale bars: 100 mm. (B) Percentage of RAM11- and HHF35-positive cells in atherosclerotic plaques. (C) Ratio of positive areas (RAM11-positive area/HHF35-
positive area). Each value represents the mean  SD of five animals. * P < 0.05, ** P < 0.01 compared with control group. (For interpretation of the references to color in this figure
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legend, the reader is referred to the web version of this article.).

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FIG. 5. Immunohistochemical analysis of PCNA expression. (A, B) Detection of PCNA in plaques. The nuclei of PCNA-positive cells are brown. Representative plaques of the control (A)
and Ctp (B) groups. Original magnification: 200. Asterisk indicates aortic lumen. Scale bars: 100 mm (A, B). (C) Analysis of the density of PCNA-positive cells. Each value represents
the average positive-cell density in a plaque and the mean  SD of five animals. * P < 0.05 compared with the control group. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.).
562 TANG ET AL.
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FIG. 6. Immunohistochemical analysis of 8-OHdG. (A, B) Detection of 8-OHdG in plaques. The nuclei of 8-OHdG-positive cells are brown. Plaques of the control (A) and Ctp (B)
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groups. Original magnification: 200. *, aortic lumen. Scale bars indicate 100 mm. (C) Analysis of the density of 8-OHdG-positive-cells. Each value represents the average positive-
cell density in a plaque and the mean  SD of five animals. * P < 0.05 compared with control group. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.).
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mechanism underlying the effects of collagen peptide remains to be
determined.
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The tripeptide components (Gly-X-Y) of Ctp promote collagen
production by human osteoblasts (13) and fibroblasts (14). How-
ever, the component(s) of Ctp that mitigates atherosclerosis is
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unknown. The effects on the physiological functions of the circu-
latory system induced by ingested collagen peptides derived from
chicken legs have been investigated (25). The administration of a
chicken collagen peptide was reported to exert an antihypertensive
effect compared with that exerted by a placebo and maintain
arterial flexibility, according to the measurements of Dbrachial-
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ankle pulse wave velocity (26). On the other hand, the tripeptide
components (Gly-X-Y) of Ctp represent a minimum essential unit of
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collagen; the molecular mass of Ctp (280 Da) is much lower than
that of the chicken collagen peptide and its absorption efficiency is
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significantly greater than that of free amino acids (12). Oral
administration of Ctp to KHC rabbits in the present study induced a
significant decrease in the area of atherosclerotic plaques and
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decreased the number of macrophages and SMCs that contribute to
the onset of atherosclerosis.
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Because hypercholesterolemia increases the production of free
reactive oxygen species (ROS) that enhance lipid oxidation (27,28),
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the decrease in serum TC induced by Ctp may mitigate oxidative
stress and contribute to the inhibition of atherosclerosis develop-
ment and progression, as described below.
Atherosclerosis is an inflammatory process driven, at least in
part, by ROS (29). Further, oxidative stress represents a mechanism
that unifies the risk factors of cardiovascular disease, including
hypercholesterolemia and diabetes mellitus, further supporting its
central role in atherosclerosis (30,31). ROS are involved in the
oxidation of LDL, which is considered a fundamental step in
atherosclerosis development and progression (32).
ROS induce extensive oxidation of DNA damage, and oxidative
damage to DNA is a prominent feature of atherosclerotic plaques
(33). When DNA sustains oxidative injury, 8-OHdG is produced and
serves as a major marker of oxidative injury (34). In the present
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study, 8-OHdG-positive cells were observed in plaques present in
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the control and Ctp groups, further implicating oxidative damage in
the formation of atherosclerotic lesions in KHC rabbits. The density
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of 8-OHdG-positive cells in the Ctp group significantly decreased in
comparison with that in the control group, suggesting that Ctp
protects against oxidative stress and subsequently inhibits the
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formation of atherosclerotic lesions.
Because monocytes and macrophages are central to the
inflammation that occurs in atherosclerosis (4,35), we performed
an immunohistochemical analysis of the cell population of
atherosclerotic lesions using antibodies specific for SMCs (HHF35)
and macrophages (RAM11). The results of these experiments were
surprising. The lesions in the control group were more cellular and
predominantly enriched in macrophages compared with those in
the Ctp group. There was an average of approximately twice the
number of RAM11-positive macrophages than HHF35-positive
SMCs in the lesions in the control group, whereas the areas con-
taining macrophages and SMCs were approximately equivalent in
the lesions in the Ctp group (Fig. 4B and C). Thus, Ctp reduced the
fraction of the lesions occupied by macrophages, suggesting that
Ctp selectively inhibits the formation of macrophage-rich lesions
via the suppression of inflammation.
In comparison with the control group, the number of PCNA-
positive proliferating cells in the lesions significantly decreased
after Ctp administration. Immunohistochemical analyses revealed
that the majority of PCNA-positive cells in the lesions were mac-
rophages in the control and Ctp (average, 77.0% and 73.3%,
respectively) groups, although a small fraction were identified as
SMCs (data not shown). This may provide a clue to explain the more
potent inhibitory effect of Ctp on macrophages that accounts for the
decreased ratio of macrophages to SMCs in the lesions. We recently
reported that Ctp inhibits the proliferation of cultured human
aortic SMCs by downregulating PCNA expression (16) and reducing
the number of HHF35-positive SMCs in atherosclerotic lesions in
the Ctp group. Therefore, Ctp may exert the same effect on SMCs in
the process of atherogenesis in KHC rabbits.
 VOL. 119, 2015
The disruption of atherosclerotic plaques, which triggers
thrombosis, is the major cause of occlusion and the majority of
acute cardiovascular events (36). Macrophages are a major source
of MMPs, which degrade most types of extracellular matrix com-
ponents and play a prominent role in weakening atherosclerotic
plaques (37). Lipid lowering and decreased oxidative stress reduces
the activity of MMPs (37). Taken together, these findings indicate
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that Ctp may stabilize atherosclerotic plaques, thereby reducing the
risk of acute cardiovascular events.
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Hypercholesterolemia is sufficient to induce lesions in the
absence of other risk factors (22). Therefore, we assume that the
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most important role of Ctp revealed here was to decrease serum TC
levels and subsequently suppress inflammatory processes,
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including inhibiting the proliferation of macrophages and SMCs
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and reducing oxidative stress induced by ROS. Our observations
provide a plausible explanation for a possible modulatory effect of
樂 22. Mitchell, R. N.: Atherosclerosis, pp. 335e344, in: Kumar, V., Abbas, A. K., and
Ctp on the stability of atherosclerotic plaques. Ctp may therefore
serve as a therapeutic drug for atherosclerotic disease. Although we
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show here that Ctp inhibited the formation of atherosclerotic le-
sions, the precise molecular mechanisms involved must be defined.
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