www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 323 (2004) 592–598

BBRC

Cigarette suppresses the expression of P4Ha and vascularcollagen production

Muthuswamy Raveendrana, Duraisamy Senthila, Budi Utamaa, Ying Shena,Donald Dudleyb, Jian Wanga, Yun Zhangc, Xing Li Wanga,*

a Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USAb Department of Obstetric and Gynecology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

c Department of Cardiology, Shandong University Medical College, Jinan, Shandong, China

Received 10 August 2004

Abstract

Objective. Collagen plays a major role in arterial wall remodeling, aneurysm formation, and atherosclerotic cap stability. Smok-

ers often have weakened arterial walls associating with aneurysm and thinned atherosclerotic plaque caps leading to rupture and

acute coronary syndromes. We hypothesize that these detrimental effects on arterial wall by tobacco are partially mediated by dis-

turbed collagen metabolism.

Methods and results. We first investigated the effect of cigarette smoke extracts (CSE) on prolyl-4-hydroxylase (P4H) expression

and collagen production in human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCSMCs).

After exposure to 0.01-U CSE for 24 h, expression of P4Ha—a rate limiting subunit of P4H enzyme responsible for the formation of

4-hydroxyproline in mature functional collagen, was significantly down-regulated according to Western blotting and quantitative

RT-PCR (HAEC p < 0.01 and HCSMC p < 0.001) when treated by CSE. The decreased P4Ha expression was corresponded with

reduced cellular collagen levels (HAEC p < 0.001 and HCSMC p < 0.001). We also found that one of the cigarette components ben-

zo(a)pyrene exerted similar effect as CSE, but not nicotine or acrolein. We further examined P4H expression in a few human ath-

erosclerotic abdominal aortas. These in vivo data demonstrated that smokers had thinner atherosclerotic cap thickness and lower

levels of P4Ha and collagen.

Conclusions. Our study suggests that cigarette may interfere with one of the key enzymes in arterial wall collagen metabolism,

which may be responsible for thin fibrous cap in atherosclerotic lesion, impaired arterial wall extensibility, and increased likelihood

of aneurysm in smokers.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Atherosclerosis; Cigarette smoke; Collagen; Prolyl-4-hydroxylase

Atherosclerosis is a progressive disease characterized

by the accumulation of lipids and fibrous elements in

media or large arteries [1]. While early lesions are

marked by an accumulation of cholesterol-engorged

macrophages, i.e., ‘‘foam cells,’’ a typical advanced ath-

erosclerotic lesion contains a lipid core, which is sur-rounded by pools of extracellular lipids, foam or

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.08.129

* Corresponding author. Fax: +1 713 798 1705.

E-mail address: xlwang@bcm.tmc.edu (X.L. Wang).

necrotic cells. This atheroma or the intima mass is cov-

ered by a layer of thin or thick fibrous cap [2,3]. The bio-

chemical and biophysical characteristics and thickness

of the cap may vary and determine the vulnerability of

the plaque to rupture. A ruptured atheromatic plaque

will expose thrombogenic material in the plaque�s coreand initiate thrombotic cascade leading to acute arterial

occlusion [4]. The constituent of fibrous cap is mainly

collagens (type I and III), which provide the cap�s bio-mechanical strength [1]. Collagen is a substrate for a

M. Raveendran et al. / Biochemical and Biophysical Research Communications 323 (2004) 592–598 593

group of enzymes including matrix metalloproteinases

(MMPs) [5]. Inappropriate activation of these enzymes

is associated with unstable atherosclerotic plaque and

rupture [6–8]. However, there is little information about

whether factors regulating collagen production play a

role in the plaque cap stability.Prolyl-4-hydroxylase (EC 1.14.11.2) (P4H) catalyzes

the formation of 4-hydroxyproline in collagens, which

is essential for the folding of the newly synthesized col-

lagen polypeptide chains into triple helical molecules

[9,10], and plays a crucial role in the synthesis of all col-

lagens [9–11]. P4H normally functions in tetramer with

two subunits—a and b, in which b isoform is produced

in abundance and a subunit is rate limiting. Cigarettesmoke, one of the strongest environmental risk factors,

has been associated with an increased risk of acute cor-

onary syndrome [12,13], thrombosis [14], and myocar-

dial infarction—the processes could be linked to acute

plaque rupture. It is tempting to speculate that smokers

may have thin and vulnerable atherosclerotic plaque

cap, therefore, easier to rupture. The reason for the thin

cap could be due to dysfunctional collagen synthesis inthese regions by cells like endothelial cells or vascular

smooth muscle cells. In the current study, we examined

the hypothesis by investigating whether and how ciga-

rette smoking would interfere in the process. We tested

the effects of cigarette smoke extract (CSE) and some

of the tobacco active components on P4H expression

and collagen production in a cell culture model. We also

examined the expression of P4Ha, collagen content, andcap thickness in advanced atheroma lesions in smokers

and non-smokers. Our data indicate that CSE and ben-

zo(a)pyrene—an active tobacco component, reduce the

P4Ha expression and collagen synthesis in CSE-treated

cells. We have also observed that smokers with athero-

sclerotic lesion have reduced expression of P4Ha, colla-gen content, and thin atherosclerotic cap comparing to

non-smokers.

Materials and methods

Cell culture. Human aortic endothelial cells (HAECs) and human

coronary artery smooth muscle cells (HCSMCs) were purchased from

Cell Applications (San Diego, CA). All other chemicals were purchased

from Sigma (St. Louis, MO), unless indicated otherwise. HAECs were

cultured in F-12K media (ATCC, Manassas, VA) containing bovine

endothelial cell growth supplement (45 lg/mL), heparin (100 lg/mL,

Acros, NJ), penicillin (50 IU/L), streptomycin (50 lg/L, Cellgro Hern-

don, VA), and 20% fetal calf serum (Invitrogen, Carlsbad, CA) in a 5%

CO2/air atmosphere. HCSMCs were cultured in smooth muscle cell

growthmedium (Cell Applications). Only cells within 3–5 passages were

used for the experiments. At 70–80% confluence, cells were treated with

CSE (0.01 cigarette equivalent unit/mL) or other active components for

24 h in the culture media described above. Experiments were repeated

three times for each experimental condition described below.

Preparation of CSE. CSE was prepared as described previously

[15]. Briefly, a research cigarette (2R4F, from Tobacco Health Re-

search, University of Kentucky) was inserted into a 10-cm length of

plastic tube that was attached to a tube submerged in a flask con-

taining 20 mL of cell culture medium. The ignited cigarette was com-

pletely consumed in puffs over 5 min. During this time the cigarette

smoke was bubbled through the medium, collecting the water-soluble

components of the cigarette smoke. The pH of the CSE was between

7.4 and 7.5. The concentration of CSE was calculated in arbitrary units

as cigarette equivalents per milliliter of the medium. CSE was used to

treat the cells within 30 min of extraction.

Determination of prolyl-4-hydroxylase protein levels. Cells of con-

trol and CSE-treated cultures were washed with ice-cold PBS and lysed

in protein lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM

EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate,

1 mM b-glycerol phosphate, 1 mM Na3VO4, 10 lg/mL of each prote-

ase inhibitor (aprotinin, leupeptin, and pepstatin), and 1 mM phenyl-

methylsulfonyl fluoride) for 1 h on ice. Protein concentration was

measured by the Bradford method. Twenty five micrograms of protein

per lane was separated by 10% SDS–polyacrylamide gels and trans-

ferred to PVDF membranes. The membrane was blocked in 5% non-

fat powdered milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, and

0.05% Tween 20). The membrane was incubated with the primary

antibodies (mouse monoclonal anti-human P4Ha antibody,

MAB2700, and anti P4Hb antibody, MAB2701, Chemicon Interna-

tional, Temecula, CA) in 2% powdered milk in TBST, and washed

extensively with TBST. The membrane was then incubated with sec-

ondary anti-mouse IgG conjugated with horseradish peroxidase

(Amersham Biosciences, Piscataway, NJ). Bands were visualized with

ECL (Amersham Biosciences) according to the manufacturer�sinstructions.

Determination of prolyl-4-hydroxylase mRNA levels. After the

experimental treatment the total RNA was isolated from control and

CSE-treated cells using the Trizol reagent (Invitrogen). Gene expres-

sion quantification was performed using a two-step RT-PCR in which

the PCR step is coupled with a 5 0 fluorogenic nuclease assay by using

the assays-on-demand gene expression protocol (Applied Biosystems,

Foster City, CA). The mRNA levels were expressed as an arbitrary

unit according to standard RNA preparation with known RNA levels.

Collagen estimation. At the end of the experimental period, colla-

gen (type I) content was estimated in cells and medium, using an

ELISA kit (Chondrex, Redmond, WA). Briefly, the media were col-

lected after the experimental period and the cells were washed with

cold 1· PBS. Then, 0.5 mL of 0.05 M acetic acid (pH 2.8–3.0 with

formic acid) was added to the cells. The cells and the medium were

subjected to pepsin and pancreatic elastase digestion. The supernatant

was used for the collagen estimation. The values of cellular collagen

contents were expressed as nanogram of collagen/microgram of total

cellular protein and the collagen content of the media was expressed as

nanogram of collagen/100 lL of the medium.

Effect of cigarette smoke contents on P4H expression. To explore

which active component of the cigarette smoke was responsible for the

reduction of P4Ha, the HAECs were treated with 3 lM benzo(a)pyr-

ene (Cat#B1760, Sigma) or nicotine hydrogen tartrate salt (1 lg/mL of

the medium, Cat#N5260,Sigma) or 50 lM acrolein (Cat#11022, Sig-

ma) for 24 h. Benzo(a)pyrene was dissolved in the dimethyl sulfoxide

(DMSO, Cat#D5879, Sigma), and nicotine hydrogen tartrate salt and

acrolein were dissolved in the cell culture medium. After the treatment,

protein and RNA were isolated from the cells as described above. Since

only benzo(a)pyrene had effect on P4Ha expression, another set of

experiments was carried out with similar conditions to measure the

collagen content in the cell and media.

Immunohistological examination of aortic atherosclerotic samples. In

order to explore whether the smoking effects on P4H observed in cell

culture model can also be found in vivo, we analyzed the expression of

P4H in 20 abdominal aorta samples with different degrees of athero-

sclerotic lesions. The samples were kindly provided from Pathobio-

logical Determinants of Atherosclerosis in Youth (PDAY) study by

Drs. Henry McGill (Southwest Foundation for Biomedical Research,

San Antonio, TX) and Arthur Zieske (University of Louisiana).

Fig. 1. Western blotting for P4Ha and b in HAECs and HCSMCs

after CSE treatment. Cells were treated with or without CSE (0.01 U)

for 24 h. Cell lysates were separated on 10% SDS–PAGE and

transferred to nitrocellulose membrane. The membrane was immu-

noblotted with either anti-human P4Ha antibody (top panel) or anti-

human P4Hb antibody (middle panel). This is a representative blot

from three independent experiments. CSE decreased the P4Ha level at

24 h, whereas P4Hb level was not altered by CSE. Western blotting

with anti-b actin antibody was carried out to assess the equal loading

(lower panel).

594 M. Raveendran et al. / Biochemical and Biophysical Research Communications 323 (2004) 592–598

Formalin-fixed samples were dehydrated in graded alcohol and

embedded in paraffin. Five longitudinal sections from each sample

were prepared, dewaxed in a decreasing series of alcohols, and used for

the following protocol. For qualitative and quantitative analysis of

P4H expression, we used the mouse ABC staining system (Santa Cruz

Biotechnology, Santa Cruz, CA) to detect the P4Ha and b. Antigen

unmasking was performed by incubating the slides in preheated

ACCU-TUF (Accurate Chemical & Scientific, Westbury, NY) for

10 min and washed with deionized water. After rehydration in Tris-

buffered saline (TBS, 0.05 M Tris–hydrochloric acid; 0.15 M sodium

chloride; pH 7.6) for 5 min at room temperature, hydrogen peroxide

1% for 10 min at room temperature was added to suppress endogenous

peroxidase activity. For the saturation of non-specific binding sites,

samples were pre-incubated with 1.5% blocking serum for 30 min at

room temperature. The preparations were incubated in a humid, dark

chamber (4 �C for 12 h) with a mouse monoclonal anti-human P4Haor b antibody dilution (1:20) in 1.5% blocking serum in PBS. A bio-

tinylated secondary antibody at a dilution of 1:50 for 30 min at room

temperature was used. From each tissue sample, three consecutive

sections were incubated on glass slides, with one negative control

(incubation with PBS without primary antibody). Slides were incu-

bated with ABC enzyme reagent for 30 min followed by washing with

PBS. Peroxidase substrate was used to develop the color intensity.

Specific P4Ha and b displays brown coloration.

After staining for P4Ha and P4Hb, slides were counter-stained

with Accustatin Trichrome stains (Masson) according to manufac-

turer�s instruction (Sigma). Collagens and muscle fibers were stained

differentially when treated sequentially with Biebrich Scarlet-Acid

Fuchsin, PTA/PMA, and aniline blue. Cytoplasm and muscle fibers

stain red whereas collagen displays blue coloration. The preparations

were examined semi-qualitatively under a microscope at a magnifica-

tion of 40–1000· for changes in P4Ha and b expression, collagen

content, and cap thickness. The expression of P4Ha or b in smokers

was compared with that in non-smokers with the similar level of ath-

erosclerotic lesion, which was classified previously according to AHA

grades [2,3,16]. For quantification of P4Ha and b expression and

collagen content, the color intensity was measured in a Leica-DMLB

(Leica Microsystems, Wetzlar, Germany) microscope using the Image-

Pro Plus software (Media Cybernetics, Silver Spring, MD). The cap

thickness was also measured as shown in Fig. 5C. The randomized

systematic subsampling was used for the measurement. Three visual

fields in each section were used. The measurement was done inde-

pendently by two examiners. The cap thickness was expressed in

nanometer.

Statistical analysis. Results are presented as means ± SEM. Stu-

dent�s t test was used to compare the difference between experimental

conditions and the two-tailed p < 0.05 was regarded as statistically

significant.

Results

Effects of CSE on the P4H expression in HAECs and

HCSMCs

As shown in Fig. 1, CSE (0.01 U for 24 h) treatment

decreased the level of P4Ha (top) in HAECs and

HCSMCs when compared to control cells. The proteinlevel of P4Hb (middle) was not affected by CSE treat-

ment in both cell types. We further determined whether

CSE depressed the P4Ha expression at transcriptional

level. We measured the mRNA levels in CSE-treated

and untreated cells. As shown in Fig. 2A, CSE decreased

the P4Ha mRNA levels significantly when comparing to

untreated cells (HAECs: p < 0.001, HCSMCs: p < 0.01).

However, P4Hb mRNA levels were not altered by CSEeither in HAECs or HCSMCs. This is consistent with

the fact that CSE down-regulates P4Ha at the transcrip-

tional level.

Effects of CSE on collagen levels in HAECs and

HCSMCs

Since P4Ha is the rate limiting subunit in the collagensynthesis, we were interested in the effects of CSE on col-

lagen production. Collagen (type I) content was ana-

lyzed in the cells and the culture medium. The cellular

collagen content was decreased (p < 0.001) significantly

in HAECs and HCSMCs when exposed to CSE (Fig.

2B). The collagen content in the media was also de-

creased (p < 0.001) in HAECs and HCSMCs in CSE-

treated cells (Fig. 2C). This indicates that CSE decreasesthe collagen synthesis in HAECs and HCSMCs.

Effects of cigarette smoke active components on P4Haexpression

Because benzo(a)pyrene, nicotine, and acrolein are

the major active components of cigarette smoke, we

investigated their effects on P4H expression in an effortto find responsible chemical component. As shown in

Fig. 3A, nicotine and acrolein had no effect on P4Haand b expression, whereas benzo(a)pyrene decreased

the P4Ha expression in HAECs but had no effect on

P4Hb expression (Fig. 3B). DMSO, a vehicle to dissolve

the benzo(a)pyrene, had no effect on P4Ha level.

We further tested the effect of benzo(a)pyrene on lev-

els of P4Ha mRNA and collagen in HAECs. As shownin Fig. 4A, benzo(a)pyrene (black column) decreased the

P4Ha mRNA levels significantly (p < 0.001) when com-

pared with the untreated HAECs (open column). The

collagen levels in the cells (Fig. 4B) and in the media

Fig. 2. Effect of CSE treatment on P4Ha, b mRNA levels and collagen contents in HAECs and HCSMCs. Cells were treated with or without CSE

(0.01 U) for 24 h. After the experimental period the total RNA was isolated using the Trizol reagent. The mRNA levels were estimated by real time

RT-PCR, and expressed as an arbitrary unit according to standard RNA preparation with known RNA levels. CSE decreased the P4Ha mRNA

levels in HAECs and HCSMCs (black column) comparing to controls (open column). The expression of P4Hb mRNA was not altered by CSE either

in HAECs or HCSMCs (black column, A). This indicates that CSE down-regulates P4Ha at the transcriptional level. Collagen levels (type I) were

estimated in the cells and the medium using an ELISA kit. The cellular collagen levels were decreased (p < 0.001) significantly in CSE-treated HAECs

and HCSMCs (black column) comparing to controls (open column, B). The collagen levels in the media were also significantly decreased (p < 0.001)

in HAECs and HCSMCs (black column) comparing to the media from untreated cells (open column, C). The values are expressed as means ± SEM

from three individual experiments. *p < 0.01, **p < 0.001, and NS p > 0.05 by independent Student�s t test.

M. Raveendran et al. / Biochemical and Biophysical Research Communications 323 (2004) 592–598 595

(Fig. 4C) were also decreased significantly (p < 0.001) by

the benzo(a)pyrene (black column) when compared with

the untreated HAECs (open column). DMSO, the sol-

vent, had no effect on the levels of P4Ha mRNA andcollagen (hatched column).

Effect of smoking on the P4H expression and collagen in

atherosclerotic lesions

In order to examine the relationship between ciga-

rette smoking and collagen production in vivo, we com-

pared the human abdominal aorta tissue withatherosclerotic lesion (AHA grade 2) between smokers

and non-smokers as shown in an example of a smoker

(male, 31 years, Fig. 5B) and a non-smoker (male, 29

years, Fig. 5A). According to the color intensity mea-

surements, the level of P4Ha in the non-smoker was

0.62 ± 0.06, but 0.28 ± 0.04 (p < 0.01) in the smoker.

The collagen level in the non-smoker was 0.34 ± 0.05,

but 0.18 ± 0.03 (p < 0.01) in the smoker. There was nosignificant difference in P4Hb levels. This smoking-de-

pendent differential P4Ha expression was further re-

flected by a thinner atherosclerotic cap in the smoker

(0.26 ± 0.01, Fig. 5E) as comparing with the non-smoker

(0.53 ± 0.06, p < 0.01, Fig. 5D).

We further examined whether atherosclerotic lesion

itself had any effect on the expressions of P4Ha and b

in an advanced atherosclerotic lesion (AHA grade 4)

in a smoker (Fig. 6). It appeared that necrotic core stim-

ulated the expression of P4H as the intense positive

stains along the edge of the necrotic core (Fig. 6A). Asexpected, the stain intensity for P4Ha (Figs. 6A and

B) was much lower than that for P4Hb (Fig. 6C and

D), which is consistent with the notion that P4Hb is

abundant. Since we did not have the atherosclerotic

sample with the same grade (AHA 4) of atherosclerotic

lesion in non-smokers, we were unable to carry out the

comparison between smokers and non-smokers. We

hypothesize that when necrotic core is formed, it releasescertain factors that stimulate collagen synthesis sur-

rounding the necrotic tissues in order to confine the

damage to a limited area as our biological defense mech-

anism. However, under the influence of cigarette smok-

ing, such compensatory mechanisms could be

attenuated. Elucidation of both processes is important

for our understanding of the atherosclerosis formation,

progression, and clinical outcomes.

Discussion

Atherosclerosis with its major complications—myo-

cardial infarction and ischemic stroke—is the leading

cause of death in the western world [17]. Cigarette

Fig. 4. Effect of benzo(a)pyrene on levels of P4Ha mRNA and collagen co

mRNA levels significantly (p < 0.001) when compared with the untreated HA

(C) were also decreased significantly (p < 0.001) by benzo(a)pyrene (black colu

vehicle for benzo(a)pyrene solution, had no effect on P4Ha mRNA expressio

means ± SEM from three individual experiments, *p < 0.001 by independent

Fig. 3. Effect of smoking contents on P4Ha and P4Hb levels in the

HAECs. To examine the effects of the active cigarette components on

the expression of P4H, HAECs were treated with nicotine (1 lg/mL of

the medium) or acrolein (50 lM) or 3 lM benzo(a)pyrene (1 lg/mL

of the medium) for 24 h and protein lysates were prepared as described

in the Materials and methods. Western blot was conducted as

described in Fig. 1. This is a representative blot from three independent

experiments. Nicotine and acrolein had no effect on P4Ha and bexpressions (A), whereas benzo(a)pyrene decreased the P4Ha expres-

sion in HAECs but had no effect on P4Hb subunit as shown in (B).

Western blotting with anti-b actin antibody was carried out to assess

the equal loading.

596 M. Raveendran et al. / Biochemical and Biophysical Research Communications 323 (2004) 592–598

smoking is a recognized risk factor for cardiovascular

disease and is known to promote the development of

atherosclerosis and thrombosis [18,19]. Endothelial dys-

function, increased pro-coagulation, hypertension, and

vasospasm have all been associated with smoking either

clinically or experimentally [18–20]. It is particularlynoticeable that smokers tend to have acute coronary

syndromes and myocardial infarction. Although exact

mechanisms are still unknown, unstable atherosclerotic

plaque and its tendency to rupture appear to be respon-

sible for the acute arterial occlusion. Several studies

have revealed that inappropriate activation of MMPs

is associated with atherosclerotic plaque rupture [6–8].

Yet, there is little information regarding whether colla-gen production also plays a role. In the present study,

we have examined the effect of CSE on collagen biosyn-

thesis. We show for the first time that CSE decreases the

P4Ha expression at transcriptional level in endothelial

cells and vascular smooth muscle cells. We have further

observed that benzo(a)pyrene—a key active tobacco

component, may be responsible for the CSE-induced

P4Ha suppression.P4H is an intracellular enzyme required for the synthe-

sis and formation of all known types of collagen [21]. It

catalyzes the hydroxylation of proline residues during

the post-translational processing of collagen. The resul-

tant hydroxyl groups of the hyroxyproline residues con-

tribute to the conformational stability of triple helical

collagen [22–24]. The active enzyme is a tetramer com-

ntent in HAECs. Benzo(a)pyrene (black column) decreased the P4HaECs (open column, A). The collagen levels in the cells (B) and the media

mn) comparing with the untreated HAECs (open column). DMSO, the

n and collagen production (hatched column). Values are expressed as

Student�s t test.

Fig. 5. Effect of smoking on P4Ha, collagen levels, and fibrous cap thickness in the human abdominal aorta atherosclerotic lesion. Human

abdominal aortic atherosclerotic lesions diagnosed as AHA grade 2 in a non-smoker (Male, 29 years, A) and a smoker (Male, 31 years, B) were

stained for P4Ha and collagen as described in the Materials and methods. The color intensity measurements showed the decreased level of P4Ha and

collagen content in the smoker comparing to the non-smoker. The specific P4Ha stained as brown (antibody based DAB staining) and collagen as

blue (Trichrome staining). Slides were with magnification 400·. The fibrous cap thickness was measured as shown in (C) and expressed in nanometer.

The slides used to measure the cap thickness are shown in (D) (non-smoker) and (E) (smoker). Thickness was measured at five randomly chosen

points around the plaque and expressed as mean ± SEM.

Fig. 6. Effect of smoking on P4Ha and P4Hb levels in the human

abdominal aorta atherosclerotic lesion. Human abdominal aortic

atherosclerotic lesion diagnosed with advanced lesion (AHA grade 4)

in a smoker was used to compare the patterns of P4Ha and bexpressions. The specific P4Ha (A,B) and P4Hb (C,D) stained as

brown color were detected using anti-human P4Ha and P4Hbantibodies. Slide was counter-stained with Trichrome. The expression

of P4Ha was lower than the P4Hb. The magnification is 100· for the

upper panel and 1000· for the lower panel.

M. Raveendran et al. / Biochemical and Biophysical Research Communications 323 (2004) 592–598 597

posed of two pairs of non-identical subunits (a2b2). Therates of synthesis of the a and b subunits are regulated dif-ferently [21]. The a subunit appears to become incorpo-

rated into the tetramer directly after its synthesis and

contains the major portion of the catalytic site [21,25].

Its expression limits the rate of active P4H formation. In

other cell systems, reduction of P4Ha mRNA results in

the decreased P4H activity and collagen production [26].

Our experiments have demonstrated that CSE can signif-

icantly depress collagen production in addition to P4Haexpression in both types of vascular wall cells, which is

consistent with the earlier observations by Jorgensen

et al. [27] in human smokers and suppression of collagen

production in cultured human lung cells [28]. However,

our study does not show whether suppressed P4Haexpression is directly responsible for CSE-induced reduc-

tion in vascular collagen. Another limitation of our study

is that we only measured type I collagen. Although type I

maybe the predominant arterial wall collagen, the type III

collagen also forms part of arterial wall extracellular ma-

trix. This clearly deserves our future investigation.

Althoughour study onhumanatheroma is preliminary

due to the small number of samples, the findings neverthe-less are consistent with the hypothesis that high incidence

of acute coronary syndrome in cigarette smokers could be

mediated by weakened plaque cap due to reduced colla-

gen production. Animalmodel is likely to provide a defin-

itive answer for the hypothesis. We are currently working

on this direction. However, there is still no widely ac-

cepted animalmodel that can consistentlymanifest unsta-

ble atherosclerotic plaque. Suitable animal smokingmodel poses yet another challenge. Recently, Nakata

and Maeda [29] reported that the atherosclerotic mice

with defective collagenmaturation by genetically induced

scurvy developed plaques with characteristics of vulnera-

bility due to a poorly formed fibrous cap. The reduced or

unstable collagen may produce a thin fibrous cap on ath-

eroma and render the lesion vulnerable to rupture. This

may be one of the reasons for the increased risk of acutecoronary syndrome [12,13] in smokers.

598 M. Raveendran et al. / Biochemical and Biophysical Research Communications 323 (2004) 592–598

In summary, our study indicates that the CSE-in-

duced inhibition of P4Ha expression and collagen pro-

duction in vascular cells may play a major role in the

development of thin fibrous cap in atherosclerotic le-

sion. Whilst other mechanisms cannot be ruled out, ben-

zo(a)pyrene—a key active tobacco component could beresponsible for the effects. To establish the molecular

mechanisms for the regulation and causal relationship

between cigarette smoking and unstable fibrous cap on

atheroma, properly developed animal models will be

needed.

Acknowledgments

The work is supported by a grant from NIH (R01-

HL066054). Dr. X.L. Wang is an AHA Established

Investigator (AHA 0440001N).

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