Vascular Biology |
From the Department of Pathology (Y.I., S.M.S.), University of Washington, Seattle, and the Department of Pathology and Laboratory Medicine (B.M.M., J.K.), University of British Columbia, Vancouver, Canada.
Correspondence to Yuji Ikari, MD, Mitsui Memorial Hospital, Division of Cardiology, 1-Kanda-Izumi-cho, Chiyoda-ku, Tokyo 101, Japan.
| Abstract |
|---|
|
|
|---|
Key Words: intima proliferating cell nuclear antigen monoclonality infants left anterior descending coronary artery
| Introduction |
|---|
|
|
|---|
As reported herein, intimal formation is a rapid process beginning just before birth and appears to be associated with a period when cell proliferation is abundant. We speculate that this process may be the first event in a clonal expansion accounting for the monoclonality of advanced lesions.1 2 Moreover, the rapid time course of intimal expansion may make the process amenable to experimental manipulations in animal models designed to determine the factors responsible for spontaneous formation of the intima.
| Methods |
|---|
|
|
|---|
We chose the proximal left anterior descending coronary artery segments, 5 mm distal to the left main bifurcation, that were sectioned transversely and fixed in 10% neutral buffered formalin. We excluded bifurcated lesions because it is known that intimal cushions start at bifurcations.10
Measurement of Intima/Media Ratio
We determined the intima as the tissue inside the internal
elastic lamina and the media as the tissue between the internal and
external elastic laminas on Verhoeffvan Giesonstained slides. We
excluded sections near branches or oblique sections. We measured the
intima/media ratio with a computer-assisted system. We did not measure
luminal area because pressure fixation was done in only a few
samples.
Antibody
We purchased monoclonal antiproliferating cell nuclear antigen
(PCNA) antibody (Ab-1) from Calbiochem.
Immunohistochemistry
After hydration, antigen retrieval was performed by microwaving
the slides for 10 minutes in an antigen unmasking solution (Vector
Laboratories). Endogenous peroxidase was blocked with 3%
H2O2 for 5 minutes at room
temperature. Blocking was performed with 10% normal horse serum in PBS
containing 1% BSA. Slides were incubated with the primary antibody for
1 hour at room temperature; the titer was 1:200 for PCNA. A
biotinylated horse anti-mouse secondary antibody was then applied for
30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC
Elite, Vector Laboratories) for 30 minutes at room temperature. Then
3,3'-diaminobenzidine with NiCl2 was added to
yield a black reaction product at 37°C for 10 minutes, and methyl
green was used as a nuclear counterstain.
Counting of PCNA-Positive Nuclei
In performing immunohistochemistry, we took care to avoid
overstaining with PCNA. We counted clearly positive nuclei as positive.
Borderline staining was designated negative. Positivity was supported
by the independent observations of 2 pathologists.
| Results |
|---|
|
|
|---|
|
At 21 weeks of gestation (Figure 2A
), the
endothelium rests on an already formed internal elastic
lamina. The vessel has an adventitia and a thin media, but no intima.
The media consists of 2 to 3 cell layers. In vessel cross sections,
smooth muscle cell nuclei are elongate, suggesting a circumferential
orientation in the media (Figure 2A
). Between 17 and 30 weeks of
gestation, the earliest time frames that we studied, we saw intimal
cells in only 1 of 17 cases (Figure 2B
). Such vessels showed
splitting of the internal elastic lamina in areas of intima formation
at 28 weeks of gestation (Figure 2B
).
|
A frequent confounding issue in the determination of the site of
intimal growth is the presence in many vessels of a second layer of
media in these young coronary arteries (Figure 2C
and 2D
). The second layer of media has been reported previously and has
been called the "musculoelastic layer."11 12 This
layer is formed before intima formation in many cases (Figure 2C
). The musculoelastic layer can be distinguished from the
outer media by the orientation and density of smooth muscle cells
(Figure 2C
). Moreover, this layer can be distinguished from true
intima because the former lies beneath the internal elastic lamina
(Figure 2C
and 2D
). Because we determined the intima as a layer
above the internal elastic lamina, the musculoelastic layer belongs to
the media.
Presence of Intima (Figure 3
)
Other than the inner media, the first detectable intima was found
at 3 months before birth (28 weeks' gestation). Of 13 specimens
between 1 and 2.5 months before birth (30 to 36 weeks' gestation),
only 2 showed intima (15%). In contrast, 2 of 6 specimens (33%) at 1
month before birth (36 to 40 weeks' gestation) showed intima. Intima
was found in 8 of 21 specimens (38%) just after birth (0 to 7 days
old). At 3 months of age, intima was detected in all specimens. Thus,
intimal formation begins spontaneously near the time of birth.
|
Intima/Media Ratio
The intima/media ratio increased with age (Figure 4
). Just after birth, the intima/media
ratio was nearly 0.1. We considered the intima/media ratio in relation
to the cause of death. The prenatal intima/media ratio of
coronary arteries from fetuses with congenital heart disease
versus other causes of death was 0.0214±0.0567 versus 0.0247±0.0670,
respectively (NS). The postnatal intima/media ratio of congenital heart
disease patients versus those with other causes of death was
0.249±0.264 versus 0.103±0.193, respectively (NS). Thus,
statistically significant differences were not observed in association
with cause of death.
|
Intimal Cell Numbers
Figure 5
shows the scatterplot of
intimal cell numbers as counted in cross sections. The newly formed
intima is rich in cells, and intimal cell number increases with
age.
|
Cell Replication in Infant Coronary Arteries
Figure 6
shows PCNA staining of
coronary arteries. A similar staining pattern was confirmed by
another proliferation marker, MIB-1. Figure 7
shows ratios of PCNA-positive cells to
total cells in each vessel wall layer. Medial replication was quite
high before birth. Intimal cells were rare, and it was not possible to
estimate a replication frequency before birth, as shown in Figure 5
.
After birth, replication in the outer media gradually
declined. However, the inner media and intima retained their cell
replication rates between 2% and 5% until 2 years of age. The
endothelium had a high rate of PCNA positivity
prenatally, which gradually diminished in a pattern similar to that in
the media. From staining the slides with cell markers, it was found
that almost all cells in the intima and media were smooth muscle
cells.
|
|
| Discussion |
|---|
|
|
|---|
The intimas of these young coronaries consisted of smooth muscle cells. We stained all of the samples with CD68, a macrophage marker; however, we did not find any CD68-positive cells. In contrast, Stary4 reported that macrophages were found in the coronary arteries of young children. We cannot exclude the possibility that macrophages existed in certain regions of the intima because we studied only the proximal left anterior descending coronary arteries. However, virtually all of the proliferating cells we observed were smooth muscle cells.
The perinatal spontaneous formation of an intima in the left anterior descending coronary artery shows a startling similarity to the histological descriptions of another spontaneously formed intima, the lining of the ductus arteriosus.14 15 Even in patients with persistent ductus arteriosus, intima formation occurs spontaneously before birth.15 The major difference is that the ductus intima begins to form earlier than in the coronary artery, presumably as a prerequisite to the requirements of postpartum ductus closure. One wonders whether formation of the left anterior descending coronary artery intima represents a similar response to the same demand of early postnatal physiology. Like the left anterior descending coronary artery, the ductus also forms an inner medial layer.15 16 17 Similarly, splitting of the internal elastic lamina has been reported during intima formation in both the human coronary artery18 and human ductus arteriosus.15 In contrast, in murine models, the ductus intima mainly forms over a period of 1 to 2 days after closure, although the first intimal cells were found at 17 days of gestation.19 Whereas there is a general impression that the ductus intima forms as a response to injury occurring during closure, ductus intima in humans forms spontaneously before birth.15 16 17
The similar spontaneous formation of the ductus intima and the coronary intima should be contrasted with what is known about experimental models of intima formation after angioplasty. Most important, there is no evidence of endothelial injury in the spontaneous process that involves the left anterior descending coronary artery, whereas endothelial denudation is believed to be critical for the response-to-injury model. In the best-studied injury model, the rat carotid artery, smooth muscle cell proliferation reaches a maximum at 48 hours in the media (46%) and at 96 hours in the intima (73%).20 Subsequently, the thymidine index declines to baseline (0.06%) by 4 weeks in the media and by 8 weeks in the intima covered by endothelium.20 Such massive levels of medial smooth muscle cell replication are not seen in the spontaneous formation of the intima. In human coronary arteries, cell proliferation as determined by PCNA staining was 3% to 5% in the media before birth and decreased with age. We also stained all of the samples with MIB-1, another proliferation marker. The staining pattern was similar to that with PCNA. Finally, formation of the second medial layer before intima formation and multiplication of the internal elastic lamina, typical of spontaneous formation but not seen in the responses to angioplasty in the rat,21 rabbit,22 monkey,23 or pig,24 have been frequently described as a characteristic feature of the advanced atherosclerotic lesion.
The spontaneous-development and response-to-injury models are similar in 1 way. Medial proliferation precedes intimal formation in both the rat model and the human coronary artery. In the rat model, antifibroblast growth factor antibody25 26 27 can inhibit medial proliferation and delay intimal formation after injury. The studies of Campbell et al28 29 30 have suggested that this medial proliferation is part of the change in smooth muscle phenotype that is required before medial cells can migrate and form an intima. If so, this part of the response to injury may be recapitulated during normal development.
Finally, rapid formation of the intima at this coronary site
offers a possible explanation for the clonality of atherosclerotic
lesions.1 2 It is possible that masses like that shown in
Figure 2
arise either by migration of a replicative medial cell
or by trapping of an intimal cell as the internal elastic lamina forms.
In either case, replicative growth of the intima continuing for 2 years
postnatally, even at levels of only 2% to 4% as shown here, would be
adequate to produce a clone as has been observed in the adult lesion.
If so, clonal expansion may be a normal early event that precedes
atherosclerosis but is neither causal, as proposed by
Benditt,31 32 Fabricant,33 and Casalone et
al,34 nor a result of mutations in the plaque, as proposed
by McCaffrey et al,35 Spandidos et al,36 and
others.
| Acknowledgments |
|---|
Received November 24, 1998; accepted February 17, 1999.
| References |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
Y. Nakashima, T. N. Wight, and K. Sueishi Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans Cardiovasc Res, July 1, 2008; 79(1): 14 - 23. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. C. Doran, N. Meller, and C. A. McNamara Role of Smooth Muscle Cells in the Initiation and Early Progression of Atherosclerosis Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 812 - 819. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Loppnow, K. Werdan, and M. Buerke Invited review: Vascular cells contribute to atherosclerosis by cytokine- and innate-immunity-related inflammatory mechanisms Innate Immunity, April 1, 2008; 14(2): 63 - 87. [Abstract] [PDF] |
||||
![]() |
F. E. Alkemade, A. C. Gittenberger-de Groot, A. E. Schiel, J. C. VanMunsteren, B. Hogers, L. S. J. van Vliet, R. E. Poelmann, L. M. Havekes, K. Willems van Dijk, and M. C. DeRuiter Intrauterine Exposure to Maternal Atherosclerotic Risk Factors Increases the Susceptibility to Atherosclerosis in Adult Life Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2228 - 2235. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Napoli, L. O. Lerman, F. de Nigris, M. Gossl, M. L. Balestrieri, and A. Lerman Rethinking Primary Prevention of Atherosclerosis-Related Diseases Circulation, December 5, 2006; 114(23): 2517 - 2527. [Full Text] [PDF] |
||||
![]() |
B. S. Pessanha, K. Potter, F. D. Kolodgie, A. Farb, R. Kutys, E. K. Mont, A. P. Burke, T. J. O'Leary, and R. Virmani Characterization of Intimal Changes in Coronary Artery Specimens with MR Microscopy Radiology, October 1, 2006; 241(1): 107 - 115. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Napoli, O. Pignalosa, F. de Nigris, and V. Sica Childhood Infection and Endothelial Dysfunction: A Potential Link in Atherosclerosis? Circulation, April 5, 2005; 111(13): 1568 - 1570. [Full Text] [PDF] |
||||
![]() |
J.-L. Hillebrands, F. A. Klatter, and J. Rozing Origin of Vascular Smooth Muscle Cells and the Role of Circulating Stem Cells in Transplant Arteriosclerosis Arterioscler. Thromb. Vasc. Biol., March 1, 2003; 23(3): 380 - 387. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Cuomo, P. Guarini, G. Gaeta, M. de Michele, F. Boeri, J. Dorn, M.G. Bond, and M. Trevisan Increased carotid intima-media thickness in children-adolescents, and young adults with a parental history of premature myocardial infarction Eur. Heart J., September 1, 2002; 23(17): 1345 - 1350. [Abstract] [Full Text] [PDF] |
||||
![]() |
W. PALINSKI and C. NAPOLI The fetal origins of atherosclerosis: maternal hypercholesterolemia, and cholesterol-lowering or antioxidant treatment during pregnancy influence in utero programming and postnatal susceptibility to atherogenesis FASEB J, September 1, 2002; 16(11): 1348 - 1360. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Iijima, M. Yoshizumi, M. Hashimoto, M. Akishita, K. Kozaki, J. Ako, T. Watanabe, Y. Ohike, B. Son, J. Yu, et al. Red Wine Polyphenols Inhibit Vascular Smooth Muscle Cell Migration Through Two Distinct Signaling Pathways Circulation, May 21, 2002; 105(20): 2404 - 2410. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Zaina, L. Pettersson, B. Ahren, L. Branen, A. B. Hassan, M. Lindholm, R. Mattsson, J. Thyberg, and J. Nilsson Insulin-like Growth Factor II Plays a Central Role in Atherosclerosis in a Mouse Model J. Biol. Chem., February 1, 2002; 277(6): 4505 - 4511. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. L. HARRIS, M. STOLL, G. T. JONES, M. A. GRANADOS, W. K. PORTEOUS, A. M. VAN RIJ, and H. J. JACOB Identification of two susceptibility loci for vascular fragility in the Brown Norway rat Physiol Genomics, August 28, 2001; 6(3): 183 - 189. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Napoli and W. Palinski Maternal hypercholesterolemia during pregnancy influences the later devolopment of atherosclerosis: clinical and pathogenic implications Eur. Heart J., January 1, 2001; 22(1): 4 - 9. [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search Copyright © 1999 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. |