International Elsevier
CARD10
361
Journal of Cardiology, 28 (1990) 361-370
01115
Coronary arterial wall and atherosclerosis in youth ( l-20 years) a histologic study in a northern Italian population
:
Annalisa Angelini ‘, Gaetano Thiene ‘, Carla Frescura ’ and Giorgio Baroldi 2 ’ Istituto di Anaromia Patologica, Universitci degli Siudi di Padova, Padoua, Italy; ’ Istiruto di Fisiologia Clinica C. N. R., Universith degli Studi di Milano, Milan, Italy (Received
22 January
1990; accepted
22 January
1990)
Angelini A, Thiene G, Frescura C, Baroldi G. Coronary arterial wall and atherosclerosis in youth (l-20 years): a histologic study in a northern Italian population. Int J Cardiol. 1990;28:361-370. Based on the workinghypothesisthat coronary atherosclerosis begins in childhood,a histologic study was carried out on the subepicardialcoronary arterial tree of 100 young persons (l-20 years), who had died from causes unrelated to the cardiovascular system. These subjects were natives of a well-defined geographic area in northern Italy, namely the region of Veneto. Intimal proliferations (musculo-elastic and fibro-elastic layers) were observed in 95.3% of the coronary arterial segments in the age group between one and five years. The more distal the coronary segments examined, the lesser was the intimal thickening. Raised mature fibrous plaques were detected in 23 segments from 15 patients (2 from subjects aged between six and 10 years; 4 between 11 and 15 years, and 9 between 16 and 20 years). Single vessel disease was present in 9, double vessel disease in 4 and triple vessel disease in 2 cases. The site most involved by plaques was the proximal part of the left anterior descending coronary artery. Only one plaque was of sufficient dimensions to be considered stenotic (50% luminal reduction). Plaques were rarely sudanophilic, and all seemed to arise in relation to previous intimal thickening. No qualitative nor quantitative sexual differences were observed. These data give rise to much concern, and one consistent with a recently observed occurrence of sudden coronary death in young people from the same geographic area. Key words: Coronary arterial disease; Juvenile atherosclerosis;
Introduction Coronary atherosclerosis, the most important cause of death in western countries, almost certainly starts in childhood [l-5]. Several morphologic studies have been undertaken in children and
Correspondence to: G. Thiene, M.D., Istituto Patologica, Via Gabelli 61, 35121 Padova, Italy.
0167-5273/90/$03.50
0 1990 Elsevier Science
di Anatomia
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Geographic heart disease
young subjects in the attempt to investigate the morphological precursors and precise onset of the disease [5-141. The recent book by Neufeld and Schneeweiss well summarizes the state of the art of the matter [15], as does the review by Davies in this issue of the Journal [16]. Many workers focused attention on fatty streaks through the macroscopic detection of sudanophilic areas, since it is held that these will later evolve into mature atherosclerotic plaque [6,7,17-191.
B.V. (Biomedical
Division)
362
Other histologic investigations disclosed that diffuse or focal intimal proliferations are present even in infancy, thus posing the question of whether a relationship exists between arterial wall thickenings in early life and adult atherosclerosis [8-10,20-271. While Haust, Stary and Strong interpreted this phenomenon as normal arterial growth and remodelling [11,20,21], Neufeld and Vladover, instead, demonstrated that structural changes in coronary arteries were related to genetic and ethnic variations, with a higher degree of early intimal thickening occurring in populations prone to coronary arterial disease [22-241. Velican and Velican [10,26,27] suggested that intimal thickening and age-related changes in the coronary arterial wall might make the intima more susceptible to and extension of the the onset, evolution atherosclerotic plaques. As coronary atherosclerosis and its complications are now a growing problem even in Italy, long considered a nation at low to medium risk for this disease, we undertook a histologic study of the prevalence and progression of early and mature coronary lesions in 100 young subjects from the same geographic area in northern Italy, whose death was unrelated to cardiac disease. The aim of this investigation was to assess the structure and composition of coronary arteries in a so-called “control” population of young people during an age period that is critical in the pathogenesis of atherosclerosis.
Clinical data, particularly risk factors for atherosclerosis, were unknown. We divided this series into four groups according to age: 1 to 5 years (23 subjects, 16 males and 7 females); 6 to 10 years (20 subjects, 12 males and 8 females); 11 to 15 years (20 subjects, 13 males and 7 females) and 16 to 20 years (37 subjects, 23 males and 14 females). Six topographic sites of the coronary arteries were selected for histological examination. These were the main stem of the left coronary artery; the proximal segment of the left anterior descending coronary artery (1 cm from the bifurcation of the left main trunk); the left circumflex coronary artery 1 cm from the bifurcation of the left main trunk; the proximal right coronary artery 1 cm from its origin from the aortic sinus; the middle segment of the right coronary artery of the acute margin, and the middle segment of the left anterior descending coronary artery at a point 3 to 5 centimetres from the bifurcation of the main trunk (Fig. 1). Thus, a total of 600 arterial segments
Materials and Methods The Veneto region in northeast Italy covers an area of 18,368 km’, and has a population of 4,370,533 inhabitants (1985 census). Almost all residents in this area are Caucasian, and the population is ethnically homogeneous. One hundred subjects (64 males and 36 females), ranging in age from l-20 years, all of whom were natives and inhabitants of the Veneto, form the basis of this study. Death was unrelated to the cardiovascular system and, in most cases, due to injury. Autopsy was performed by one of us (G.T.) at the Institute of Pathological Anatomy of the University of Padova.
Fig. 1. Diagram illustrating the topographic sites within the coronary arteries from where the samples were taken. 1= Left main trunk; 2 = proximal left anterior descending coronary artery; 3 = left circumflex coronary artery; 4 = proximal right coronary artery; 5 = middle right coronary artery; 6 = middle left anterior descending coronary artery.
were examined. Two facing samples, each 2 mm thick, were taken at each site. One of these was embedded in paraffin, and cut into 6 pm thick sections, which were stained with haematoxylin eosin, Weigert-Van Gieson, and the Alcian-periodic acid Schiff stain. The other sample was frozen, cut, and stained with Sudan III. The remainder of the subepicardial coronary arterial tree was grossly inspected by taking cross sections at 3-5 mm intervals to rule out the presence of an otherwise major disease.
A
Fig. 2. Histology of proximal left anterior descending coronary artery. (A) Normal wall, with intact internal elastic lamina and no intimal proliferation in an 18-month-old boy. (B) Focal intimal proliferation of smooth muscle cells (arrows) with split and disrupted internal elastic lamina (musculo-elastic layer) in a 20-month-old boy. Weigert-Van Gieson stain. Original magnification X 64.
Fig. 3. Musculo-elastic layer in the proximal right coronary artery of a 4-year-old girl (arrows). The intimal thickening, characterized by a proliferation of smooth muscle cells within a split and fragmented internal elastic membrane, is almost semicircumferential. Weigert-Van Gieson stain. Original magnification X 16.
Intimal proliferations were defined according to the following histologic features: “Musculo-ela.stic intimal thickening” was characterized by focal or diffuse intimal proliferation of smooth muscle cells within a split and fragmented internal elastic membrane (musculo-elastic layer) (Fig. 2, 3), and further deposition of collagen and elastin (fibroelastic layer) (Fig. 4A) [28]. “Fibrous plaque”, was a focal raised lesion consisting of an intimal smooth muscle cell proliferation covered by a fibrous cap and enmeshed in a connective tissue matrix in the presence or absence of intra- or extra-cellular lipid deposition (Fig. 4B). In each slide, maximal intimal thickening was measured with the aid of a micrometer eyepiece. The circumferential extension of the intimal proliferation was scored as follows: absent; focal (less than 25% of circumference) (Fig. 2); semicircumferential (more than 25% and less than 75%) (Fig. 3); and diffuse or circumferential (more than 75%) (Fig. 5A). The presence or absence of a positive lipid stain was also noted. The mean maximal intimal thickening + standard deviation for each site in all the age groups was calculated, and results were analyzed according to Student’s t-test for unpaired data. In the proximal left anterior descending coronary artery, where the highest number of fibrous
364
Fig. 4. (A) Focal fibro-elastic layer characterized by smooth muscle cells proliferation within elastic and collagen fibrils in the proximal left anterior descending coronary artery of a 14-year-old boy. Weigert-Van Gieson stain, Original magnificationx64. (B) Fibrous plaque in the proximal left anterior descending coronary artery of a 9-year-old boy. The plaque displays a musculo-elastic thickening at its base. The lesion is focal with smooth muscle cells enmeshed in a loose connective tissue matrix. Weigert-Van Gieson stain. Original magnification X 48.
plaques were found, we also distributed the maximal intimal thickenings observed into 15 progressive classes, each with a range of 80 ,um (80, 160, 240.. .1200 pm). Except in one segment, luminal narrowing (in terms of diameter and cross-sectional area) was never significant enough to require definition and calculation.
RC?SUltS The distribution of intimal thickenings according to the surface involved in the 4 age groups is reported in Table 1.
Fig. 5. (A) Circumferential fibro-elastic proliferation of middle right coronary artery in a Ill-year-old boy. Weigert-Van Gieson stain. Original magnification X 16. (B) Stenotic fibrous plaque in the proximal left anterior descending coronary artery of a 16-year-old boy. Weigert-Van G&on stain. original magnification x 16.
Intimal thickening was observed in 95.3% of the coronary arterial segments in the age group from one to five years. These lesions were focal in
TABLE 1 Percentage distribution in circumferentia& extension intimal thickening in the four age groups.
of the
Age groups
Absent
Focal
SeDlitiC.
arc.
l-5 6-10 11-15 16-20
4.7 2.6 2.7 4.0
21.1 17.8 16.9 15.0
4.1 6.2 8.9 6.0
69.5 73.4 71.5 75.0
Semi&c. = semicircular; Circ. = circular.
365 TABLE
2
Number
of patients
with atherosclerotic
As
Patients
groups
N
l- 5 6-10 11-15 16-20
23 20 20 31
plaques.
With plaques 0 2 4 9
(%)
(0) (10) (20) (24)
21.1%, semicircumferential in 4.7%, and circumferential in 69.5% of the cases. No significant increases in extension were detected in the older age groups. For example, in the subjects aged from 16 to 20 years, intimal proliferations occurred in 96% of the arterial segments, and were focal in 15%, semicircumferential in 6X, and circumferential in 75% (P = n.s.). No clear-cut prevalence of musculo-elastic as opposed to fibro-elastic layers was observed in relation to age and vascular topography. Mature fibrous plaques were observed in 23 coronary arterial segments from 15 patients. In every case, they seemed to arise from an underlying musculo-elastic or fibro-elastic layer. No plaques were found in subjects aged from one to five years. Four were detected in two of the patients aged from 6 to 10 years, six in 4 patients aged from 11 to 15 years, and 13 in 9 patients aged from 16 to 20 years (Table 2). Therefore, 24% of the subjects aged from 16 to 20 years had one or more plaques (P < 0.01). These focal or semilunar lesions were located in the proximal left anterior descending coronary artery in 12 instances; in the proximal right coronary artery in 6; in the left circumflex branch in 3; and in the main stem of the left coronary artery in 2. Single vessel disease was present in 9 patients, double vessel disease in 4, and triple vessel disease in 2. Sudan staining was positive in only 3 of the 23 plaques. The most severe plaque was found in a 16-year-old boy, and consisted of a mild (50% luminal reduction) eccentric stenosis at the site of the proximal left anterior descending coronary artery (Fig. 5B).
Mean maximal intimal thickening at the 6 different sites for each age group is reported in Table 3. The more distal the coronary segment examined, the lesser was the intimal thickening. Hence, proximal segments with a larger diameter showed thicker proliferations, while distal segments with smaller diameters presented thinner ones. The middle segment of the left anterior descending coronary artery was the anatomic site showing least thickening. Although intimal thickening in each segment tended to increase with age (Table 3). this trend was not statistically significant except within the proximal anterior descending coronary artery. A significant difference in mean maximal intimal thickening was observed at this site between the youngest (one to five years) and the oldest (16 to 20 years) groups of patients (P < 0.01). Maximal intimal thickening ranged from 0 to 1200 pm_ By dividing the observed maximal intimal proliferations into 15 progressive classes, each with a range of 80 pm, a Gaussian distribution was found at all the topographic sites, with the apex of the bell-shaped curve corresponding to the intervals of 81-160 pm or 161-240 pm. This was independent of age. A bimodal lesion distribution, in contrast, was seen at the proximal anterior descending coronary artery in the three oldest age groups (six to 20 years overall - Fig. 6). This finding could be explained by the onset of fibrous plaques in some subjects. No qualitative or quantitative sexual differences were observed in either musculo-elastic thickening or mature atherosclerotic lesions. Mural thrombosis and aggregations of platelets were never observed in correspondence with any type of intimal lesion. Only one plaque appeared “inflamed”, having a mononuclear cell infiltrate mainly located in the intima. None of the plaques showed complications such as fissuring, thrombosis and calcification. Discussion The causes and progression of the atherosclerotic plaque are still controversial. Our data confirm that coronary atherosclerosis can take place early in the life cycle of man [29-311, and that
43.05 f
6-MLAD
27.04
78.58
87.63 98.62
71.05 *
96.05 f 47.47
54.35
128.89 & 85.64
141.33 f
202.16 + 152.00
M/F 264.42 + 292.86 + 232.17 + 150.71 f 129.82 f 159.43 f 125.33 f 135.00 f 79.70 f 125.OOf 64.83 it 81.71 f
163.58 182.55 179.11 74.86 104.54 93.36 75.95 106.58 44.25 61.03 55.33 30.60 96.81
103.05 + 111.62
106.37 + 64.23
197.47* 119.44
211.58 f
234.74 f 139.38
244.94+181.10
M/F 240.82 f 211.08 252.50 + 125.45 229.23 * 156.82 246.67 f 103.28 211.92~101.02 210.83 f 96.15 196.92 f 134.82 198.67 f 87.92 88.15 f 55.74 145.83 5 68.29 81.08+ 75.16 150.67 f 165.27
85.26 88.26 + 65.89
148.20+
239.59 f 138.98
192.26 + 105.11
319.83 + 233.30 *
283.97 f 169.56
Total
297.45 f 257.00* 320.22 f 319.08 + 208.39 f 161.33 f 241.55 f 236.00+ 153.52 f 138.00 f 97.52 f 70.50 f
M/F
Age group 16-20 years
172.24 168.82 230.56 248.84 116.75 72.79 133.03 155.38 78.67 99.60 0.39 54.64
* P < 0.01. F = female; LC = left circumflex; LMT = left main trunk; M = male; MLAD = middle left anterior descending; MR = middle right; PLAD = proximal left anterior descending; PR = proximal right.
90.95 f
119.05 f 136.11
4-PR
5-MR
113.19*
3-LC
86.71 *
177.335
2-PLAD
275.42 + 166.28
Total
246.69 111.01 85.62 97.71 89.43 82.62 156.31 62.96 78.22 86.58 30.50 13.21
Total
303.62 f 164.17+ 177.60 + 176.67 + 125.13 * 83.33 + 125.88* 100.83 f 89.80 f 93.00 f 46.06+ 35.00 f
M/F
Total
269.05 f 216.16
Age group 11-15 years
Age group 6-10 years
Age group 1-5 years
l-LMT
Level
Maximal intimal thickening at the six levels in the four age groups.
TABLE 3
12
1-5 yrs AGE
6-10 yrs
GROUP
AGE
800
400
12OOp-
0
400
800
400
11-15 yrs AGE
GROUP
12oop-
16-20 yrs
GROUP
AGE
800
1200pm
0
400
GROUP
800
Fig. 6. Histograms in which the maximal intimal thickening, observed at the level of proximal left anterior descending coronary in the various age groups, is distributed into 15 progressive classes, each with a range of 80 pm. Note the bimodal distribution older groups, due to the onset of fibrous plaques.
musculo-elastic intimal thickening may have an important role in its genesis [32-361. We observed that a raised fibrous plaque, which by definition is the basic lesion of atherosclerosis, evolves from the musculo-elastic and fibro-elastic layers. This aspect of gradual transition from a vascular smooth muscle cell lesion into fibrosis is definitely in keeping with the current idea that atherosclerosis derives from proliferation of smooth muscle cells, migration and differentiation [37-391. Our study disclosed that early intimal proliferations were almost a constant finding in coronary arteries. Indeed, in the various ages studied, only 2.6 to 4.7% of the segments were spared by this process. We had previously observed that coronary arteries are almost intact at birth, and that pro-
12OOpm
artery in the
liferative lesions occur in the first year of life [40], being nearly absent in the middle cerebral and renal arteries. These lesions were randomly distributed in the different age groups (l-20 years) and sexes, in agreement with previous reports [22-25,41,42] showing no sex and age differences in coronary intimal thickenings in children belonging to populations with low incidence of coronary arterial disease. From a qualitative point of view, we observed the entire spectrum of proliferations in our study. Mature plaques, however, appeared between the ages of 6 and 10 years, and increased in frequency in the older subjects. Such lesions were rarely positive when stained for lipid and appeared as fibrous plaques. This seems to suggest that, at
368
least in our series, the nature of the proliferative processes occurring in youth is morphologically different, even as far as plaques are concerned, from those occurring later in life. These lesions, in fact, are very similar to those described in hypertensive patients [43], and in some animal models where high blood pressure is the only risk factor of atherosclerosis [44]. The localization of the mature intimal lesions is peculiar, since the initial segment of the left anterior descending coronary artery was almost invariably involved. Fibrous plaques developed where proliferation of smooth muscle cells was more exuberant. Our findings gave the impression that certain early thickenings had undergone further proliferation leading to fibrous deposition and formation of plaques, and that the proximal left anterior descending coronary artery was the segment most prone to formation of such lesions. The pathobiological determinants of the transition from an apparently benign, early proliferation of intimal smooth muscle to an ominous plaque are unknown. Biochemical and structural studies of intimal smooth muscle cells demonstrated that cells from intimal thickenings have the phenotype” as the medial same “contractile smooth muscle cells, while smooth muscle cells from intimal thickenings adjacent to atheromatous plaques have a “synthetic phenotype” [38]. There must be some key factors, either individual or topographic, which trigger such a transformation. It is well established that some mechanical factors, such as velocity of flow and low wall shear stress, lead to the onset of atherosclerotic plaques in coronary arteries by inducing injury to the vascular endothelium. This seems especially true for the left anterior descending coronary artery [45,46]. Without doubt, individual risk factors such as high blood pressure, familial disease, and hypercholesterolemia might have played a key role in triggering growth of plaques but, unfortunately, we were unable to collect such information in our study population. It is interesting to note, nonetheless, that, in our recent study of sudden coronary death in young people in the same geographic area [47], we found single vessel disease in most cases with an obstructive atherosclerotic plaque located at the proximal left anterior de-
scending coronary artery. These plaques were mainly fibrotic, free of complications such as fissuring, hemorrhage or thrombosis, and resembled those described above. Whether this type of fibrous plaque is typical of young people in general, or is peculiar to our population, remains to be ascertained. According to the observation made in this investigation, and in a study addressing selected groups (normal subjects and patients with different patterns of ischemic heart disease) of variously aged Italian adults [48], as well as the above-mentioned report on sudden death in young people [47], this type of fibrous plaque seems to constitute the first stage of the mature atherosclerotic lesion. In conclusion, our findings show that coronary intimal proliferation was almost the rule after one year of age, and appeared thicker in the proximal coronary arterial segments. Moreover, fibrous plaques evolved from early intimal thickenings, and developed mainly in the proximal left anterior descending coronary artery. In addition, 24% of subjects aged from 16 to 20 years had one or more plaques, only one of which was stenotic. Plaques were rarely sudanophilic and all seemed to arise from previous intimal thickenings. Whether the latter are indeed lesions, or represent a normal process of modelling, remains unresolved.
Acknowledgments The authors are deeply indebted to Mauro Pagetta for the skillful histologic preparations, and to Chiara Carturan for computering and typing the manuscript. This study was supported by grants from National Research Council, Rome (Special Project FATMA) and from Regione Veneto, Venice (Juvenile Sudden Death Research Project), Italy.
References 1 Holman RL. Atherosclerosis. A pediatric nutrition problem? Am J Clin Nutr 1961;9:565-569. 2 Strong JP, McGill HD Jr. The pediatric aspects of atherosclerosis. J Atheroscler Res 1969;9:251-265. 3 McGandy RB. Adolescence and the onset of atherosclerosis. Bull NY Acad Med 1971;47:590-600. 4 Kennel WB, Dawber IR. Atherosclerosis as a pediatric problem. J Pediatr 1972;80:544-554.
369 5 Strong JP, McGill HC Jr. The natural history of coronary atherosclerosis. Am J Path01 1962;40:37-49. 6 McGill HC Jr. Fatty streaks in the coronary arteries and aorta. Lab Invest 1968;18:560-564. 7 Guzman MA, McMahan CA, McGill HC Jr, Strong P, Tejada C, Restrepo C, Eggen DA, Robertson WB, Solberg LA. Selected methodologic aspects of the International Atherosclerosis Project. Lab Invest 1968;18:479-497. 8 Moon H, Rinehart JF. Histogenesis of coronary arteriosclerosis. Circulation 1952;6:481-488. 9 Velican D, Velican C. Atherosclerotic involvement of the coronary arteries of adolescents and young adults. Atherosclerosis 1980;36:449-460. 10 Velican C, Velican D. The precursors of coronary atherosclerotic plaques in subjects up to 40 years old. Atherosclerosis 1980;37:33-46. 11 Haust MD. Atherosclerosis in childhood. Perspect Pediatric Path01 1978;4:155-216. 12 Enos WF, Holmes RH, Beyer J. Coronary disease among United States soldiers killed in action in Korea. J Am Med Assoc 1953;152:1090-1093. 13 McNamara JJ, Molot MA. Stremple JF, Cutting RT. Coronary artery disease in combat casualties in Vietnam. J Am Med Assoc 1971;216:1185-1187. 14 Wissler RW, McAllister HA Jr, Vesselinovitch DA. Histopathological study of the fatty streak in aortas and coronary arteries of young American military personnel. Am J Path01 1975;78:64a. 15 Neufeld HN, Schneeweiss A. Coronary artery disease in infants and children. Lea & Febiger, Philadelphia, 1983. 16 Davies H. Atherogenesis and the coronary arteries of childhood. Int J Cardiol 1990;28:283-292. 17 Klotz 0, Manning MF. Fatty streaks in the intima of arteries. J Path01 Bacterial 1911;16:211-220. 18 McGill HC Jr. The geographic pathology of atherosclerosis. Lab Invest 1968;18:463-635. 19 Tejada C, Strong JP, Montenegro MR, Restrepo C, Solberg LA. Distribution of coronary and aortic atherosclerosis by geographic location. race and sex. Lab Invest 1968:18:509526. 20 Haust MD. The morphogenesis and fate of potential and early atherosclerotic lesions in man. Hum Path01 1971;230:1-29. 21 Stary HC, Strong JP. The fine structure of non atherosclerotic intimal thickening, of developing and of regressing atherosclerotic lesions at the bifurcation of the left coronary artery. Adv Exp Med Biol 1976;67:89-108. 22 Neufeld HN, Wagenvoort CA, Edwards JE. Coronary arteries in fetuses, infants, juveniles and young adults. Lab Invest 1962;11:837-844. 23 Neufeld HN, Vlodaver Z. Structural changes of coronary arteries in young age groups (abstract). Eur J Cardiol 1971;2/3:56. 24 Vlodaver Z, Kahn HA, Neufeld HN. The coronary arteries in early life in three different ethnic groups. Circulation 1964;29:541-550. 25 Shomagel HE. Intimal thickening in the coronary arteries in infants. Arch Path01 1956;62:427-432.
26 Velican C, Velican D. Intimal thickening in developing coronary arteries and its relevance to atherosclerotic involvement. Atherosclerosis 1976;23:345-355. 27 Velican D, Velican C. Study of fibrous plaques occurring in the coronary arteries of children. Atherosclerosis 1979:33: 201-215. 28 Wolkoff K. Uber die Atherosclerose der Coronararterien des Herzens. Beitr Path01 Anat 1929;82:555-596. 29 Daoud AS. Jarmolych J, Fani K, Zumbo 0. Possible medial origin of intimal smooth muscle cells in preatheroma phase of arteriosclerosis. Fed Proc 1964:23:195-203. 30 Friedman M. The pathogenesis of coronary plaques, thromboses, and hemorrhages: an evaluative review. Circulation 1975;51/52 (Suppl 11):34-40. 31 Wissler RW, Vesselinovitch D, Komatsu A. Bridenstine RT. The arterial wall and atherosclerosis in youth. In: Biology of the arterial wall “Interaction in the arterial wall and atherosclerosis” 8th Int. Symp on Atherosclerosis, Siena, October 7-8, 1988:265-269. 32 Velican C, Velican D. Study of coronary intimal thickening. Atherosclerosis 1985;56:331-334. 33 Tracy R, I&sling GE. Age and fibroplasia as preconditions for atheronecrosis in human coronary arteries. Arch Path01 Lab Med 1987;111:957-963. 34 Becker A. Coronary atherosclerosis revisited. A pathologist’s view. Acta Med Stand 1984;694(suppl):69-76. 35 Svindland A. The localization of sudanophilic and fibrous plaques in the main left coronary bifurcation. Atherosclerosis 1983;48:139-145. 36 Grottum P, Svindland A, Walloe L. Localization of atherosclerotic lesions in the bifurcation of the main left coronary artery. Atherosclerosis 1983;47:55-62. 37 Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med 1986;314:488-500. 38 Campbell GR, Campbell JH, Manderson JA, Horrigan S, Rennick RE. Arterial smooth muscle. A multifunctional mesenchymal cell. Arch Path01 Lab Med 1988;112:977-986. 39 Wissler RW. The arterial medial cell; smooth muscle or multifunctional mesenchyme? J Atheroscler Res 1968:8: 201-213. 40 Thiene G, Angelini A. Carini A. Cefis F, Frescura C. Baroldi G. Juvenile atherosclerosis in northern Italy: a postmortem study. In: Doyle, E.F., Engle, M.A., Gersony, W.M., Rashkind WJ, Talner NS. eds. Pediatric cardiology. New York: Springer-Verlag, 1986;1189-1194. 41 Lober PH. Pathogenesis of coronary sclerosis. Arch Path01 1953;55:357-383. 42 Moon HD. Coronary arteries of infants. Am J Med Sci 1957;16:263-267. 43 Chobanian AV. The arterial smooth muscle cell in systemic hypertension. Am J Cardiol 1987;60:941-981. 44 Pauletto P, Pessina AC, Pagnan A, Thiene G, Semplicini A, Vescovo G, Mazzucato A, Scamrapieco G, Angelini A. Dal Palh C. Evidence of reduced atherosclerotic lesions in broad breasted white turkeys treated with oxprenolol. Artery 1983;12:220-233. 45 Fox B, Seed WA. Location of early atheroma in the human coronary arteries. J Biochem Eng 1981;103:208-213.
370 46 Sabbah HN, Khaja F, Hawkins ET, Brymer JF, McFarland TM, Van Der Bel-Khan J, Doerger PT, Stein PD. Relation of atherosclerosis to arterial wall shear in the left anterior descending coronary artery of man. Am Heart J 1986;112:453-458. 47 Corrado D, Thiene G, Pennelli N. Sudden death as the first
manifestation of coronary artery disease in young people ( 5 35 years). Eur Heart J 1988;9:139-144. 48 Baroldi G, Silver MD, Mariani F, Giuliano G. Correlation of morphologic variables in the coronary atherosclerotic plaque with clinical patterns of ischemic heart disease. Am J Cardiovasc Path01 1988;2:159-172.
CARD10
361
Journal of Cardiology, 28 (1990) 361-370
01115
Coronary arterial wall and atherosclerosis in youth ( l-20 years) a histologic study in a northern Italian population
:
Annalisa Angelini ‘, Gaetano Thiene ‘, Carla Frescura ’ and Giorgio Baroldi 2 ’ Istituto di Anaromia Patologica, Universitci degli Siudi di Padova, Padoua, Italy; ’ Istiruto di Fisiologia Clinica C. N. R., Universith degli Studi di Milano, Milan, Italy (Received
22 January
1990; accepted
22 January
1990)
Angelini A, Thiene G, Frescura C, Baroldi G. Coronary arterial wall and atherosclerosis in youth (l-20 years): a histologic study in a northern Italian population. Int J Cardiol. 1990;28:361-370. Based on the workinghypothesisthat coronary atherosclerosis begins in childhood,a histologic study was carried out on the subepicardialcoronary arterial tree of 100 young persons (l-20 years), who had died from causes unrelated to the cardiovascular system. These subjects were natives of a well-defined geographic area in northern Italy, namely the region of Veneto. Intimal proliferations (musculo-elastic and fibro-elastic layers) were observed in 95.3% of the coronary arterial segments in the age group between one and five years. The more distal the coronary segments examined, the lesser was the intimal thickening. Raised mature fibrous plaques were detected in 23 segments from 15 patients (2 from subjects aged between six and 10 years; 4 between 11 and 15 years, and 9 between 16 and 20 years). Single vessel disease was present in 9, double vessel disease in 4 and triple vessel disease in 2 cases. The site most involved by plaques was the proximal part of the left anterior descending coronary artery. Only one plaque was of sufficient dimensions to be considered stenotic (50% luminal reduction). Plaques were rarely sudanophilic, and all seemed to arise in relation to previous intimal thickening. No qualitative nor quantitative sexual differences were observed. These data give rise to much concern, and one consistent with a recently observed occurrence of sudden coronary death in young people from the same geographic area. Key words: Coronary arterial disease; Juvenile atherosclerosis;
Introduction Coronary atherosclerosis, the most important cause of death in western countries, almost certainly starts in childhood [l-5]. Several morphologic studies have been undertaken in children and
Correspondence to: G. Thiene, M.D., Istituto Patologica, Via Gabelli 61, 35121 Padova, Italy.
0167-5273/90/$03.50
0 1990 Elsevier Science
di Anatomia
Publishers
Geographic heart disease
young subjects in the attempt to investigate the morphological precursors and precise onset of the disease [5-141. The recent book by Neufeld and Schneeweiss well summarizes the state of the art of the matter [15], as does the review by Davies in this issue of the Journal [16]. Many workers focused attention on fatty streaks through the macroscopic detection of sudanophilic areas, since it is held that these will later evolve into mature atherosclerotic plaque [6,7,17-191.
B.V. (Biomedical
Division)
362
Other histologic investigations disclosed that diffuse or focal intimal proliferations are present even in infancy, thus posing the question of whether a relationship exists between arterial wall thickenings in early life and adult atherosclerosis [8-10,20-271. While Haust, Stary and Strong interpreted this phenomenon as normal arterial growth and remodelling [11,20,21], Neufeld and Vladover, instead, demonstrated that structural changes in coronary arteries were related to genetic and ethnic variations, with a higher degree of early intimal thickening occurring in populations prone to coronary arterial disease [22-241. Velican and Velican [10,26,27] suggested that intimal thickening and age-related changes in the coronary arterial wall might make the intima more susceptible to and extension of the the onset, evolution atherosclerotic plaques. As coronary atherosclerosis and its complications are now a growing problem even in Italy, long considered a nation at low to medium risk for this disease, we undertook a histologic study of the prevalence and progression of early and mature coronary lesions in 100 young subjects from the same geographic area in northern Italy, whose death was unrelated to cardiac disease. The aim of this investigation was to assess the structure and composition of coronary arteries in a so-called “control” population of young people during an age period that is critical in the pathogenesis of atherosclerosis.
Clinical data, particularly risk factors for atherosclerosis, were unknown. We divided this series into four groups according to age: 1 to 5 years (23 subjects, 16 males and 7 females); 6 to 10 years (20 subjects, 12 males and 8 females); 11 to 15 years (20 subjects, 13 males and 7 females) and 16 to 20 years (37 subjects, 23 males and 14 females). Six topographic sites of the coronary arteries were selected for histological examination. These were the main stem of the left coronary artery; the proximal segment of the left anterior descending coronary artery (1 cm from the bifurcation of the left main trunk); the left circumflex coronary artery 1 cm from the bifurcation of the left main trunk; the proximal right coronary artery 1 cm from its origin from the aortic sinus; the middle segment of the right coronary artery of the acute margin, and the middle segment of the left anterior descending coronary artery at a point 3 to 5 centimetres from the bifurcation of the main trunk (Fig. 1). Thus, a total of 600 arterial segments
Materials and Methods The Veneto region in northeast Italy covers an area of 18,368 km’, and has a population of 4,370,533 inhabitants (1985 census). Almost all residents in this area are Caucasian, and the population is ethnically homogeneous. One hundred subjects (64 males and 36 females), ranging in age from l-20 years, all of whom were natives and inhabitants of the Veneto, form the basis of this study. Death was unrelated to the cardiovascular system and, in most cases, due to injury. Autopsy was performed by one of us (G.T.) at the Institute of Pathological Anatomy of the University of Padova.
Fig. 1. Diagram illustrating the topographic sites within the coronary arteries from where the samples were taken. 1= Left main trunk; 2 = proximal left anterior descending coronary artery; 3 = left circumflex coronary artery; 4 = proximal right coronary artery; 5 = middle right coronary artery; 6 = middle left anterior descending coronary artery.
were examined. Two facing samples, each 2 mm thick, were taken at each site. One of these was embedded in paraffin, and cut into 6 pm thick sections, which were stained with haematoxylin eosin, Weigert-Van Gieson, and the Alcian-periodic acid Schiff stain. The other sample was frozen, cut, and stained with Sudan III. The remainder of the subepicardial coronary arterial tree was grossly inspected by taking cross sections at 3-5 mm intervals to rule out the presence of an otherwise major disease.
A
Fig. 2. Histology of proximal left anterior descending coronary artery. (A) Normal wall, with intact internal elastic lamina and no intimal proliferation in an 18-month-old boy. (B) Focal intimal proliferation of smooth muscle cells (arrows) with split and disrupted internal elastic lamina (musculo-elastic layer) in a 20-month-old boy. Weigert-Van Gieson stain. Original magnification X 64.
Fig. 3. Musculo-elastic layer in the proximal right coronary artery of a 4-year-old girl (arrows). The intimal thickening, characterized by a proliferation of smooth muscle cells within a split and fragmented internal elastic membrane, is almost semicircumferential. Weigert-Van Gieson stain. Original magnification X 16.
Intimal proliferations were defined according to the following histologic features: “Musculo-ela.stic intimal thickening” was characterized by focal or diffuse intimal proliferation of smooth muscle cells within a split and fragmented internal elastic membrane (musculo-elastic layer) (Fig. 2, 3), and further deposition of collagen and elastin (fibroelastic layer) (Fig. 4A) [28]. “Fibrous plaque”, was a focal raised lesion consisting of an intimal smooth muscle cell proliferation covered by a fibrous cap and enmeshed in a connective tissue matrix in the presence or absence of intra- or extra-cellular lipid deposition (Fig. 4B). In each slide, maximal intimal thickening was measured with the aid of a micrometer eyepiece. The circumferential extension of the intimal proliferation was scored as follows: absent; focal (less than 25% of circumference) (Fig. 2); semicircumferential (more than 25% and less than 75%) (Fig. 3); and diffuse or circumferential (more than 75%) (Fig. 5A). The presence or absence of a positive lipid stain was also noted. The mean maximal intimal thickening + standard deviation for each site in all the age groups was calculated, and results were analyzed according to Student’s t-test for unpaired data. In the proximal left anterior descending coronary artery, where the highest number of fibrous
364
Fig. 4. (A) Focal fibro-elastic layer characterized by smooth muscle cells proliferation within elastic and collagen fibrils in the proximal left anterior descending coronary artery of a 14-year-old boy. Weigert-Van Gieson stain, Original magnificationx64. (B) Fibrous plaque in the proximal left anterior descending coronary artery of a 9-year-old boy. The plaque displays a musculo-elastic thickening at its base. The lesion is focal with smooth muscle cells enmeshed in a loose connective tissue matrix. Weigert-Van Gieson stain. Original magnification X 48.
plaques were found, we also distributed the maximal intimal thickenings observed into 15 progressive classes, each with a range of 80 ,um (80, 160, 240.. .1200 pm). Except in one segment, luminal narrowing (in terms of diameter and cross-sectional area) was never significant enough to require definition and calculation.
RC?SUltS The distribution of intimal thickenings according to the surface involved in the 4 age groups is reported in Table 1.
Fig. 5. (A) Circumferential fibro-elastic proliferation of middle right coronary artery in a Ill-year-old boy. Weigert-Van Gieson stain. Original magnification X 16. (B) Stenotic fibrous plaque in the proximal left anterior descending coronary artery of a 16-year-old boy. Weigert-Van G&on stain. original magnification x 16.
Intimal thickening was observed in 95.3% of the coronary arterial segments in the age group from one to five years. These lesions were focal in
TABLE 1 Percentage distribution in circumferentia& extension intimal thickening in the four age groups.
of the
Age groups
Absent
Focal
SeDlitiC.
arc.
l-5 6-10 11-15 16-20
4.7 2.6 2.7 4.0
21.1 17.8 16.9 15.0
4.1 6.2 8.9 6.0
69.5 73.4 71.5 75.0
Semi&c. = semicircular; Circ. = circular.
365 TABLE
2
Number
of patients
with atherosclerotic
As
Patients
groups
N
l- 5 6-10 11-15 16-20
23 20 20 31
plaques.
With plaques 0 2 4 9
(%)
(0) (10) (20) (24)
21.1%, semicircumferential in 4.7%, and circumferential in 69.5% of the cases. No significant increases in extension were detected in the older age groups. For example, in the subjects aged from 16 to 20 years, intimal proliferations occurred in 96% of the arterial segments, and were focal in 15%, semicircumferential in 6X, and circumferential in 75% (P = n.s.). No clear-cut prevalence of musculo-elastic as opposed to fibro-elastic layers was observed in relation to age and vascular topography. Mature fibrous plaques were observed in 23 coronary arterial segments from 15 patients. In every case, they seemed to arise from an underlying musculo-elastic or fibro-elastic layer. No plaques were found in subjects aged from one to five years. Four were detected in two of the patients aged from 6 to 10 years, six in 4 patients aged from 11 to 15 years, and 13 in 9 patients aged from 16 to 20 years (Table 2). Therefore, 24% of the subjects aged from 16 to 20 years had one or more plaques (P < 0.01). These focal or semilunar lesions were located in the proximal left anterior descending coronary artery in 12 instances; in the proximal right coronary artery in 6; in the left circumflex branch in 3; and in the main stem of the left coronary artery in 2. Single vessel disease was present in 9 patients, double vessel disease in 4, and triple vessel disease in 2. Sudan staining was positive in only 3 of the 23 plaques. The most severe plaque was found in a 16-year-old boy, and consisted of a mild (50% luminal reduction) eccentric stenosis at the site of the proximal left anterior descending coronary artery (Fig. 5B).
Mean maximal intimal thickening at the 6 different sites for each age group is reported in Table 3. The more distal the coronary segment examined, the lesser was the intimal thickening. Hence, proximal segments with a larger diameter showed thicker proliferations, while distal segments with smaller diameters presented thinner ones. The middle segment of the left anterior descending coronary artery was the anatomic site showing least thickening. Although intimal thickening in each segment tended to increase with age (Table 3). this trend was not statistically significant except within the proximal anterior descending coronary artery. A significant difference in mean maximal intimal thickening was observed at this site between the youngest (one to five years) and the oldest (16 to 20 years) groups of patients (P < 0.01). Maximal intimal thickening ranged from 0 to 1200 pm_ By dividing the observed maximal intimal proliferations into 15 progressive classes, each with a range of 80 pm, a Gaussian distribution was found at all the topographic sites, with the apex of the bell-shaped curve corresponding to the intervals of 81-160 pm or 161-240 pm. This was independent of age. A bimodal lesion distribution, in contrast, was seen at the proximal anterior descending coronary artery in the three oldest age groups (six to 20 years overall - Fig. 6). This finding could be explained by the onset of fibrous plaques in some subjects. No qualitative or quantitative sexual differences were observed in either musculo-elastic thickening or mature atherosclerotic lesions. Mural thrombosis and aggregations of platelets were never observed in correspondence with any type of intimal lesion. Only one plaque appeared “inflamed”, having a mononuclear cell infiltrate mainly located in the intima. None of the plaques showed complications such as fissuring, thrombosis and calcification. Discussion The causes and progression of the atherosclerotic plaque are still controversial. Our data confirm that coronary atherosclerosis can take place early in the life cycle of man [29-311, and that
43.05 f
6-MLAD
27.04
78.58
87.63 98.62
71.05 *
96.05 f 47.47
54.35
128.89 & 85.64
141.33 f
202.16 + 152.00
M/F 264.42 + 292.86 + 232.17 + 150.71 f 129.82 f 159.43 f 125.33 f 135.00 f 79.70 f 125.OOf 64.83 it 81.71 f
163.58 182.55 179.11 74.86 104.54 93.36 75.95 106.58 44.25 61.03 55.33 30.60 96.81
103.05 + 111.62
106.37 + 64.23
197.47* 119.44
211.58 f
234.74 f 139.38
244.94+181.10
M/F 240.82 f 211.08 252.50 + 125.45 229.23 * 156.82 246.67 f 103.28 211.92~101.02 210.83 f 96.15 196.92 f 134.82 198.67 f 87.92 88.15 f 55.74 145.83 5 68.29 81.08+ 75.16 150.67 f 165.27
85.26 88.26 + 65.89
148.20+
239.59 f 138.98
192.26 + 105.11
319.83 + 233.30 *
283.97 f 169.56
Total
297.45 f 257.00* 320.22 f 319.08 + 208.39 f 161.33 f 241.55 f 236.00+ 153.52 f 138.00 f 97.52 f 70.50 f
M/F
Age group 16-20 years
172.24 168.82 230.56 248.84 116.75 72.79 133.03 155.38 78.67 99.60 0.39 54.64
* P < 0.01. F = female; LC = left circumflex; LMT = left main trunk; M = male; MLAD = middle left anterior descending; MR = middle right; PLAD = proximal left anterior descending; PR = proximal right.
90.95 f
119.05 f 136.11
4-PR
5-MR
113.19*
3-LC
86.71 *
177.335
2-PLAD
275.42 + 166.28
Total
246.69 111.01 85.62 97.71 89.43 82.62 156.31 62.96 78.22 86.58 30.50 13.21
Total
303.62 f 164.17+ 177.60 + 176.67 + 125.13 * 83.33 + 125.88* 100.83 f 89.80 f 93.00 f 46.06+ 35.00 f
M/F
Total
269.05 f 216.16
Age group 11-15 years
Age group 6-10 years
Age group 1-5 years
l-LMT
Level
Maximal intimal thickening at the six levels in the four age groups.
TABLE 3
12
1-5 yrs AGE
6-10 yrs
GROUP
AGE
800
400
12OOp-
0
400
800
400
11-15 yrs AGE
GROUP
12oop-
16-20 yrs
GROUP
AGE
800
1200pm
0
400
GROUP
800
Fig. 6. Histograms in which the maximal intimal thickening, observed at the level of proximal left anterior descending coronary in the various age groups, is distributed into 15 progressive classes, each with a range of 80 pm. Note the bimodal distribution older groups, due to the onset of fibrous plaques.
musculo-elastic intimal thickening may have an important role in its genesis [32-361. We observed that a raised fibrous plaque, which by definition is the basic lesion of atherosclerosis, evolves from the musculo-elastic and fibro-elastic layers. This aspect of gradual transition from a vascular smooth muscle cell lesion into fibrosis is definitely in keeping with the current idea that atherosclerosis derives from proliferation of smooth muscle cells, migration and differentiation [37-391. Our study disclosed that early intimal proliferations were almost a constant finding in coronary arteries. Indeed, in the various ages studied, only 2.6 to 4.7% of the segments were spared by this process. We had previously observed that coronary arteries are almost intact at birth, and that pro-
12OOpm
artery in the
liferative lesions occur in the first year of life [40], being nearly absent in the middle cerebral and renal arteries. These lesions were randomly distributed in the different age groups (l-20 years) and sexes, in agreement with previous reports [22-25,41,42] showing no sex and age differences in coronary intimal thickenings in children belonging to populations with low incidence of coronary arterial disease. From a qualitative point of view, we observed the entire spectrum of proliferations in our study. Mature plaques, however, appeared between the ages of 6 and 10 years, and increased in frequency in the older subjects. Such lesions were rarely positive when stained for lipid and appeared as fibrous plaques. This seems to suggest that, at
368
least in our series, the nature of the proliferative processes occurring in youth is morphologically different, even as far as plaques are concerned, from those occurring later in life. These lesions, in fact, are very similar to those described in hypertensive patients [43], and in some animal models where high blood pressure is the only risk factor of atherosclerosis [44]. The localization of the mature intimal lesions is peculiar, since the initial segment of the left anterior descending coronary artery was almost invariably involved. Fibrous plaques developed where proliferation of smooth muscle cells was more exuberant. Our findings gave the impression that certain early thickenings had undergone further proliferation leading to fibrous deposition and formation of plaques, and that the proximal left anterior descending coronary artery was the segment most prone to formation of such lesions. The pathobiological determinants of the transition from an apparently benign, early proliferation of intimal smooth muscle to an ominous plaque are unknown. Biochemical and structural studies of intimal smooth muscle cells demonstrated that cells from intimal thickenings have the phenotype” as the medial same “contractile smooth muscle cells, while smooth muscle cells from intimal thickenings adjacent to atheromatous plaques have a “synthetic phenotype” [38]. There must be some key factors, either individual or topographic, which trigger such a transformation. It is well established that some mechanical factors, such as velocity of flow and low wall shear stress, lead to the onset of atherosclerotic plaques in coronary arteries by inducing injury to the vascular endothelium. This seems especially true for the left anterior descending coronary artery [45,46]. Without doubt, individual risk factors such as high blood pressure, familial disease, and hypercholesterolemia might have played a key role in triggering growth of plaques but, unfortunately, we were unable to collect such information in our study population. It is interesting to note, nonetheless, that, in our recent study of sudden coronary death in young people in the same geographic area [47], we found single vessel disease in most cases with an obstructive atherosclerotic plaque located at the proximal left anterior de-
scending coronary artery. These plaques were mainly fibrotic, free of complications such as fissuring, hemorrhage or thrombosis, and resembled those described above. Whether this type of fibrous plaque is typical of young people in general, or is peculiar to our population, remains to be ascertained. According to the observation made in this investigation, and in a study addressing selected groups (normal subjects and patients with different patterns of ischemic heart disease) of variously aged Italian adults [48], as well as the above-mentioned report on sudden death in young people [47], this type of fibrous plaque seems to constitute the first stage of the mature atherosclerotic lesion. In conclusion, our findings show that coronary intimal proliferation was almost the rule after one year of age, and appeared thicker in the proximal coronary arterial segments. Moreover, fibrous plaques evolved from early intimal thickenings, and developed mainly in the proximal left anterior descending coronary artery. In addition, 24% of subjects aged from 16 to 20 years had one or more plaques, only one of which was stenotic. Plaques were rarely sudanophilic and all seemed to arise from previous intimal thickenings. Whether the latter are indeed lesions, or represent a normal process of modelling, remains unresolved.
Acknowledgments The authors are deeply indebted to Mauro Pagetta for the skillful histologic preparations, and to Chiara Carturan for computering and typing the manuscript. This study was supported by grants from National Research Council, Rome (Special Project FATMA) and from Regione Veneto, Venice (Juvenile Sudden Death Research Project), Italy.
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