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Role of wall factors in the pathogenesis of coronary atherosclerosis
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
R O L E OF W A L L FACTORS IN T H E P A T H O G E N E S I S OF CORONARY ATHEROSCLEROSIS AN APPROACH BASED UPON QUANTITATIVE HISTOLOGICAL CHANGES IN THE NON-ATHEROMATOUS INTIMAL THICKENING AND IN THE TUNICA MEDIA J. LOPES DE FARIA Institute of Pathology, University of Campinas, Campinas, Estado de Sao Paulo (Brazil)
(]Revised, received August 25th, ]967)
SUMMARYAND CONCLUSIONS A quantitative histological study was made of the intimal thickening and of the tunica media of the anterior descending branch of the left coronary arteries from 23 healthy patients, ranging in age from 2 months to 70 years. In all patients, but one, there was no intimal lipidic deposition at the level of the counts. The intimal thickening presented a precocious phase (in the younger patients) and a late phase (in the older patients), Tile latter was characterized, in comparison with the former, b y a decrease in the volume percentage of elastic fibers, and an increase in the volume percentage of metachromatic substance and collagenous fibers. Possibly there was also a decrease of muscle cells. The volumetric relation of all elements in the thickening was calculated in precocious and late thickenings, and indicated by an index. A highly significant difference (P < 0.001) was found between the two indices. Possibly, the precocious phase of the intimal thickening is physiological, related to arterial growth, and the late phase pathological and preatheromatous in nature. In the media, there was in the older group of patients, in comparison with the younger group, a lower volume percentage of muscle cells and a larger volume percentage of metachromatic substance and of collagenous plus reticular fibers. The relation of all elements in the media, as revealed b y the index, showed a highly significant difference between the values of group 1 and 2 (P < 0.00l). The increase of metachromatic substance in the media of the coronary arteries sustains the view t h a t this increase is a reparative phenomenon and secondary to decrease of muscle cells. As the changes in the arterial wall preceded the lipidic deposition in the tunica intima, they m a y play a role in this deposition.
j . Atheroscler. Res., 1968, 8:291-302
292
j . LOPES DE FARIA
INTRODUCTION
The inner lining of the coronary arteries presents a precocious non-lipidic thickening, seen in infants 1-4 and even before birth 5. Such thickening consists of muscle cells, collagen and elastic fibersl, 6. It cannot be sharply delineated from the arteriosclerotic process and its nature is unknown. The thickening is considered to be a physiological adaptive phenomenon 7 or a pathological change, preceding the lipidic deposition2-~, s. With advancing age the aortic media presents qualitative and quantitative histological changes 9. In the coronary arteries, however, the medial changes are less understood, and only a few observations on qualitative changes have been reported. Fibrosis in the media of the arteries of papillary muscles 10, decrease of elastic fibers, and increase of collagen elements n-13 and of acid mucopolysaccharides 14 have been described. The role of medial changes in the pathogenesis of atherosclerosis is a matter of dispute 9. In this investigation, not previously performed, a quantitative histological study of the coronary arteries in two groups of patients of different ages has been done. The purpose of the research is to study the changes in the intimal thickening and in the media that precede the lipidic deposition. MATERIAL AND
METHODS
The coronary arteries of 23 healthy patients who died suddenly in accidents, most of whom were white males, aged 2 months to 70 years, were examined. The cases were divided into group 1, younger patients ranging in age from 2 months to 14 years, and group 2, older patients aged 19 years and over (Tables 1 and 2). The autopsies were performed at the State Medicolegal Institute between 6 and 15 h after death. Gross findings in the heart and ascending aorta were not recorded, except for intimal lipoidosis. The latter was present in the aorta of both groups and in the coronary arteries of 6 patients of group 2. At the level of the counts, however, only 1 case (no. 3/64) presented lipoidosis and atherosclerotic plaques. Examination for lipids was made grossly after staining with Sudan IV and microscopically after carbowax embedding and staining with Sudan IV-propylene glycolla. The methods used were the same as previously reported 9, but with a few modifications. All steps in the preparatory procedure were standardized because this was essential for the quantitative evaluation. The coronary arteries were fixed in 20 % neutral formalin with 0.7 % sodium chloride 16 as follows: a block constituted by the heart, the ascending aorta, the aortic arch and the initial portion of descending aorta was removed. A cannula was introduced in an arterial branch arising from the arch; the other branches and the descending aorta were clamped. Through the cannula the formalin solution was injected under a pressure of 650-750 mm Hg for infants and children and 800-950 for adolescents and adults. Such pressure was maintained for 1 h in order to distend the walls of the coronary arteries uniformly and to avoid j . Atheroscler. Res., 1968, 8:291-302
ROLE OF WALL FACTORS IN PATHOGENESIS
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retraction artefacts. Then the block was immersed and held in the formalin solution for 48 h. One or two transverse specimens were taken from the middle third (in Cases 7, 14 and 22 also from the upper third) of the anterior descending branch of the left coronary artery, placed in Bouin's fluid overnight and then embedded in paraffin as usual; for the counts in the media the specimen was taken only from the middle third of the vessel. One to three series, each of 12 serial sections 6 # thick, were made. One to three groups (older patients) or three to eight groups (younger patients) each of three serial sections were stained for quantitative study. This variation in the number of groups was due to the changing thickness of the media and intima. Such sampling size permitted counting of the total number of points necessary to estimate the volumetric proportion of the components within the required confidence limits (relative error was about 5 %)17. In each group the three sections were stained respectively by Masson's trichrome with aniline blue is, toluidine blue and Weigert's elastic methods. The toluidine blue staining was performed according to LISON19, using a 0.1 ~o buffered solution at p H 4.2 4.5 for 5 min. A red or violet stain detected within 48 h after the staining was taken as positive metachromasia 20. After this time there was fading of the staining. The quantitative histological technique employed was the point-counting method which measures the volumetric ratios of components of tissue. A full account of it is given by WEIBEL 17. We used an objective 100 (oil) and a Kpl 8 X eyepiece of Zeiss with a quadratic lattice having 400 points. Only the intermediate points, formed by crossing of the thinner lines, were used (maximum 100 points). The ratios were estimated b y counting the hits on each component. The volume percentages of the muscle cells, metachromatic substance, collagenous and/or reticular fibers and elastic fibers were each calculated in approximately the same area of the intimal thickenings and media of each group of serial sections. All nuclei were counted as muscular nuclei. As in the former report 9, an index (I) was calculated in the intimal layer and media for the ratio of the amount of metachromatic substance (Ams) plus collagenous and/or reticular fibers (Acf) to the number of muscular cells (Amc) plus elastic fibers (Aef) as follows: Ams + Acf I-Amc + Aef For the intimal counts in the arteries of group 1, the sample was 42-160 microscopic fields (420-2050 points); it was 29-64 (626-1500 points) in the arteries of group 2. For the medial count the sample was 85-229 (1240 4240 points) and 30-79 (14602610 points) for group 1 and 2 respectively. In the wider thickenings (group 2) the counting was not in the whole height of them, but preferably in the deeper, less fibrous part, adjacent to the media. In Case no. 3/64 the areas with lipidic deposition were excluded as far as possible from the count. J. Atheroscler. Res., 1968, 8:291-302
296
j.
LOPES DE FARIA
The volume percentage of the elements in each individual was calculated, performing in all microscopic fields the differential count in the areas of the intima and media with the lattice of the eyepiece superimposed. A statistical evaluation of the results was made by calculating the average values, standard deviations and tile significance of the differences between the values of group 1 and group 2 according to the Kruskal-Wallis one-way analysis of variance b y ranks 21.
RESULTS
Microscopical examination Qualitative data Group J. At the level of the countings the coronary vessel presented the structure of a muscular artery and the intima was delineated from the media by means of the internal elastic membrane. The latter revealed short interruptions. The intima showed a thickening, which changed in width and involved the whole lumen or part of it in the younger patients. The thickening consisted of longitudinal muscular cells, longitudinal thick, membrane-like, elastic fibers (thinner than the inner elastic membrane), collagenous and reticular fibers (Masson's trichrome) and inconspicuous metachromatic substance. The collagenous elements were diffusely scattered and did not form a subendothelial layer as in coronary trunks 1 (Figs. 1 and 3). At the level of the interruptions, the muscle cells in the media were continuous to those in the intima. Occasionally there were segments of thick elastic fibers in the subintimal part of the media, recording fragments of the inner elastic membrane (Fig. 4). In the tunica media itself the muscle cells were densely arranged, the collagen fibers few in number, the elastic fibers thin and rare and the metachromasia (toluidine blue) slight, or not observed in a few areas. Group 2. In comparison with group 1 the intimal thickening was wider (up to about 1.5 times the media thickness), and involved the whole lumen. The interruptions in the internal elastic membrane were more frequent and often longer. The thickening consisted of the same elements as in group 1 with more metachromatic substance and collagenous fibers, and fewer elastic fibers and nmscle cells. The collagenous fibers were also scattered diffusely and did not form a subendothelial layer, even in the oldest patients (Figs. 2 and 5). In Masson's trichrome stain the deeper part of the intima, adjacent to the media, was frequently more cellular than the superficial part. The media presented a loose arrangement of the muscle cells, increase of the metachromatic substance and of reticular plus coltagenous fibers. These changes, however, were not present to the same degree in the whole media; they were slight in certain areas and conspicuous in others (Figs. 6 and 7). The latter areas revealed J. Athetoscler. Res., 1968, 8:291 302
ROLE OF WALL FACTORS IN PATHOGENESIS OF CORONARY ATHEROSCLEROSIS
297
Fig. 1. Coronary artery of 8-year-old patient (necropsy no. 8/64). At the top, notice the slight intimal thickening with the red staining diffusely scattered due to collagen. (Adventitia at the bottom.) Van Gieson's stain. • 320.
Fig. 2. Coronary artery of 43-year-old patient (necropsy no. 2/65). At the top, notice the blue stained intimal thickening. (Adventitia stained blue, at the bottom; media, red.) Masson's trichrome stain. • 320.
j . Atheroscler. Res., 1968, 8:291 302
298
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Fig. 3. Coronary artery of 12-year-old patient (necropsy no. 4164.) Notice the intimal thickening (top). Weigert's elastic tissue stain. • 640.
Fig. 4. Same artery and stain as shown in Fig. 3. Slight intimal thickening. Notice the elastic fibers in the media (arrow), recalling fragments of the internal elastic membrane. • 640.
Fig. 5. Coronary artery of 35-year-old patient (necropsy no. 1/64). Intimal thickening (top) with less elastic fibers as compared with Fig. 3. Interruption of the internal elastic membrane at the central part. Weigert's elastic tissue stain. • 256.
J. Atheroscler. Res., 1968, 8:291-302
ROLE OF WALL FACTORS IN PATHOGENESIS OF CORONARY ATHEROSCLEROSIS
299
Fig. 6. C o r o n a r y a r t e r y of 35-year-old p a t i e n t (necropsy no. 1/64). Notice t h e slight m e t a c h r o m a t i c s t a i n i n g in t h e media. (The i n n e r layer, a t t h e top, p r e s e n t s m o r e m e t a c h r o m a t i c s u b s t a n c e . ) Toluidine blue stain. • 320.
Fig. 7. S a m e a r t e r y as s h o w n in Fig. 6 in a n o t h e r area. C o n s p i c u o u s m e t a c h r o m a t i c s t a i n i n g in t h e media. • 320.
J. Atheroscler. Res., 1968, 8 : 2 9 1 - 3 0 2
300
J. LOPES DE FARIA
Fig. 8. Coronary artery of 24-year-old patient (necropsy no. 15/64). Beneath the thickened intima, notice the media with light spaces due to disappearance of muscle cells and accumulation of m e t a c h r o m a t i c substance. Weigert's elastic tissue stain. • 256.
apparent disappearance of muscle cells, accumulation of metachromatic substance and collagen deposition (Fig. 8).
Quantitative data The data from both groups of patients are presented in Tables 1 and 2. Intimal thickening. A comparison of Table 1 (younger patients) with Table 2 (older patients) shows that in the intima of the coronary arteries in the older patients there is a lower volume percentage of muscle cells (P < 0.10) and of elastic fibers (P < 0.01), and a higher volume percentage of metachromatic substance (P < 0.05) and of collagenous plus reticular fibers (P < 0.01). Tunica media. As shown in Table 1 compared with Table 2, in the media of the coronary arteries the volume percentage of muscle cells is smaller (P < 0.001) whereas the volume percentage of metaehromatic substance (P < 0.001) and the volume percentage of collagenous plus reticular fibers is greater (P < 0.01) in the older patients. The relation of the elements, both in the intimal thickening and in the media, as revealed b y the indices, showed a highly significant difference between the values of groups 1 a n d 2 (P < 0.001). DISCUSSION
The results show that the non-atheromatous intimal thickening of the coron a r y arteries presents a precocious phase and a late phase. The latter was seen in the older patients and was characterized, as compared with the precocious phase, b y a decrease of elastic fibers and an increase of metachromatic substance and collagenous elements. The non-significant decrease of muscle cells in older patients (P < 0.10) m a y be due to the difficulty of distinguishing fibroblasts from muscle cells, so that all cellular elements in the intima were counted as muscle cells. j . Atheroscler. Res., 1968, 8 : 2 9 1 - 3 0 2
ROLE OF WALL FACTORS IN PATHOGENESIS OF CORONARYATHEROSCLEROSIS
301
There was a modification in the structure of the precocious thickening toward arteriosclerosis (fibrosis, diminution of elastic fibers). This indicates t h a t the late phase of the intimal thickening is the non-atheromatous phase of atheroma, or a " p r e a t h e r o m a "'22. The late phase m a y be a reparative process, secondary to disappearance of muscle cells and elastic fibers. Atherosclerotic plaque formation as a reparative phenomenon has been suggested recently b y others23, 24. The precocious phase of the intimal thickening m a y be physiological and due to growth of the media in thickness. The latter assumption is supported b y the impossibility of delimiting the lower part of the intima from the media at the level of the internal elastic m e m b r a n e interruptions (Fig. 5), and b y occasional finding of fragments of this m e m b r a n e within the media (Fig. 4). Intimal thickening as an expression of arterial growth has been suggested b y others25, 26. The increase of m e t a c h r o m a t i c substance in the media is to be considered a reparative and not a regressive phenomenon, as suggested b y previous observations27-29. More severe changes in certain areas in the media (Fig. 7) m a y have significance in the pathogenesis of the focal nature of atheroma. Research is required to determine which element is primarily damaged in the intimal thickening and in the media. Our previous results on nutritive disturbance in arterial walls, m a i n l y anoxic in nature 2v-29, suggest t h a t the muscle cells in both layers are involved. The role of muscle cells in atherogenesis has been emphasized b y others6, 3~ As the intimal and medial changes preceded the lipidic deposition in the intima, they m a y play a role, as arterial wall factors, in this deposition.
ACKNOWLEDGEMENTS
This work was supported b y a grant from the F u n d a c a o de A m p a r o a Pesquisa do E s t a d o de Sao Paulo (reed. 6/62 and 64/269) and partially made in the D e p a r t m e n t of Histology and E m b r y o l o g y , F a c u l t y of Medicine, University of Sao Paulo (Prof. Dr. L. C. JUNQUEIRA).
REFERENCES i WOLKOFF, K,,
~ber die histologische Struktur der Coronararterien des menschlichen Herzens,
Virchows Arch. path. Anat., 1923, 241: 42.
2 LEVENE,C., The early lesions of atheroma in the coronary arteries, J. Path. Bact., 1956, 72: 79. MOON, H. D., Coronary arteries in fetuses, infants and juveniles, Circulation, 1957, 16: 263. 4 GILLOT, P., Les alt4rations histologiques des coronaires feetaIes et infantiles, Acta cardiol. (Brux.), 1962, 17: 145. 5 NEUI~ELD,H. N., C. A. WAGENVOORTANn J. E. EDWARDS,Coronary arteries in fetuses, infants, juveniles and young adults, Lab. Invest., 1962, 11: 837. 6 ROBERTSON, W. B., J. C. CrEER, J. P. STRONGAND H. C. McGILL JR., The fate of the fatty streak, Exp. molec. Path., 1963, Suppl. 1: 28. 7 SCHORNAGEL, H. E., Intimal thickening in the coronary arteries of infants, Arch. Path., 19S6, 62 : 427. 80SBORN, Cr. R., The Incubation Period of Coronary Thromboses, Butterworths, London, 1963. j . Atheroscler. Res., 1968, 8:291-302
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j. LOPES DE FARIA
9 DE FARIA, J. L., Role of medial changes in t h e pathogenesis of the diffuse intimal thickening and in atherogenesis. An approach based upon q u a n t i t a t i v e histological changes in t h e media of the ascending aorta, J. Atheroscler. Res., 1965, 5: 509. 10 UEOKA, W., Beitr•ge zur histologischen K e n n t n i s der Aa. Coronariae cordis des Menschen, Nagoya J. reed. Sci., 1934, 8: 36. 11 GROSS, L., E. Z. EPSTEIN AND M. A. KUGEL, Histology of the coronary arteries and their branches in the h u m a n heart, Amer. J. Path., 1934, 10: 253. 12 HIERONYMI, G., Uber den altersbedingten Formwandel elastischer und muskul~rer Arterien, S.-B. dkad. Wiss. Heidelberg, math.-nat. Kl., 1956, 3: 221. 13 GARDIOL, D., Aspects histologiques et histochimiques de la sGnescence des artGres de gros et de m o y e n calibre, Ann. Anat. path., 1964, 9: 269. 14 PAPACHARALAMPOUS, N. X., Altersbedingte histologische u n d histochemische VerAnderungen der Coronargef~sse, Virehows Arch. path. Anat., 1964, 338: 187. 15 WOOLFREY, B. F. AND H. PEARSON, A reliable m e t h o d for the histologic evaluation of tissue lipid. The joint use of sudan IV-propylene glycol, phosphin 3 (an aqueous fluorochrome), and t h e cryostat, Amer. J. clin. Path., 1962, 37: 437. 16 BIROE, W. J. AND F. D. TIBmTS, The use of sodium containing fixatives in minimizing cellular distortion in histochemical and cytochemical preparations, J. Histochem. Cytochem., 1961, 9: 409. iv WEIBEL, E. R., Principles and m e t h o d s for the morphometric s t u d y of the lung and other organs, Lab. Invest., 1963, 12: 131. 18 GRIDLEY, M. F., Manual of Histologic and Special Staining Technics, Armed Forces I n s t i t u t e of Pathology, W a s h i n g t o n , D.C., 1957. 19 LlsoN, L., Histochimieet Cytochimie Animales, Vol. 2, 3rd ed., Gauthie~Villars, Paris, 1960, p. 743. 20 KRAMER, H. AND G. M. WINDRUM, The m e t a c h r o m a t i c staining reaction, J. Histochem. Cytochem., 1955, 3: 227. 21 SIEGEL, S., Nonparametric Statistics for the Behavioral Sciences, MacGraw-Hill, New York, 1956. 22 DAOUD, A., J. JARMOLYCH, A. ZUMBO, K. FANI AND R. FLORENTIN, P r e a t h e r o m a phase of coronary atherosclerosis in m a n , Exp. molec. Path., 1964, 3: 475. 23 HAss, G. M., Vascular structure in relation to h u m a n and experimental arteriosclerosis, Nat. Acad. Sci., Nat. Res. Council, Washington, D.C., 1954, pp. 24-32. 24 MURRAY, M., G_ R. SCFIRODT AND I~. F. ]~ERG, The enzymatic induction of atherosclerosis, Arch. Path., 1965, 79: 144. 25 GILLMAN, T., Reduplication, remodeling, regenetion, repair, and degeneration of arterial elastic m e m b r a n e s . Some implications for the pathogenesis of arterial diseases, Arch. Path., 1959, 67: 624. 26 WRIGHT, I., The microscopical appearances of h u m a n peripheral arteries during growth and aging, J. clin. Path., 1963, 16: 499. 27 DE FARIA, J. L., Medionekrose der grossen u n d mittelgrossen Arterien nach o r t h o s t a t i s c h e m Kollaps des Kaninchens. Z u m Problem der Medionecrosis aortae des Menschen u n d der s p o n t a n e n Aortensklerose des Kaninchens, Beitr. path. Anat., 1955, 115: 373. 28 DE FARIA, J. L., Aortenmedionekrose mit sekund~irer Arteriosklerose bei den kongenitalen zyanotischen Herzkrankheiten, Beitr. path. Anat., 1961, 125: 129. 29 DE FARIA, J. L., Histopathological changes in the coronary arteries following shock or hypotension in man. Their relation to thrombosis and atherogenesis, Path. Microbiol., 1963, 26: 385. 30 PARKER, F. AND G. F. ODLAND, A light microscopic, histochemical and electron microscopic s t u d y of experimental atherosclerosis in rabbit coronary artery and a comparison with rabbit aorta atherosclerosis, Amer. J. Path., 1966, 48: 451.
J. Atheroseler. Res., 1968, 8 : 2 9 1 - 3 0 2
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
R O L E OF W A L L FACTORS IN T H E P A T H O G E N E S I S OF CORONARY ATHEROSCLEROSIS AN APPROACH BASED UPON QUANTITATIVE HISTOLOGICAL CHANGES IN THE NON-ATHEROMATOUS INTIMAL THICKENING AND IN THE TUNICA MEDIA J. LOPES DE FARIA Institute of Pathology, University of Campinas, Campinas, Estado de Sao Paulo (Brazil)
(]Revised, received August 25th, ]967)
SUMMARYAND CONCLUSIONS A quantitative histological study was made of the intimal thickening and of the tunica media of the anterior descending branch of the left coronary arteries from 23 healthy patients, ranging in age from 2 months to 70 years. In all patients, but one, there was no intimal lipidic deposition at the level of the counts. The intimal thickening presented a precocious phase (in the younger patients) and a late phase (in the older patients), Tile latter was characterized, in comparison with the former, b y a decrease in the volume percentage of elastic fibers, and an increase in the volume percentage of metachromatic substance and collagenous fibers. Possibly there was also a decrease of muscle cells. The volumetric relation of all elements in the thickening was calculated in precocious and late thickenings, and indicated by an index. A highly significant difference (P < 0.001) was found between the two indices. Possibly, the precocious phase of the intimal thickening is physiological, related to arterial growth, and the late phase pathological and preatheromatous in nature. In the media, there was in the older group of patients, in comparison with the younger group, a lower volume percentage of muscle cells and a larger volume percentage of metachromatic substance and of collagenous plus reticular fibers. The relation of all elements in the media, as revealed b y the index, showed a highly significant difference between the values of group 1 and 2 (P < 0.00l). The increase of metachromatic substance in the media of the coronary arteries sustains the view t h a t this increase is a reparative phenomenon and secondary to decrease of muscle cells. As the changes in the arterial wall preceded the lipidic deposition in the tunica intima, they m a y play a role in this deposition.
j . Atheroscler. Res., 1968, 8:291-302
292
j . LOPES DE FARIA
INTRODUCTION
The inner lining of the coronary arteries presents a precocious non-lipidic thickening, seen in infants 1-4 and even before birth 5. Such thickening consists of muscle cells, collagen and elastic fibersl, 6. It cannot be sharply delineated from the arteriosclerotic process and its nature is unknown. The thickening is considered to be a physiological adaptive phenomenon 7 or a pathological change, preceding the lipidic deposition2-~, s. With advancing age the aortic media presents qualitative and quantitative histological changes 9. In the coronary arteries, however, the medial changes are less understood, and only a few observations on qualitative changes have been reported. Fibrosis in the media of the arteries of papillary muscles 10, decrease of elastic fibers, and increase of collagen elements n-13 and of acid mucopolysaccharides 14 have been described. The role of medial changes in the pathogenesis of atherosclerosis is a matter of dispute 9. In this investigation, not previously performed, a quantitative histological study of the coronary arteries in two groups of patients of different ages has been done. The purpose of the research is to study the changes in the intimal thickening and in the media that precede the lipidic deposition. MATERIAL AND
METHODS
The coronary arteries of 23 healthy patients who died suddenly in accidents, most of whom were white males, aged 2 months to 70 years, were examined. The cases were divided into group 1, younger patients ranging in age from 2 months to 14 years, and group 2, older patients aged 19 years and over (Tables 1 and 2). The autopsies were performed at the State Medicolegal Institute between 6 and 15 h after death. Gross findings in the heart and ascending aorta were not recorded, except for intimal lipoidosis. The latter was present in the aorta of both groups and in the coronary arteries of 6 patients of group 2. At the level of the counts, however, only 1 case (no. 3/64) presented lipoidosis and atherosclerotic plaques. Examination for lipids was made grossly after staining with Sudan IV and microscopically after carbowax embedding and staining with Sudan IV-propylene glycolla. The methods used were the same as previously reported 9, but with a few modifications. All steps in the preparatory procedure were standardized because this was essential for the quantitative evaluation. The coronary arteries were fixed in 20 % neutral formalin with 0.7 % sodium chloride 16 as follows: a block constituted by the heart, the ascending aorta, the aortic arch and the initial portion of descending aorta was removed. A cannula was introduced in an arterial branch arising from the arch; the other branches and the descending aorta were clamped. Through the cannula the formalin solution was injected under a pressure of 650-750 mm Hg for infants and children and 800-950 for adolescents and adults. Such pressure was maintained for 1 h in order to distend the walls of the coronary arteries uniformly and to avoid j . Atheroscler. Res., 1968, 8:291-302
ROLE OF WALL FACTORS IN PATHOGENESIS
OF CORONARY ATHEROSCLEROSIS
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R O L E OF W A L L FACTORS I N P A T H O G E N E S I S OF C O R O N A R Y A T H E R O S C L E R O S I S
295
retraction artefacts. Then the block was immersed and held in the formalin solution for 48 h. One or two transverse specimens were taken from the middle third (in Cases 7, 14 and 22 also from the upper third) of the anterior descending branch of the left coronary artery, placed in Bouin's fluid overnight and then embedded in paraffin as usual; for the counts in the media the specimen was taken only from the middle third of the vessel. One to three series, each of 12 serial sections 6 # thick, were made. One to three groups (older patients) or three to eight groups (younger patients) each of three serial sections were stained for quantitative study. This variation in the number of groups was due to the changing thickness of the media and intima. Such sampling size permitted counting of the total number of points necessary to estimate the volumetric proportion of the components within the required confidence limits (relative error was about 5 %)17. In each group the three sections were stained respectively by Masson's trichrome with aniline blue is, toluidine blue and Weigert's elastic methods. The toluidine blue staining was performed according to LISON19, using a 0.1 ~o buffered solution at p H 4.2 4.5 for 5 min. A red or violet stain detected within 48 h after the staining was taken as positive metachromasia 20. After this time there was fading of the staining. The quantitative histological technique employed was the point-counting method which measures the volumetric ratios of components of tissue. A full account of it is given by WEIBEL 17. We used an objective 100 (oil) and a Kpl 8 X eyepiece of Zeiss with a quadratic lattice having 400 points. Only the intermediate points, formed by crossing of the thinner lines, were used (maximum 100 points). The ratios were estimated b y counting the hits on each component. The volume percentages of the muscle cells, metachromatic substance, collagenous and/or reticular fibers and elastic fibers were each calculated in approximately the same area of the intimal thickenings and media of each group of serial sections. All nuclei were counted as muscular nuclei. As in the former report 9, an index (I) was calculated in the intimal layer and media for the ratio of the amount of metachromatic substance (Ams) plus collagenous and/or reticular fibers (Acf) to the number of muscular cells (Amc) plus elastic fibers (Aef) as follows: Ams + Acf I-Amc + Aef For the intimal counts in the arteries of group 1, the sample was 42-160 microscopic fields (420-2050 points); it was 29-64 (626-1500 points) in the arteries of group 2. For the medial count the sample was 85-229 (1240 4240 points) and 30-79 (14602610 points) for group 1 and 2 respectively. In the wider thickenings (group 2) the counting was not in the whole height of them, but preferably in the deeper, less fibrous part, adjacent to the media. In Case no. 3/64 the areas with lipidic deposition were excluded as far as possible from the count. J. Atheroscler. Res., 1968, 8:291-302
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The volume percentage of the elements in each individual was calculated, performing in all microscopic fields the differential count in the areas of the intima and media with the lattice of the eyepiece superimposed. A statistical evaluation of the results was made by calculating the average values, standard deviations and tile significance of the differences between the values of group 1 and group 2 according to the Kruskal-Wallis one-way analysis of variance b y ranks 21.
RESULTS
Microscopical examination Qualitative data Group J. At the level of the countings the coronary vessel presented the structure of a muscular artery and the intima was delineated from the media by means of the internal elastic membrane. The latter revealed short interruptions. The intima showed a thickening, which changed in width and involved the whole lumen or part of it in the younger patients. The thickening consisted of longitudinal muscular cells, longitudinal thick, membrane-like, elastic fibers (thinner than the inner elastic membrane), collagenous and reticular fibers (Masson's trichrome) and inconspicuous metachromatic substance. The collagenous elements were diffusely scattered and did not form a subendothelial layer as in coronary trunks 1 (Figs. 1 and 3). At the level of the interruptions, the muscle cells in the media were continuous to those in the intima. Occasionally there were segments of thick elastic fibers in the subintimal part of the media, recording fragments of the inner elastic membrane (Fig. 4). In the tunica media itself the muscle cells were densely arranged, the collagen fibers few in number, the elastic fibers thin and rare and the metachromasia (toluidine blue) slight, or not observed in a few areas. Group 2. In comparison with group 1 the intimal thickening was wider (up to about 1.5 times the media thickness), and involved the whole lumen. The interruptions in the internal elastic membrane were more frequent and often longer. The thickening consisted of the same elements as in group 1 with more metachromatic substance and collagenous fibers, and fewer elastic fibers and nmscle cells. The collagenous fibers were also scattered diffusely and did not form a subendothelial layer, even in the oldest patients (Figs. 2 and 5). In Masson's trichrome stain the deeper part of the intima, adjacent to the media, was frequently more cellular than the superficial part. The media presented a loose arrangement of the muscle cells, increase of the metachromatic substance and of reticular plus coltagenous fibers. These changes, however, were not present to the same degree in the whole media; they were slight in certain areas and conspicuous in others (Figs. 6 and 7). The latter areas revealed J. Athetoscler. Res., 1968, 8:291 302
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Fig. 1. Coronary artery of 8-year-old patient (necropsy no. 8/64). At the top, notice the slight intimal thickening with the red staining diffusely scattered due to collagen. (Adventitia at the bottom.) Van Gieson's stain. • 320.
Fig. 2. Coronary artery of 43-year-old patient (necropsy no. 2/65). At the top, notice the blue stained intimal thickening. (Adventitia stained blue, at the bottom; media, red.) Masson's trichrome stain. • 320.
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Fig. 3. Coronary artery of 12-year-old patient (necropsy no. 4164.) Notice the intimal thickening (top). Weigert's elastic tissue stain. • 640.
Fig. 4. Same artery and stain as shown in Fig. 3. Slight intimal thickening. Notice the elastic fibers in the media (arrow), recalling fragments of the internal elastic membrane. • 640.
Fig. 5. Coronary artery of 35-year-old patient (necropsy no. 1/64). Intimal thickening (top) with less elastic fibers as compared with Fig. 3. Interruption of the internal elastic membrane at the central part. Weigert's elastic tissue stain. • 256.
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Fig. 6. C o r o n a r y a r t e r y of 35-year-old p a t i e n t (necropsy no. 1/64). Notice t h e slight m e t a c h r o m a t i c s t a i n i n g in t h e media. (The i n n e r layer, a t t h e top, p r e s e n t s m o r e m e t a c h r o m a t i c s u b s t a n c e . ) Toluidine blue stain. • 320.
Fig. 7. S a m e a r t e r y as s h o w n in Fig. 6 in a n o t h e r area. C o n s p i c u o u s m e t a c h r o m a t i c s t a i n i n g in t h e media. • 320.
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Fig. 8. Coronary artery of 24-year-old patient (necropsy no. 15/64). Beneath the thickened intima, notice the media with light spaces due to disappearance of muscle cells and accumulation of m e t a c h r o m a t i c substance. Weigert's elastic tissue stain. • 256.
apparent disappearance of muscle cells, accumulation of metachromatic substance and collagen deposition (Fig. 8).
Quantitative data The data from both groups of patients are presented in Tables 1 and 2. Intimal thickening. A comparison of Table 1 (younger patients) with Table 2 (older patients) shows that in the intima of the coronary arteries in the older patients there is a lower volume percentage of muscle cells (P < 0.10) and of elastic fibers (P < 0.01), and a higher volume percentage of metachromatic substance (P < 0.05) and of collagenous plus reticular fibers (P < 0.01). Tunica media. As shown in Table 1 compared with Table 2, in the media of the coronary arteries the volume percentage of muscle cells is smaller (P < 0.001) whereas the volume percentage of metaehromatic substance (P < 0.001) and the volume percentage of collagenous plus reticular fibers is greater (P < 0.01) in the older patients. The relation of the elements, both in the intimal thickening and in the media, as revealed b y the indices, showed a highly significant difference between the values of groups 1 a n d 2 (P < 0.001). DISCUSSION
The results show that the non-atheromatous intimal thickening of the coron a r y arteries presents a precocious phase and a late phase. The latter was seen in the older patients and was characterized, as compared with the precocious phase, b y a decrease of elastic fibers and an increase of metachromatic substance and collagenous elements. The non-significant decrease of muscle cells in older patients (P < 0.10) m a y be due to the difficulty of distinguishing fibroblasts from muscle cells, so that all cellular elements in the intima were counted as muscle cells. j . Atheroscler. Res., 1968, 8 : 2 9 1 - 3 0 2
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There was a modification in the structure of the precocious thickening toward arteriosclerosis (fibrosis, diminution of elastic fibers). This indicates t h a t the late phase of the intimal thickening is the non-atheromatous phase of atheroma, or a " p r e a t h e r o m a "'22. The late phase m a y be a reparative process, secondary to disappearance of muscle cells and elastic fibers. Atherosclerotic plaque formation as a reparative phenomenon has been suggested recently b y others23, 24. The precocious phase of the intimal thickening m a y be physiological and due to growth of the media in thickness. The latter assumption is supported b y the impossibility of delimiting the lower part of the intima from the media at the level of the internal elastic m e m b r a n e interruptions (Fig. 5), and b y occasional finding of fragments of this m e m b r a n e within the media (Fig. 4). Intimal thickening as an expression of arterial growth has been suggested b y others25, 26. The increase of m e t a c h r o m a t i c substance in the media is to be considered a reparative and not a regressive phenomenon, as suggested b y previous observations27-29. More severe changes in certain areas in the media (Fig. 7) m a y have significance in the pathogenesis of the focal nature of atheroma. Research is required to determine which element is primarily damaged in the intimal thickening and in the media. Our previous results on nutritive disturbance in arterial walls, m a i n l y anoxic in nature 2v-29, suggest t h a t the muscle cells in both layers are involved. The role of muscle cells in atherogenesis has been emphasized b y others6, 3~ As the intimal and medial changes preceded the lipidic deposition in the intima, they m a y play a role, as arterial wall factors, in this deposition.
ACKNOWLEDGEMENTS
This work was supported b y a grant from the F u n d a c a o de A m p a r o a Pesquisa do E s t a d o de Sao Paulo (reed. 6/62 and 64/269) and partially made in the D e p a r t m e n t of Histology and E m b r y o l o g y , F a c u l t y of Medicine, University of Sao Paulo (Prof. Dr. L. C. JUNQUEIRA).
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