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Histopathological comparison between endofibrosis of the high-performance cyclist and atherosclerosis in the external iliac artery
Histopathological comparison between endofibrosis of the high-performance cyclist and atherosclerosis in the external iliac artery Aryan Vink, MD, PhD,a Mart H. Bender, MD,b Goof Schep, MD, PhD,c Dick F. van Wichen, BSc,a Roel A. de Weger, PhD,a Gerard Pasterkamp, MD, PhD,d and Frans L. Moll, MD, PhD,e Utrecht and Veldhoven, The Netherlands Introduction: High performance athletes, predominantly professional cyclists, can develop symptomatic arterial flow restriction in one or both legs during exercise. The ischemic symptoms are caused by endofibrosis and/or kinking of the external iliac artery. Because these athletes are young and have no classic risk factors for atherosclerosis, endofibrosis and atherosclerosis are considered different disease entities. We compared histology of endofibrotic lesions from young sportsmen with atherosclerotic lesions of the external iliac artery in elderly individuals. Methods and Results: Nineteen external iliac endarterectomy specimens from 18 cyclists (age 29 ⴞ 8 years) were compared with 42 external iliac segments from 22 elderly individuals (82 ⴞ 10 years). Ten arteries from elderly individuals revealed an intimal area that was >25% of the area encompassed by the internal elastic lamina and were considered atherosclerotic lesions. Stenosis was higher in patients (65% [interquartile range 50-75]) than in controls (11% [7-24]) (P < .0001). The endofibrotic lesions revealed loose connective tissue with moderate to high cellularity. Both in endofibrosis and atherosclerosis, most cells in the lesion were smooth muscle actin positive. In the endofibrosis specimens, loose fibers of collagen were observed, whereas in the atherosclerotic lesions collagen was mostly densely packed. Calcification of the lesion was not observed in endofibrotic lesions, whereas calcium deposition was observed in 80% of atherosclerotic lesions. Lymphocytes were present in 21% of endofibrotic lesions and in 80% of atherosclerotic cases. Macrophages were observed in 16% of endofibrotic lesions and in all atherosclerotic plaques. Luminal thrombosis was observed in one case of endofibrosis. Conclusion: In the external iliac artery, atherosclerotic lesions and endofibrotic lesions of high performance cyclists have distinct morphologic characteristics. Endofibrosis in the external iliac artery may serve as soil for luminal thrombosis. ( J Vasc Surg 2008;48:1458-63.) Clinical Relevance: Endofibrosis can be a serious clinical problem in high-performance athletes. Because endofibrosis predominantly occurs in sportsmen, most surgeons do not frequently see patients with endofibrosis. The results of the present study show that endofibrosis has distinct histologic characteristics as compared to atherosclerosis. In clinical practice, it is important to recognize patients with endofibrotic lesions, because endofibrosis and atherosclerosis require different diagnostic and therapeutic approaches.
Since 1985, reports have been published about claudication at exercise in highly trained cyclists.1,2 The observed claudication in these athletes is associated with stenosis and/or kinking of the external iliac artery. A possible treatment for these complaints is surgery. The surgically removed specimens revealed intimal thickening consisting of loose connective tissue, a phenomenon since then called endofibrosis.3 The young sportsmen that develop endofibrosis mostly have no classic risk factors for the development of atherosclerosis. Therefore, other factors than the traditional athFrom the Department of Pathology, University Medical Center, Utrecht,a Department of Surgery,b Department of Sports Medicine, Maxima Medical Center, Veldhoven,c Department of Cardiology,d Department of Surgery, University Medical Center, Utrecht.e Competition of interest: none. Correspondence: Aryan Vink, MD, University Medical Center Utrecht, Department of Pathology, Room H04-312, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands (e-mail: [email protected]). 0741-5214/$34.00 Copyright © 2008 Published by Elsevier Inc. on behalf of The Society for Vascular Surgery. doi:10.1016/j.jvs.2008.07.057
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erosclerotic risk factors are involved in the development of endofibrosis. It has been suggested that arterial injury as a result of hemodynamic injury secondary to shear stresses of very high blood flow in combination with the mechanical stress to the artery due to the athlete’s body position leads to intimal thickening.4 Because endofibrosis and atherosclerosis have different pathophysiologic mechanisms, they are considered as different disease entities. Thus far, however, studies that compare histologic aspects of endofibrosis and atherosclerosis in the external iliac artery are lacking. In the present study, we compared the histology of endofibrosis and atherosclerosis in the external iliac artery. METHODS Endarterectomy and control specimens. Nineteen endarterectomy specimens from 18 patients were collected (15 male, 3 female; age 29 ⫾ 8 years). All patients were professional or top class competition amateur cyclists with complaints of claudication during exercise. Magnetic resonance angiography in these patients revealed a stenosis in the external iliac artery. Patients with claudication solely
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due to kinking of the iliac artery without significant stenosis were not included.5 In most cases, a patch of the great saphenous vein was placed. Median length of the endarterectomy specimens was 15 mm (interquartile range [IQR] 15-40). After fixation in 4% formalin, endarterectomy specimens were dissected in 3-mm segments. All segments were embedded in paraffin and used for immunohistochemical staining. As control specimens, 42 arterial segments of the external iliac artery were collected post mortem from 22 individuals (11 men and 11 women, age 82 ⫾ 10 years, history of cardiovascular disease and risk factors unknown) who donated their body to medical science. Arteries were pressure fixed in 4% formalin after death and a 5 mm part of the external iliac artery was dissected.6 This segment was dissected in two parts that were both used for immunohistochemical staining. Immunohistochemical staining. Paraffin sections were stained with hematoxylin and eosin (H&E), Elastica von Giesson, and Sirius red. Immunohistochemical stainings were performed with CD68 (to detect macrophages; dilution 1:800; Novocastra Laboratories Ltd, Newcastle upon Tyne, UK), CD45 (to detect lymphocytes; dilution 1:200; DAKO, Glostrup, Denmark) and alpha actin (to detect myofibroblasts and smooth muscle cells; dilution 1:8000; Sigma, St. Louis, Mo), as described previously.7 In addition, the endarterectomy specimens from the patients were stained with an antibody against collagen 4 (dilution 1:100; Eurodiagnostica, Arnhem, The Netherlands) after pre-treatment with pepsin and an antibody against MIB-1 (dilution 1:100; DAKO) after boiling in a citrate buffer. The signal of all immunohistochemical stainings was visualized using diaminobenzidine. Sections were counterstained with hematoxylin. In the control arteries, percentage stenosis was determined using the Elastica von Giesson staining. The size of the intima/plaque was calculated by subtracting the luminal area from the area encompassed by internal elastic lamina (IEL area). Stenosis was defined as (intimal area/ IEL area) ⫻ 100%.6 The number of cells per mm2 was analyzed semi quantitatively and divided into 3 groups: low, moderate, and high. The Sirius red staining was analyzed with polarized light to detect collagen. The CD68 and CD45 stainings were analyzed semi quantitatively: negative, no positive cells; minor, few positive cells; moderate, groups of 10 cells present; heavy, abundant staining. Presence of an extracellular lipid core was scored as percentage of the total intimal/plaque area. RESULTS Patients. In the patients, median stenosis was 65% (IQR 50-75) (Fig 1). The endarterectomy specimens of all patients with endofibrosis revealed a loose connective tissue matrix with moderate to high cellularity (Fig 2, A and C). The cells were spindle shaped or stellate and mostly not arranged in bundles. Most of the cells stained positive with alpha actin and could, therefore, be classified as smooth muscle cells or myofibroblasts. In between the myofibro-
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Fig 1. Stenosis (%) of external iliac arteries of controls and patients (p ⬍ .0001). Horizontal line indicates the median value.
blasts, loose collagen fibers were observed that stained in the Sirius red staining and in most cases also in the immunohistochemical collagen 4 staining. An area with denselypacked collagen was observed in 7/19 (37%) cases. This area with densely-packed collagen was at most 40% of the lesion. In 15/19 (79%) cases, a newly formed elastic lamina was present in the lesion, most often near the intimal layer. An occluding thrombus superimposed on the endofibrosis was observed in one case. This thrombus showed signs of organization with presence of fibroblasts and microvessels. Minor CD68 staining was observed in 1 (5%) and moderate CD68-staining in 2 (11%) cases (Fig 3). Negative staining of CD68 was observed in 16/19 (84%) lesions. In 4 (21%) lesions, minor staining of CD45-positive lymphocytes was observed, whereas in the other 15 (79%) lesions no lymphocytes were observed. Calcium deposition and an extra cellular atheroma were not observed in the endofibrotic lesions. In most specimens, no MIB-1 staining was observed. Results are summarized in Tables I and II. Controls. Percentage stenosis was calculated in the arterial sections from the controls to get insight in the natural development of stenosis in the external iliac arteries. Median stenosis of the arterial sections obtained from the elderly individuals was 11 % (IQR 7-24) (Fig 1). Ten arteries obtained from 7 individuals revealed a stenosis ⱖ25% and were considered significant atherosclerotic lesions. Percentage stenosis was significantly higher in patients than in controls (P ⬍ .0001, Mann-Whitney test; Fig 1). The lesions with stenosis ⱖ25% revealed low to moderate cellularity (Fig 2, B and D). However, predominantly in the fibrous cap of the atherosclerotic lesions also some areas with high cellularity were observed. Most of the cells in the lesions stained positive for alpha actin. The matrix of these lesions showed moderate to abundant presence of collagen of which at least 50% was densely packed in all cases. In 4/10 cases, doubling of the internal elastic lamina was observed. The newly formed elastic lamina was located in the neighborhood of the original internal elastic lamina. In all cases, CD68 staining was present (minor staining in 2 (20%), moderate in 1 (10%), and heavy in 7 (70%)). In 8 (80%) cases, CD45 staining was observed (minor in 5
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Fig 2. Histological comparison between endofibrosis and atherosclerosis in the external iliac artery. A and C, Hematoxylin and eosin (H&E) stainings of endofibrotic lesion in a professional cyclist. A, Overview of the wall of the artery showing the media (m) and the endofibrotic lesion (e). Bar ⫽ 250 m. C, Higher magnification of the endofibrotic lesion showing a loose connective tissue matrix with moderate to high cellularity consisting of spindle and stellate shaped cells. The cells are not orderly arranged. Bar ⫽ 100 m. B and D, H&E stainings of atherosclerotic lesion. B, Overview of the wall of the artery showing the media (m) and the atherosclerotic plaque. The plaque consists of an extracellular atheroma (a), a socalled shoulder with macrophages (s) and a fibrous cap (c). Bar ⫽ 250 m. D, Higher magnification of the fibrous cap of the atherosclerotic plaque showing low cellularity with spindle cells arranged in bundles. The extra cellular matrix consists of densely-packed collagen. Bar ⫽ 100 m.
Fig 3. Macrophages in endofibrotic lesion. A, Endofibrotic lesion with moderate to high cellularity. Foam cells are present near the lumen at the top of the photo. B, CD68 immunohistochemical staining in adjacent section, identifying the cells as macrophage foam cells (brown staining). Bar ⫽ 125 m.
(50%), moderate in 1 (10%), and heavy in 2 (20%)). Calcium deposition was observed in 8 (80%) cases (minor in 1 (10%) and moderate in 7 (70%)). An extra cellular lipid pool (atheroma) was observed in 7 (70%) cases (⬍40% of total plaque
area in 3 (30%) and ⱖ40% in 4 (40%)) (Fig 2, B). No recent or organized thrombus was observed, however in 1 case amorphous material was found, suggesting presence of an old thrombus. Results are summarized in Tables I and II.
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Table I. Characteristics of endofibrosis, atherosclerosis and adaptive intimal thickening in the external iliac artery Intimal lesions in elderly individuals
CD68 staining No Minor Moderate Heavy CD45 staining No Minor Moderate Heavy Calcium deposition No Minor Moderate Heavy Atheroma No ⬍40% ⱖ40%
Endofibrosis n (percentage)
AS ⱖ25% (atherosclerosis) n (percentage)
AS ⬍25% (adaptive intimal thickening) n (percentage)
16 (84%) 1 (5%) 2 (11%) 0 (0%)
0 (0%) 2 (20%) 1 (10%) 7 (70%)
6 (19%) 13 (41%) 10 (31%) 3 (9%)
15 (79%) 4 (21%) 0 (0%) 0 (0%)
2 (20%) 5 (50%) 1 (10%) 2 (20%)
17 (53%) 14 (44%) 1 (3%) 0 (0%)
19 (100%) 0 (0%) 0 (0%) 0 (0%)
2 (20%) 1 (10%) 7 (70%) 0 (0%)
25 (78%) 2 (6%) 3 (9%) 2 (6%)
19 (100%) 0 (0%) 0 (0%)
3 (30%) 3 (30%) 4 (40%)
29 (91%) 1 (3%) 2 (6%)
Table II. Summary of histopathological comparison between endofibrosis and atherosclerosis in the external iliac artery
Connective tissue Collagen Cellularity Cellular form Arrangement of cells Smooth muscle actin Newly formed elastic lamina Macrophages Lymphocytes Calcification Atheroma
Endofibrosis
Atherosclerosis
Loose Loose fibers Moderate to high Spindle or stellate Loose, not organized Most cells positive Near the intimal layer – or ⫹ – or ⫹ – –
Compact Densely packed Low to moderate Spindle Organized in bundles Most cells positive Doubling original internal elastic lamina ⫹, ⫹⫹ or ⫹⫹⫹ –, ⫹ or ⫹⫹ –, ⫹ or ⫹⫹ –, ⫹ or ⫹⫹
The arterial segments with stenosis ⬍25% on average revealed low to moderate cellularity (high cellularity was observed in a few cases). Most of the cells in the lesions stained positive for alpha actin. Most lesions showed moderate to high collagen amount of which at least 50% was densely packed in most cases. Doubling of the internal elastic lamina was observed in 20 (63%) cases. The newly formed elastic lamina was located in the neighborhood of the original internal elastic lamina. CD68 staining was present in 26 (81%) cases (minor in 13 (41%), moderate in 10 (31%) and heavy in 3 (9%)). In 15 (47%) cases, CD45 staining was present (minor in 14 (44%) and moderate in 1 (3%)). Calcium deposition was observed in 7 (22%) cases (minor in 2 (6%), moderate in 3 (9%), and heavy in 2 (6%)). An atheroma was observed in 3 (9%) cases (⬍40% of total plaque area in 1 (3%) and ⱖ40% in 2 (6%) cases)). No recent or organized thrombus was observed, however, in 2 cases amorphous material suggesting an old thrombus was found. Results are summarized in Table I.
DISCUSSION To the best of our knowledge this is the first study in which histology of endofibrosis and atherosclerosis in the external iliac artery is compared. Although reports have been published about histology of endofibrosis (mostly based on small numbers), its histology was not directly compared to atherosclerotic lesions in the external iliac artery.2,3,8-10 In high performance athletes, flow restriction in the external iliac artery is mostly caused by endofibrosis, kinking of the artery or both.4,5 In the present study, we have selected patients in which the arterial flow restriction was predominantly caused by endofibrosis. We made this selection because in these cases the endofibrosis was expected to be most pronounced. To gain insight in the natural development of atherosclerosis in the external iliac artery, control segments were obtained from elderly individuals. Because not all control arteries revealed significant stenosis of
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the lumen, we decided to split the control arteries in two groups (stenosis ⬍ and ⱖ25%). In this way, arteries with “adaptive intimal thickening” and atherosclerosis were separated. We realize that this is an arbitrary cut off point only based on percentage stenosis. In the group with ⬍25% stenosis, 3/32 (9%) arteries revealed an atheroma and could, therefore, be considered atherosclerotic lesions.11 In all patients with endofibrotic lesions, loose connective tissue was observed with moderate to high cellularity, which is consistent with earlier reports.3,8,9 Stellate cells were predominantly observed in endofibrotic lesions and not in atherosclerotic lesions. In the endofibrosis specimens, predominantly loose fibers of collagen were observed, whereas in the controls collagen was mostly densely packed. Calcification of the lesion was not observed in endofibrotic lesions, whereas 80% of atherosclerotic lesions revealed calcification. Thus, cellularity, presence of stellate cells, large areas of densely-packed collagen and presence of calcium deposition may help to discriminate between endofibrosis and atherosclerosis. Presence of inflammation is another factor that may discriminate between atherosclerosis and endofibrosis. In 21% of cases with endofibrosis, we observed only minor presence of lymphocytes, whereas in the atherosclerotic lesions lymphocytes were observed in 80% of cases. Minor or moderate presence of macrophages was observed in 16% of endofibrotic lesions. In earlier reports, macrophages have not been described in endofibrosis.3,9 All cases of atherosclerosis revealed macrophages with heavy staining in 70% of cases. This observation is consistent with the current understanding of the pathophysiologic mechanism of atherosclerosis that inflammation plays a crucial role in all stages of atherosclerotic lesion development.12 In contrast to atherosclerosis, the development of intimal lesions due to hemodynamic injury and mechanical stress in endofibrosis is mostly not accompanied by influx of macrophages. Our observation suggests that involvement of inflammation in the development of endofibrosis is absent or limited. Presence of inflammatory cells could, therefore, help to discriminate between endofibrosis and atherosclerosis. Luminal thrombosis in atherosclerosis is considered a result of rupture of a vulnerable atherosclerotic plaque consisting of a large atheroma and a thin fibrous cap with abundant inflammation in the cap and shoulders of the plaque.12 When the thin fibrous cap ruptures, the highly thrombogenic atheroma comes in contact with the blood, leading to thrombosis. In addition, plaque erosion with superficial inflammation of the atherosclerotic lesion also is a substrate for luminal thrombosis in atherosclerosis. In the present study, we observed an occluding thrombus superimposed on an endofibrotic lesion. This observation is consistent with two earlier reports in which luminal thrombosis has been described in 3 cases of endofibrosis.9,10 These results suggest that not only advanced atherosclerotic plaques, but also endofibrosis may serve as soil for luminal thrombosis. The fact that endofibrosis can lead to luminal thrombosis adds new knowledge to the current
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understanding of the development of acute arterial occluding luminal thrombosis. In the present study, we considered atherosclerosis and endofibrosis as two different disease entities. However, a clear distinction between these two cannot always be made. The natural development of endofibrosis is unknown. Possibly the loose connective tissue organizes in time to a lesion that more resembles “adaptive intimal thickening” or atherosclerosis in elderly individuals. The fact that we observed macrophages in 16% of endofibrotic lesions suggests that in time an influx of macrophages in endofibrosis may occur. This development of endofibrotic lesions with an influx of macrophages results in lesions in which a clear distinction between endofibrosis and atherosclerosis is more difficult to make. The difference in histology between the control group with lesions with stenosis ⬍25% (“adaptive intimal thickening”) and endofibrotic lesions was less well pronounced. Because we aimed to determine the natural development of atherosclerosis in the external iliac artery, segments from elderly individuals were used as controls. However, the fact that the age of the controls was significantly higher than the patients may have influenced the results and could, therefore, be considered a limitation of the study. The fact that the endofibrotic lesions were obtained during endarterectomy and the control specimens post mortem might have influenced our results. However, the results of a recent study showed no difference in expression profile on a protein level between atherosclerotic lesions obtained during autopsy and surgery.13 The cause of death and risk factors of cardiovascular disease of the controls were unknown. Therefore, the influence of specific risk factors to the development of atherosclerotic disease in the controls could not be determined. Because we selected cases in which luminal stenosis was mostly caused by endofibrosis and, therefore, treated by endarterectomy, we did not study the media and adventitia of the external iliac arteries. It has been reported in a study in four women that next to endofibrosis, medial hypertrophy and adventitial hyperplasia are also phenomenon that can be observed in athletes with stenotic iliac arteries.9 In summary, the histology of external iliac artery endofibrosis has distinct characteristics as compared to atherosclerotic lesions. External iliac artery endofibrosis can serve as soil for luminal thrombosis. These results may help to gain insight in the development of endofibrosis in the external iliac artery in high performance athletes. AUTHOR CONTRIBUTIONS Conception and design: AV, MB, GS, FM Analysis and interpretation: AV, MB, GS, RW, GP, FM Data collection: AV, MB, GS, DW, RW, GP, FM Writing the article: AV, MB Critical revision of the article: GS, DW, RW, GP, FM Final approval of the article: AV, MB, GS, DW, RW, GP, FM Statistical analysis: AV, MB Obtained funding: Not applicable Overall responsibility: AV
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REFERENCES 1. Walder J, Mosimann F, Van Melle G, Mosimann R. A propos de l’endofibrose iliaque chez deux coureurs cyclistes. Helv Chir Acta 1985;51:793-5. 2. Chevalier JM, Enon B, Walder J, Barral X, Pillet J, Megret A, et al. Endofibrosis of the external iliac artery in bicycle racers: an unrecognized pathological state. Ann Vasc Surg 1986;1:297-303. 3. Rousselet MC, Saint-Andre JP, L’Hoste P, Enon B, Megret A, Chevalier JM. Stenotic intimal thickening of the external iliac artery in competition cyclists. Hum Pathol 1990;21:524-9. 4. Bender MH, Schep G, de Vries WR, Hoogeveen AR, Wijn PF. Sportsrelated flow limitations in the iliac arteries in endurance athletes: aetiology, diagnosis, treatment, and future developments. Sports Med 2004; 34:427-42. 5. Schep G, Bender MH, van de Tempel G, Wijn PF, de Vries WR, Eikelboom BC. Detection and treatment of claudication due to functional iliac obstruction in top endurance athletes: a prospective study. Lancet 2002;359:466-73. 6. Vink A, Schoneveld AH, Poppen M, de Kleijn DP, Borst C, Pasterkamp G. Morphometric and immunohistochemical characterization of the intimal layer throughout the arterial system of elderly humans. J Anat 2002;200:97-103. 7. Vink A, Schoneveld AH, Richard W, de Kleijn DP, Falk E, Borst C, Pasterkamp G. Plaque burden, arterial remodeling, and plaque vulner-
8. 9.
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ability: determined by systemic factors? J Am Coll Cardiol 2001; 38:718-23. Abraham P, Chevalier JM, Loire R, Saumet JL. External iliac artery endofibrosis in a young cyclist. Circulation 1999;100:e38. Kral CA, Han DC, Edwards WD, Spittell PC, Tazelaar HD, Cherry KJ Jr. Obstructive external iliac arteriopathy in avid bicyclists: new and variable histopathologic features in four women. J Vasc Surg 2002;36: 565-70. Scavèe V, Stainier L, Deltombe T, Theys S, Delos M, Trigaux JP, Schoevaerdts JC. External iliac artery endofibrosis: a new possible predisposing factor. J Vasc Surg 2003;38:180-2. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol 2000;20:1262-75. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation 1995;92:657-71. Sluimer JC, Kisters N, Cleutjens KB, Volger OL, Horrevoets AJ, van den Akker LH, et al. Dead or alive: gene expression profiles of advanced atherosclerotic plaques from autopsy and surgery. Physiol Genomics 2007;30:335-41.
Submitted Jun 5, 2008; accepted Jul 16, 2008.
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Since 1985, reports have been published about claudication at exercise in highly trained cyclists.1,2 The observed claudication in these athletes is associated with stenosis and/or kinking of the external iliac artery. A possible treatment for these complaints is surgery. The surgically removed specimens revealed intimal thickening consisting of loose connective tissue, a phenomenon since then called endofibrosis.3 The young sportsmen that develop endofibrosis mostly have no classic risk factors for the development of atherosclerosis. Therefore, other factors than the traditional athFrom the Department of Pathology, University Medical Center, Utrecht,a Department of Surgery,b Department of Sports Medicine, Maxima Medical Center, Veldhoven,c Department of Cardiology,d Department of Surgery, University Medical Center, Utrecht.e Competition of interest: none. Correspondence: Aryan Vink, MD, University Medical Center Utrecht, Department of Pathology, Room H04-312, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands (e-mail: [email protected]). 0741-5214/$34.00 Copyright © 2008 Published by Elsevier Inc. on behalf of The Society for Vascular Surgery. doi:10.1016/j.jvs.2008.07.057
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erosclerotic risk factors are involved in the development of endofibrosis. It has been suggested that arterial injury as a result of hemodynamic injury secondary to shear stresses of very high blood flow in combination with the mechanical stress to the artery due to the athlete’s body position leads to intimal thickening.4 Because endofibrosis and atherosclerosis have different pathophysiologic mechanisms, they are considered as different disease entities. Thus far, however, studies that compare histologic aspects of endofibrosis and atherosclerosis in the external iliac artery are lacking. In the present study, we compared the histology of endofibrosis and atherosclerosis in the external iliac artery. METHODS Endarterectomy and control specimens. Nineteen endarterectomy specimens from 18 patients were collected (15 male, 3 female; age 29 ⫾ 8 years). All patients were professional or top class competition amateur cyclists with complaints of claudication during exercise. Magnetic resonance angiography in these patients revealed a stenosis in the external iliac artery. Patients with claudication solely
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due to kinking of the iliac artery without significant stenosis were not included.5 In most cases, a patch of the great saphenous vein was placed. Median length of the endarterectomy specimens was 15 mm (interquartile range [IQR] 15-40). After fixation in 4% formalin, endarterectomy specimens were dissected in 3-mm segments. All segments were embedded in paraffin and used for immunohistochemical staining. As control specimens, 42 arterial segments of the external iliac artery were collected post mortem from 22 individuals (11 men and 11 women, age 82 ⫾ 10 years, history of cardiovascular disease and risk factors unknown) who donated their body to medical science. Arteries were pressure fixed in 4% formalin after death and a 5 mm part of the external iliac artery was dissected.6 This segment was dissected in two parts that were both used for immunohistochemical staining. Immunohistochemical staining. Paraffin sections were stained with hematoxylin and eosin (H&E), Elastica von Giesson, and Sirius red. Immunohistochemical stainings were performed with CD68 (to detect macrophages; dilution 1:800; Novocastra Laboratories Ltd, Newcastle upon Tyne, UK), CD45 (to detect lymphocytes; dilution 1:200; DAKO, Glostrup, Denmark) and alpha actin (to detect myofibroblasts and smooth muscle cells; dilution 1:8000; Sigma, St. Louis, Mo), as described previously.7 In addition, the endarterectomy specimens from the patients were stained with an antibody against collagen 4 (dilution 1:100; Eurodiagnostica, Arnhem, The Netherlands) after pre-treatment with pepsin and an antibody against MIB-1 (dilution 1:100; DAKO) after boiling in a citrate buffer. The signal of all immunohistochemical stainings was visualized using diaminobenzidine. Sections were counterstained with hematoxylin. In the control arteries, percentage stenosis was determined using the Elastica von Giesson staining. The size of the intima/plaque was calculated by subtracting the luminal area from the area encompassed by internal elastic lamina (IEL area). Stenosis was defined as (intimal area/ IEL area) ⫻ 100%.6 The number of cells per mm2 was analyzed semi quantitatively and divided into 3 groups: low, moderate, and high. The Sirius red staining was analyzed with polarized light to detect collagen. The CD68 and CD45 stainings were analyzed semi quantitatively: negative, no positive cells; minor, few positive cells; moderate, groups of 10 cells present; heavy, abundant staining. Presence of an extracellular lipid core was scored as percentage of the total intimal/plaque area. RESULTS Patients. In the patients, median stenosis was 65% (IQR 50-75) (Fig 1). The endarterectomy specimens of all patients with endofibrosis revealed a loose connective tissue matrix with moderate to high cellularity (Fig 2, A and C). The cells were spindle shaped or stellate and mostly not arranged in bundles. Most of the cells stained positive with alpha actin and could, therefore, be classified as smooth muscle cells or myofibroblasts. In between the myofibro-
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Fig 1. Stenosis (%) of external iliac arteries of controls and patients (p ⬍ .0001). Horizontal line indicates the median value.
blasts, loose collagen fibers were observed that stained in the Sirius red staining and in most cases also in the immunohistochemical collagen 4 staining. An area with denselypacked collagen was observed in 7/19 (37%) cases. This area with densely-packed collagen was at most 40% of the lesion. In 15/19 (79%) cases, a newly formed elastic lamina was present in the lesion, most often near the intimal layer. An occluding thrombus superimposed on the endofibrosis was observed in one case. This thrombus showed signs of organization with presence of fibroblasts and microvessels. Minor CD68 staining was observed in 1 (5%) and moderate CD68-staining in 2 (11%) cases (Fig 3). Negative staining of CD68 was observed in 16/19 (84%) lesions. In 4 (21%) lesions, minor staining of CD45-positive lymphocytes was observed, whereas in the other 15 (79%) lesions no lymphocytes were observed. Calcium deposition and an extra cellular atheroma were not observed in the endofibrotic lesions. In most specimens, no MIB-1 staining was observed. Results are summarized in Tables I and II. Controls. Percentage stenosis was calculated in the arterial sections from the controls to get insight in the natural development of stenosis in the external iliac arteries. Median stenosis of the arterial sections obtained from the elderly individuals was 11 % (IQR 7-24) (Fig 1). Ten arteries obtained from 7 individuals revealed a stenosis ⱖ25% and were considered significant atherosclerotic lesions. Percentage stenosis was significantly higher in patients than in controls (P ⬍ .0001, Mann-Whitney test; Fig 1). The lesions with stenosis ⱖ25% revealed low to moderate cellularity (Fig 2, B and D). However, predominantly in the fibrous cap of the atherosclerotic lesions also some areas with high cellularity were observed. Most of the cells in the lesions stained positive for alpha actin. The matrix of these lesions showed moderate to abundant presence of collagen of which at least 50% was densely packed in all cases. In 4/10 cases, doubling of the internal elastic lamina was observed. The newly formed elastic lamina was located in the neighborhood of the original internal elastic lamina. In all cases, CD68 staining was present (minor staining in 2 (20%), moderate in 1 (10%), and heavy in 7 (70%)). In 8 (80%) cases, CD45 staining was observed (minor in 5
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Fig 2. Histological comparison between endofibrosis and atherosclerosis in the external iliac artery. A and C, Hematoxylin and eosin (H&E) stainings of endofibrotic lesion in a professional cyclist. A, Overview of the wall of the artery showing the media (m) and the endofibrotic lesion (e). Bar ⫽ 250 m. C, Higher magnification of the endofibrotic lesion showing a loose connective tissue matrix with moderate to high cellularity consisting of spindle and stellate shaped cells. The cells are not orderly arranged. Bar ⫽ 100 m. B and D, H&E stainings of atherosclerotic lesion. B, Overview of the wall of the artery showing the media (m) and the atherosclerotic plaque. The plaque consists of an extracellular atheroma (a), a socalled shoulder with macrophages (s) and a fibrous cap (c). Bar ⫽ 250 m. D, Higher magnification of the fibrous cap of the atherosclerotic plaque showing low cellularity with spindle cells arranged in bundles. The extra cellular matrix consists of densely-packed collagen. Bar ⫽ 100 m.
Fig 3. Macrophages in endofibrotic lesion. A, Endofibrotic lesion with moderate to high cellularity. Foam cells are present near the lumen at the top of the photo. B, CD68 immunohistochemical staining in adjacent section, identifying the cells as macrophage foam cells (brown staining). Bar ⫽ 125 m.
(50%), moderate in 1 (10%), and heavy in 2 (20%)). Calcium deposition was observed in 8 (80%) cases (minor in 1 (10%) and moderate in 7 (70%)). An extra cellular lipid pool (atheroma) was observed in 7 (70%) cases (⬍40% of total plaque
area in 3 (30%) and ⱖ40% in 4 (40%)) (Fig 2, B). No recent or organized thrombus was observed, however in 1 case amorphous material was found, suggesting presence of an old thrombus. Results are summarized in Tables I and II.
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Table I. Characteristics of endofibrosis, atherosclerosis and adaptive intimal thickening in the external iliac artery Intimal lesions in elderly individuals
CD68 staining No Minor Moderate Heavy CD45 staining No Minor Moderate Heavy Calcium deposition No Minor Moderate Heavy Atheroma No ⬍40% ⱖ40%
Endofibrosis n (percentage)
AS ⱖ25% (atherosclerosis) n (percentage)
AS ⬍25% (adaptive intimal thickening) n (percentage)
16 (84%) 1 (5%) 2 (11%) 0 (0%)
0 (0%) 2 (20%) 1 (10%) 7 (70%)
6 (19%) 13 (41%) 10 (31%) 3 (9%)
15 (79%) 4 (21%) 0 (0%) 0 (0%)
2 (20%) 5 (50%) 1 (10%) 2 (20%)
17 (53%) 14 (44%) 1 (3%) 0 (0%)
19 (100%) 0 (0%) 0 (0%) 0 (0%)
2 (20%) 1 (10%) 7 (70%) 0 (0%)
25 (78%) 2 (6%) 3 (9%) 2 (6%)
19 (100%) 0 (0%) 0 (0%)
3 (30%) 3 (30%) 4 (40%)
29 (91%) 1 (3%) 2 (6%)
Table II. Summary of histopathological comparison between endofibrosis and atherosclerosis in the external iliac artery
Connective tissue Collagen Cellularity Cellular form Arrangement of cells Smooth muscle actin Newly formed elastic lamina Macrophages Lymphocytes Calcification Atheroma
Endofibrosis
Atherosclerosis
Loose Loose fibers Moderate to high Spindle or stellate Loose, not organized Most cells positive Near the intimal layer – or ⫹ – or ⫹ – –
Compact Densely packed Low to moderate Spindle Organized in bundles Most cells positive Doubling original internal elastic lamina ⫹, ⫹⫹ or ⫹⫹⫹ –, ⫹ or ⫹⫹ –, ⫹ or ⫹⫹ –, ⫹ or ⫹⫹
The arterial segments with stenosis ⬍25% on average revealed low to moderate cellularity (high cellularity was observed in a few cases). Most of the cells in the lesions stained positive for alpha actin. Most lesions showed moderate to high collagen amount of which at least 50% was densely packed in most cases. Doubling of the internal elastic lamina was observed in 20 (63%) cases. The newly formed elastic lamina was located in the neighborhood of the original internal elastic lamina. CD68 staining was present in 26 (81%) cases (minor in 13 (41%), moderate in 10 (31%) and heavy in 3 (9%)). In 15 (47%) cases, CD45 staining was present (minor in 14 (44%) and moderate in 1 (3%)). Calcium deposition was observed in 7 (22%) cases (minor in 2 (6%), moderate in 3 (9%), and heavy in 2 (6%)). An atheroma was observed in 3 (9%) cases (⬍40% of total plaque area in 1 (3%) and ⱖ40% in 2 (6%) cases)). No recent or organized thrombus was observed, however, in 2 cases amorphous material suggesting an old thrombus was found. Results are summarized in Table I.
DISCUSSION To the best of our knowledge this is the first study in which histology of endofibrosis and atherosclerosis in the external iliac artery is compared. Although reports have been published about histology of endofibrosis (mostly based on small numbers), its histology was not directly compared to atherosclerotic lesions in the external iliac artery.2,3,8-10 In high performance athletes, flow restriction in the external iliac artery is mostly caused by endofibrosis, kinking of the artery or both.4,5 In the present study, we have selected patients in which the arterial flow restriction was predominantly caused by endofibrosis. We made this selection because in these cases the endofibrosis was expected to be most pronounced. To gain insight in the natural development of atherosclerosis in the external iliac artery, control segments were obtained from elderly individuals. Because not all control arteries revealed significant stenosis of
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the lumen, we decided to split the control arteries in two groups (stenosis ⬍ and ⱖ25%). In this way, arteries with “adaptive intimal thickening” and atherosclerosis were separated. We realize that this is an arbitrary cut off point only based on percentage stenosis. In the group with ⬍25% stenosis, 3/32 (9%) arteries revealed an atheroma and could, therefore, be considered atherosclerotic lesions.11 In all patients with endofibrotic lesions, loose connective tissue was observed with moderate to high cellularity, which is consistent with earlier reports.3,8,9 Stellate cells were predominantly observed in endofibrotic lesions and not in atherosclerotic lesions. In the endofibrosis specimens, predominantly loose fibers of collagen were observed, whereas in the controls collagen was mostly densely packed. Calcification of the lesion was not observed in endofibrotic lesions, whereas 80% of atherosclerotic lesions revealed calcification. Thus, cellularity, presence of stellate cells, large areas of densely-packed collagen and presence of calcium deposition may help to discriminate between endofibrosis and atherosclerosis. Presence of inflammation is another factor that may discriminate between atherosclerosis and endofibrosis. In 21% of cases with endofibrosis, we observed only minor presence of lymphocytes, whereas in the atherosclerotic lesions lymphocytes were observed in 80% of cases. Minor or moderate presence of macrophages was observed in 16% of endofibrotic lesions. In earlier reports, macrophages have not been described in endofibrosis.3,9 All cases of atherosclerosis revealed macrophages with heavy staining in 70% of cases. This observation is consistent with the current understanding of the pathophysiologic mechanism of atherosclerosis that inflammation plays a crucial role in all stages of atherosclerotic lesion development.12 In contrast to atherosclerosis, the development of intimal lesions due to hemodynamic injury and mechanical stress in endofibrosis is mostly not accompanied by influx of macrophages. Our observation suggests that involvement of inflammation in the development of endofibrosis is absent or limited. Presence of inflammatory cells could, therefore, help to discriminate between endofibrosis and atherosclerosis. Luminal thrombosis in atherosclerosis is considered a result of rupture of a vulnerable atherosclerotic plaque consisting of a large atheroma and a thin fibrous cap with abundant inflammation in the cap and shoulders of the plaque.12 When the thin fibrous cap ruptures, the highly thrombogenic atheroma comes in contact with the blood, leading to thrombosis. In addition, plaque erosion with superficial inflammation of the atherosclerotic lesion also is a substrate for luminal thrombosis in atherosclerosis. In the present study, we observed an occluding thrombus superimposed on an endofibrotic lesion. This observation is consistent with two earlier reports in which luminal thrombosis has been described in 3 cases of endofibrosis.9,10 These results suggest that not only advanced atherosclerotic plaques, but also endofibrosis may serve as soil for luminal thrombosis. The fact that endofibrosis can lead to luminal thrombosis adds new knowledge to the current
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understanding of the development of acute arterial occluding luminal thrombosis. In the present study, we considered atherosclerosis and endofibrosis as two different disease entities. However, a clear distinction between these two cannot always be made. The natural development of endofibrosis is unknown. Possibly the loose connective tissue organizes in time to a lesion that more resembles “adaptive intimal thickening” or atherosclerosis in elderly individuals. The fact that we observed macrophages in 16% of endofibrotic lesions suggests that in time an influx of macrophages in endofibrosis may occur. This development of endofibrotic lesions with an influx of macrophages results in lesions in which a clear distinction between endofibrosis and atherosclerosis is more difficult to make. The difference in histology between the control group with lesions with stenosis ⬍25% (“adaptive intimal thickening”) and endofibrotic lesions was less well pronounced. Because we aimed to determine the natural development of atherosclerosis in the external iliac artery, segments from elderly individuals were used as controls. However, the fact that the age of the controls was significantly higher than the patients may have influenced the results and could, therefore, be considered a limitation of the study. The fact that the endofibrotic lesions were obtained during endarterectomy and the control specimens post mortem might have influenced our results. However, the results of a recent study showed no difference in expression profile on a protein level between atherosclerotic lesions obtained during autopsy and surgery.13 The cause of death and risk factors of cardiovascular disease of the controls were unknown. Therefore, the influence of specific risk factors to the development of atherosclerotic disease in the controls could not be determined. Because we selected cases in which luminal stenosis was mostly caused by endofibrosis and, therefore, treated by endarterectomy, we did not study the media and adventitia of the external iliac arteries. It has been reported in a study in four women that next to endofibrosis, medial hypertrophy and adventitial hyperplasia are also phenomenon that can be observed in athletes with stenotic iliac arteries.9 In summary, the histology of external iliac artery endofibrosis has distinct characteristics as compared to atherosclerotic lesions. External iliac artery endofibrosis can serve as soil for luminal thrombosis. These results may help to gain insight in the development of endofibrosis in the external iliac artery in high performance athletes. AUTHOR CONTRIBUTIONS Conception and design: AV, MB, GS, FM Analysis and interpretation: AV, MB, GS, RW, GP, FM Data collection: AV, MB, GS, DW, RW, GP, FM Writing the article: AV, MB Critical revision of the article: GS, DW, RW, GP, FM Final approval of the article: AV, MB, GS, DW, RW, GP, FM Statistical analysis: AV, MB Obtained funding: Not applicable Overall responsibility: AV
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Submitted Jun 5, 2008; accepted Jul 16, 2008.
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