Author + information
- Received July 23, 2018
- Revision received August 21, 2018
- Accepted August 30, 2018
- Published online December 12, 2018.
- Sho Torii, MD, PhDa,
- Jihad A. Mustapha, MDb,
- Jagat Narula, MD, PhDc,
- Hiroyoshi Mori, MDa,
- Fadi Saab, MDb,
- Hiroyuki Jinnouchi, MDa,
- Kazuyuki Yahagi, MDa,
- Atsushi Sakamoto, MDa,
- Maria E. Romero, MDa,
- Navneet Narula, MDd,
- Frank D. Kolodgie, PhDa,
- Renu Virmani, MDa,∗ ( and )
- Aloke V. Finn, MDa
- aCVPath Institute, Gaithersburg, Maryland
- bAdvanced Cardiac and Vascular Amputation Prevention Centers, Grand Rapids, Michigan
- cIcahn School of Medicine at Mount Sinai, New York, New York
- dNew York University School of Medicine, New York, New York
- ↵∗Address for correspondence:
Dr. Renu Virmani, CVPath Institute, 19 Firstfield Road, Gaithersburg, Maryland 20878.
Objectives The aim of this study was to comprehensively evaluate the pathology of the lower extremity arteries across their entire length in subjects dying with abundant risk factors and to evaluate the clinical and imaging implications of the pathological characteristics.
Background Lower extremity peripheral arterial disease is a major cause of cardiovascular morbidity, but a systematic characterization of the pathology has never been undertaken.
Methods Twelve legs were obtained from 8 cadavers with histories of coronary risk factors (median age 82 years, 6 men); 8 of 12 legs were evaluated using computed tomography before the major peripheral arteries were dissected along their entire length. Dissected arteries were cut serially at 3 to 4 mm, and a total of 2,987 sections were examined.
Results Luminal irregularities and stenosis were more commonly seen in computed tomography images of above-the-knee (AK) arteries. Atherosclerotic lesions were histologically confirmed and were more common in AK (95.7%) than below-the-knee (BK) (56.8%) arteries. Occluded vessels were observed at 18 sites, including 8 AK and 10 BK arteries. Pathologically, acute thrombus was observed in all 8 AK sites, of which 3 were associated with plaque rupture and 5 were related to calcified nodules. The 10 occluded BK arteries revealed chronic total occlusions, of which half were embolic in origin and half were associated with atherosclerotic lesions. Intimal (75.3%) and medial (86.2%) calcifications were commonly encountered. Proportionate to the neointimal atherosclerosis, intimal calcification was more severe in AK arteries; the severity of medial calcification was no different between AK and BK arteries. Calcification was significantly greater in arteries excised from subjects with compared with those without diabetes.
Conclusions Atherosclerosis occurs more commonly in AK arteries and luminal occlusion from acute thrombosis secondary to rupture or calcified nodules. BK occlusion was chronic in nature, and at least half of lesions were embolic in origin. Medial calcification was similarly common in AK and BK arteries but more prevalent in subjects with diabetes.
- chronic total occlusion
- computed tomography angiography
- medial calcification
- peripheral artery disease
Peripheral artery disease (PAD) of the lower extremities is the most prevalent cardiovascular disease worldwide (1). PAD has commonly been believed to be caused by atherosclerotic disease, and risk factors for limb events have been considered to be similar to those for coronary events. Diabetes mellitus (DM), chronic renal disease, and smoking (2,3) are widely associated with PAD. Diagnosis and management of atherosclerosis is critical for limiting morbidity and disability and preventing limb ischemia. Long chronic total occlusion (CTO) and severe vascular calcification including bone formation have been described in the lower extremities (4). However, although the pathology of atherosclerosis in the coronary arteries has been well studied (5,6), the pathology of lower extremity vascular disease remains poorly understood. Although several studies have evaluated histological lesions of lower extremities collected from patients undergoing open endarterectomy (7–9), proximal iliofemoral artery segments collected from autopsy (10–12), and above-the-knee (AK) and below-the-knee (BK) amputations for critical limb ischemia (CLI) (13–16); no report has described serially sectioned pathological characterization of PAD. None of the reports have hitherto compared peripheral artery pathology using noninvasive imaging modalities. The aim of the present study was to characterize atherosclerotic lesions and calcification of the lower extremity dissected from deceased subjects who were known to have risk factors for coronary artery disease. We performed computed tomographic (CT) imaging of the dissected vessels to establish imaging-pathology correlation.
Detailed methods are described in the Online Appendix.
Twelve legs dismembered from 8 cadavers at the hip joint were obtained from donors through Science Care (Phoenix, Arizona). Included in this study were deceased subjects ≥70 years of age with known histories of smoking, hypertension, hyperlipidemia, DM, and chronic kidney disease. To enrich the population being studied for the extent and type of atherosclerotic disease in peripheral arteries, we chose to study subjects with abundant risk factors as also the presence of calcification observed on radiograms of the legs. Cadavers with any history of vascular intervention and/or orthopedic treatment of the lower extremity were excluded from the study. This study was approved by the Institutional Review Board at CVPath Institute.
CT imaging of the dissected vessel along its length from 8 legs (5 cadavers) was performed at Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, before dissection. Following the imaging evaluation, the arteries were perfusion-fixed at 100 mm Hg using 10% neutral buffered formalin, harvested, and immersed in the same fixative until histological evaluation upon paraffin or plastic embedding.
The lower extremity arteries, dissected along their entire length, were radiographed and photographed before serial sectioning for comparison with CT imaging and histology. Six vessels (from 5 cadavers) of the total 12 vessels (from 8 cadavers) were decalcified in ethylenediaminetetraacetic acid. These 6 arterial trees were sequentially cut at 3- to 4-mm intervals and embedded in paraffin as previously described, followed by 4- to 5-μm sectioning and histochemical staining (6). The remaining 6 vessels (from 3 cadavers) were not decalcified, wherein the segments with heavy calcification on radiography were embedded in Spurr resin and ground by Exakt technique to 30 to 60 μm in thickness, and the segments without calcification on radiography were routinely embedded in paraffin for subsequent histochemical staining.
Classification of atherosclerotic plaques and calcification
Peripheral atherosclerotic plaques were classified using the modified American Heart Association classification proposed for coronary atherosclerotic lesions as previously described (Figure 1) (5). Histologically, different degrees of intimal and medial calcification and the size of the calcium were assessed as previously described (17–19). The microcalcification was defined as calcium particles varying from ≥0.5 but <15 μm in diameter, punctate calcification when >15 μm but <1 mm, and fragment calcification ≥1 mm. Sheet calcification was noted when >1 quadrant of the vessel demonstrated calcification. Nodular calcification was reported if the nodular calcium deposits in the atherosclerotic lesion had not disrupted through the lesion’s luminal surface (Figures 2 and 3⇓⇓). The extent of circumferential intimal and medial calcification was also evaluated. Any calcification (intimal and medial) that was ≥0.5 mm and was visible on radiography was included and assessed as circumferential degree (arc) of involvement (Online Figure 1).
Coregistration of imaging modalities with histology
The lower extremity CT and radiographic images were coregistered with the histological sections by measuring the distance from the severance of the femoral artery to the CT or radiographic location of calcium and anatomic landmarks such as the vessel branches. Assessment of both calcium and anatomic landmarks is essential because arterial shrinkage occurs upon removal of the artery and perfusion fixation.
Results for continuous variables with normal distribution are expressed as mean ± SD. Normality of distribution was tested using the Shapiro-Wilk test. Variables with non-normal distribution are expressed as median (interquartile range). Categorical data were analyzed using chi-square or Fisher exact tests. The Student t test was used to define the significance of differences for continuous variables with normal distribution, whereas comparisons of variables with nonparametric distribution were performed using the Kruskal-Wallis test. For vessel-level analysis, the generalized estimating equation method was used. Continuous variables were tested using the b method with γ with the log-link or Poisson model, as appropriate. Categorical data were tested using the generalized estimating equation method with an ordinal logistic model or tubular Fisher exact test, as appropriate. A p value of <0.05 was considered to indicate statistical significance. JMP version 13.0 (SAS Institute, Cary, North Carolina) and SPSS version 19 (IBM, Armonk, New York) were used for statistical analyses.
A total of 2,987 atherosclerotic peripheral artery sections (1,006 AK and 1,981 BK sections) from 12 legs belonging to 8 subjects (6 men and 2 women) with a median age of 82 years, were evaluated. The case demographics and lesion characteristics are shown in Tables 1 and 2⇓⇓.
CT and roentgenographic characterization of the lower extremity arteries
Focal stenosis was observed in all legs, with total occlusion in 8 AK and 10 BK arteries (Figure 4). Calcification was observed in all legs, with inability of imaging to differentiate between intimal and medial calcification. However, ex vivo radiography of the dissected vessels showed higher density of the intimal calcification more so than the medial calcification. The extent of calcification was most marked in the middle to distal superficial femoral artery (SFA). CT images demonstrated the presence of mainly eccentric plaques rather than concentric. Most were located in the distal SFA and popliteal arteries.
Eight leg arteries involving 155 histological sections were coregistered with CT images. The location of calcification on CT imaging was compared with the histological sections, and the total area and largest dimension of calcium in millimeters were measured using ZEN software (Zeiss, Oberkochen, Germany). The cutoff values of length and area of calcification for the detection of plaque calcification on CT imaging was determined to be 1.5 mm (sensitivity 97.1%, specificity 96.7%) and 0.5 mm2 (sensitivity 100%, specificity 96.7%), respectively (Online Figure 2).
Histopathologic characteristics of lower extremity plaques
The plaque type distribution was significantly different between the AK and BK arteries. Excluding adaptive intimal thickening, prevalence of atherosclerotic lesions was more common in AK (95.7%) compared with BK (56.8%) arteries (p < 0.0001) (Table 2), with a higher incidence of lipid-rich plaque, including pathological intimal thickening and fibroatheroma, and plaque rupture in AK compared with BK arteries (Figure 5). Acute thrombotic events were exclusively observed in 8 AK arteries (6 SFA and 2 popliteal from 6 legs of 5 cadavers). Of these 8 lesions, 5 had underlying calcified nodules and 3 had plaque rupture (Table 1, Online Table 2). In contrast, CTOs were more common in BK arteries (10 arteries from 9 legs of 6 cadavers) compared with AK arteries. Of the 10 CTO lesions, 5 (50%) were occluded because of atherosclerotic plaques such as healed plaque rupture in 1 and fibrocalcific plaque in 4, whereas the other half were embolic in nature, probably derived from the upstream thrombotic lesions (Online Table 3, Figures 6 and 7⇓⇓, Online Figures 3 to 6). As expected, intimal calcification in CTO lesions was significantly higher in atherosclerotic (26.3% [3.9% to 47.2%]) than embolic (1.0% [0.1% to 3.2%]) CTOs (p = 0.016). However, the severity of medial calcification was comparable (1.3% [1.1% to 10.0%] vs. 2.0% [0.6% to 3.0%], respectively, p = 0.75). CT images correlated with severe calcification in atherosclerotic CTO by histology, compared with embolic CTO, which showed absence of calcium by CT imaging with negative remodeling.
Intimal and medial calcification and bone formation in the lower extremities
The degree of intimal calcification assessed both in nondecalcified and decalcified sections was significantly higher in AK arteries (15.1% [9.6% to 17.5%]) compared with BK arteries (1.6% [0.2% to 10.6%]) (p = 0.04). However, the extent of medial calcification was similar in AK (2.4% [1.0% to 4.8%]) and BK (2.3% [1.2% to 4.0%]) (p = 0.71) arteries (Table 2). Type of calcification was separately assessed in nondecalcified sections from 6 legs. Of the 1,578 nondecalcified sections examined, intimal (72.7%) and medial (86.2%) calcification was commonly seen. Atherosclerotic nodular calcification was more frequent in AK (6.6%) compared with BK (0.5%) arteries (Figure 8, Online Table 1).
Comparing the degree of calcification between patients with DM (6 legs from 4 patients) and those without DM (6 legs from 4 patients) assessed both with nondecalcified and decalcified sections, the prevalence of intimal fragmented, sheet, and nodular calcification was significantly greater in DM (40.8%) than non-DM (21.1%), as was the extent of medial calcification (18.1% vs. 7.3%, respectively) (Figure 9). Bone formation was observed in 10 of 12 legs (83%) from 7 cadavers, with a significantly higher prevalence in AK arteries (8 legs from 7 cadavers) compared with BK arteries (4 legs from 4 cadavers) (p = 0.02) (Tables 1 and 2). There was a significant correlation between atherosclerotic intimal calcification and percentage stenosis, but no correlation was observed between medial calcification and the extent of luminal stenosis (Online Figure 7).
We report a detailed distribution of atherosclerotic plaque types in the lower extremity and the differences in the type and extent of intimal and medial calcification in AK versus BK arterial lesions using serial sectioning of the lower extremity arteries cut at 3- to 4-mm intervals. The principal findings of the present study are as follows: 1) AK arterial lesions were predominantly atherosclerotic in nature compared with BK arteries; 2) acute thrombotic events were observed exclusively in AK arterial lesions and were associated predominantly with calcified nodules; 3) BK lesions demonstrated mostly CTO, and the etiology was equally divided between distal emboli and atherosclerosis; 4) intimal atherosclerotic and medial calcification were equally common; 5) intimal atherosclerotic calcification was more severe in AK lesions with a higher prevalence of bone formation; and 6) subjects with DM had more advanced calcification in both intima and media compared with those without DM.
Plaque characteristics in lower extremities
Previous autopsy studies have demonstrated differences in plaque characteristics in the SFA and other vascular beds (aorta, coronary, and carotid arteries) (8,10,11). However, plaque characteristics in the lower extremities have not been systematically studied, as only limited areas of SFA, popliteal, anterior tibial artery, posterior tibial artery, and peroneal arteries were sampled in the previous studies, and most of these involve amputations, when the disease is in the end stage (15,16). In the present study, the arteries of the lower extremity were serially sectioned from proximal SFA to distal BK vessels. When the results were compared with our previous reports of prevalence of various types of atherosclerotic plaque morphology in coronary arteries (19), the AK lesions demonstrated a higher incidence of fibrocalcific plaque than in coronary arteries, whereas BK lesions demonstrated a higher incidence of fibrous plaque.
Acute thrombotic lesions were exclusively observed in AK lesions, and the majority of thrombosis were caused by calcified nodules (62.5%), while the remaining occurred from plaque rupture. The incidence of calcified nodules is higher in the lower extremity than in coronary arteries (2% to 7%) (5) and carotid arteries (4% to 14%) (20). The SFA is the most dynamically active vessel in the body (21), and this may contribute to the higher prevalence of calcified nodules, resulting from breaks in the sheet calcium.
In the present study, we found a total of 10 CTO lesions from 36 BK vessels, and the etiology was embolic from upstream AK lesions in 50% and in the rest from local atherosclerotic disease, suggesting that thrombotic events in AK arteries likely contribute to the distal CTO lesions. Therefore, it is likely that distal occlusive emboli as well as SFA lesions are involved in the etiology of CLI, and these findings support the role of antithrombotic agents for prevention of acute limb events as reported recently (22).
Intimal and medial calcification in lower extremities
Fibrocalcific plaques with severe intimal calcification were more frequent in AK compared with BK arteries and are also more frequent than what has been reported for coronary arteries (17,23). In contrast, the degree of medial calcification was similar in AK and BK arteries in the present study. These results are different from previous histological studies that reported a higher incidence of medial calcification BK compared with AK arteries in amputated legs (15). This is not surprising, as the disease severity is greater in patients with CLI with amputation than in those without CLI studied in the present work. In addition, more than one-half of the vessels evaluated in amputations involved only BK lesions, thus limiting the comparability. Medial calcification is believed to be the main etiology of vessel stiffness, decrease of vessel elasticity and compliance that may lead to reduced perfusion (24). However, the impact of medial calcification during progression of PAD has not been directly correlated with intimal calcification, because of the inability to differentiate between the 2 using currently available imaging modalities (25). Further studies are needed to correlate pathology, molecular biology, functional, and imaging studies.
DM is a well-known independent risk factor in patients with PAD (2), but the prevalence of intimal and medial calcification has been not reported. In this study, the presence of more advanced calcification in subjects with DM was significantly higher than in those without DM. In a previous study, we reported the type of coronary calcification in 121 patients with sudden coronary death and demonstrated a significant reciprocal increase in sheet calcification with escalating glycated hemoglobin levels (18). Further studies are needed to evaluate the correlation between the severity of calcification in lower extremities and severity of DM.
Focal bone formation was observed in 10 of 12 limbs (83%) and was more common in AK arteries. The prevalence of bone formation in the present study was higher than that in previous studies that evaluated amputated CLI legs (6.0% to 18.8% [13,15]). Likely the prevalence of bone formation was underestimated in these studies involving amputations because only spot sectioning was performed. Bone formation always occurred in the presence of high calcification, mostly in lesions with presence of nodular calcification.
There were several limitations of the present study. Although all the legs arteries were coregistered with radiographs and histology, CT imaging (or CT angiography) was performed in selected cases (n = 8). We do not have the cross-sectional CT imaging analysis available to coregister with histological cross sections. The number of subjects evaluated was relatively small, and therefore the prevalence and the proportions of the plaque types, calcification, and the acute and chronic thrombotic occlusions may not offer accurate estimates. Selection bias may also arise, as only legs with calcification in AK arteries were evaluated, according to the inclusion criteria. However, we believe that systematic sectioning at every 3 to 4 mm of the 3-foot-long multiple arterial system starting from the SFA and extending to the foot should be fairly representative. We also believe that the number of sections evaluated is large enough to understand the natural history of the lower extremity in high-risk subjects. Our study, however, shows that there is extensive intimal and medial calcification in subjects with advancing age at high risk for coronary artery disease, attention must be paid to the prevention of PAD thorough risk factor control, and that imaging of the peripheral arteries for pathological characterization would pose a major challenge.
Atherosclerosis and intimal-medial calcification in peripheral lower extremity vessels are commonly observed in asymptomatic high-risk subjects. The etiology of thrombotic disease is significantly different for AK and BK arteries, with the former driven by a high prevalence of intimal atherosclerotic calcification and from embolic disease in the latter. Greater understanding of the etiology of PAD is needed in the aging population, with an emphasis on prevention.
COMPETENCY IN MEDICAL KNOWLEDGE: Atherosclerosis and intimal and medial calcification in peripheral lower extremity vessels are commonly observed in asymptomatic high-risk subjects. The etiology of thrombotic disease is significantly different for AK than BK arteries, with the former driven by calcified nodule or plaque rupture and from embolic disease in the latter. Medial calcification was similarly common in AK and BK arteries but more prevalent in subjects with DM.
TRANSLATIONAL OUTLOOK: Medial calcification is believed to be the main etiology of vessel stiffness, decrease of vessel elasticity and compliance that may lead to reduced perfusion. However, the impact of medial calcification during progression of PAD has not been directly correlated with intimal calcification, because of the inability to differentiate between the 2 using currently available imaging modalities. Further studies are needed to correlate pathology, molecular biology, functional, and imaging studies.
Drs. Torii and Mustapha contributed equally to this work and are joint first authors. Cardiovascular Systems, Inc. provided the funding for this study. Dr. Torii has received research grants from SUNRISE lab. Dr. Mustapha has received honoraria from Bard Peripheral Vascular, Boston Scientific, Cardiovascular Systems, Inc., Cook Medical, Medtronic, Spectranetics, and Terumo. Dr. Saab has received honoraria from Bard Peripheral Vascular, Boston Scientific, Cardiovascular Systems, Inc., Cook Medical, Medtronic, Spectranetics, and Terumo. Drs. Virmani and Finn have received institutional research support from Abbott Vascular, Boston Scientific, Cook Medical, Cardiovascular Systems, Inc., Medtronic, and Terumo. Dr. Virmani is a consultant for 480 Biomedical, Abbott Vascular, Medtronic, and W.L. Gore. All other authors have reported that they have no no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- critical limb ischemia
- computed tomographic
- chronic total occlusion
- diabetes mellitus
- peripheral artery disease
- superficial femoral artery
- Received July 23, 2018.
- Revision received August 21, 2018.
- Accepted August 30, 2018.
- 2018 American College of Cardiology Foundation
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