Abstract
Aims: Though the number of patients with peripheral arterial disease (PAD) and critical limb ischemia (CLI) is increasing, few histopathological studies of PAD, particularly that involving below-the-knee arteries, has been reported. We analyzed the pathology of anterior tibial artery (ATA) and posterior tibial artery (PTA) specimens obtained from patients who underwent lower extremity amputation due to CLI
Methods: Dissected ATAs and PTAs were subjected to ex-vivo soft X-ray radiography, followed by pathological examination using 860 histological sections. This protocol was approved by the Ethics Review Board of Nihon University Itabashi Hospital (RK-190910-01) and Kyorin University Hospital (R02-179).
Results: The calcified area distribution was significantly larger in PTAs than in ATAs on soft X-ray radiographic images (ATAs, 48.3% ±19.2 versus PTAs, 61.6% ±23.9; p＜0.001). Eccentric plaque with necrotic core and macrophage infiltration were more prominent in ATAs than in PTAs (eccentric plaque: ATAs, 63.7% versus PTAs, 49.1%; p＜0.0001, macrophage: ATAs, 0.29% [0.095 - 1.1%] versus PTAs, 0.12% [0.029 - 0.36%]; p＜0.001), histopathologically. Thromboembolic lesions were more frequently identified in PTAs than in ATAs (ATAs, 11.1% versus PTAs 15.8%; p＜0.05). Moreover, post-balloon injury pathology differed between ATAs and PTAs.
Conclusions: Histological features differed strikingly between ATAs and PTAs obtained from CLI patients. Clarifying the pathological features of CLI would contribute to establishing therapeutic strategies for PAD, particularly disease involving below-the knee-arteries.
Introduction
Peripheral artery disease (PAD) is caused by narrowing and stricture of the arteries in the lower extremities1). It is usually the result of atherosclerosis involving the vessel wall, but can also be caused by embolism, thrombosis or vasculitis2). Symptoms include fatigue when walking, and atypical leg pain referred to as intermittent claudication3). The number of patients with PAD is rising and it is now estimated to be exceed 200 million worldwide. One of the most serious complications of PAD is critical limb ischemia (CLI)3). Approximately 1 to 3% of PAD patients develop CLI, and the associated mortality rate can be as high as 20% within just 6 months of diagnosis, even possibly exceeding 50% at 5 years4). Prevention of major amputation in CLI is the most important goal. Achieving revascularization employing bypass surgery or endovascular therapy (EVT) can, if successful, prevent major amputation, improve quality of life and prolong survival in patients with CLI5).
There have been major advances in the devices used for EVT. Covered stents, drug-eluting stents, cutting balloons and drug-coated balloons have been introduced6). Previously, the only EVT technique available for below-the-knee (BK) -arteries was balloon angioplasty. However, a report comparing old balloon angioplasty and drug-eluting stents in BK-arteries showed the latter to be associated with lower restenosis after EVT7). Elucidating the pathology of a calcified lesion is crucial for successful EVT. Pre-dilation with a high-pressure balloon should be attempted before stenting for calcified lesions because the minimum stent area after the procedure is predictive of the long-term stent patency of lower extremity arterial lesions8).
There are a few prior histopathological reports focus on PAD9-11). However, according to our literature search, no past studies have assessed differences between the anterior tibial artery (ATA) and posterior tibial artery (PTA). Since the indications for EVT have been extended to lesions of the BK-arteries, histopathological examination of these arteries, as well as above-the-knee (AK)-arteries, is essential for optimal treatment of PAD. Understanding the histopathology of BK-arteries is anticipated to play an important role in establishing innovative therapeutic strategies for PAD.
Aim
The aim of this study was to compare plaque characteristics and the distribution of calcification between ATA and PTA in CLI patients.
Materials and Methods
Study Population
In this retrospective study, 30 tibial arteries obtained from 15 patients were examined. All patients had undergone lower extremity amputation due to CLI (Rutherford categories 5 to 6) at Nihon University Itabashi Hospital during the period from July 2019 to July 2021 and at Kyorin University Hospital during the period from November 2020 to November 2021. Ankle brachial index and / or angiography and / or computed tomographic angiography had been performed to diagnose PAD. Study protocols were approved by the Ethics Review Board of Nihon University Itabashi Hospital (RK-190910-01) and Kyorin University Hospital (R02-179).
Soft X-Ray Image and Section Preparation
ATAs and PTAs were dissected from lower leg amputation specimens with CLI. Peripheral arteries were fixed in 10% buffered formalin after removal from lower extremities and the vessel lumen was gently flushed with saline. Ex vivo soft X-ray radiographs (LORAD M-4, HITACHI, Tokyo, Japan) were obtained using whole vessel specimens under a tube voltage of 26 kV, tube current of 10 mA, and focus of 0.1 mm (Fig.1). Decalcification in 10% ethylenediaminetetraacetic acid disodium solution was then carried out for 7 days.
The area of calcification, extracted employing the constant threshold value, was measured, and the value obtained was divided by the total vascular area. The threshold value was determined for each image to extract the highest intensity areas considered to be calcification and to distinguish between non-calcified vessel walls and adipose tissue. After decalcification, they were cut transversely at approximately 5 mm intervals and all sections were then embedded in paraffin. Histological sections four micrometers in thickness were stained with hematoxylin-eosin, Masson’s trichrome and elastica van Gieson. Histological sections were captured using a microscopic system equipped with a digital camera (Digital microscopic system DP-70, Olympus Corporation Japan). Morphometrical analysis was calculated employing image analysis software, ImageJ 35 (U.S. National Institutes of Health, Bethesda, Maryland, USA).
Histopathological Assessments
Plaques were classified using the modified American Heart Association (AHA) classification proposed for coronary atherosclerotic lesions9, 12) (Fig.2). We classified the plaques as pathological intimal thickening, fibrous cap atheroma, and fibrocalcific plaque. Pathological intimal thickening was defined as the presence of an extracellular lipid pool with a decreasing number of smooth muscle cells but abundance of proteoglycan. Fibrous cap atheroma was identified by the presence of necrotic debris containing a core covered with a fibrous cap. Fibrocalcific plaque was characterized by collagen-rich intima with few smooth muscle cells and extracellular lipid and these lesions lacked a necrotic core. Furthermore, we classified thromboembolic lesions as being defined by complete occlusion due to emboli by organized thrombi, findings distinct from the tissue characteristics of the surrounding intima.
Plaques without thromboembolic lesions were classified into eccentric and concentric plaques. If the ratio of the maximum to minimum thickness from the external elastic lamina to the lumen in the cross section was greater than 1.5, the plaque was defined as eccentric; if the ratio was less than 1.5, the plaque was defined concentric. Calcification was defined as an area of basophilic staining in the form of plates or nodules on HE stained sections (Fig.3). The extent of calcification in the intima and media was quantitatively evaluated morphometrically. Moreover, Calcification was classified according to the size as speckled calcification, fragment calcification, and sheet calcification. Speckled calcification was defined as calcification less than 1 mm in diameter, fragment calcification was defined as calcification greater than 1 mm and less than one quadrant of the vessel, and sheet calcification was defined as calcification greater than one quadrant of the vessel.
The number of tears in the vessel wall was calculated for each section of specimens from patients who underwent EVT. To determine the degree of vascular damage caused by EVT, the depths of the tear caused by EVT in each section were classified into 3 groups as within the intima, reached the media, or invaded the adventitia. Furthermore, the morphology of tears caused by EVT was classified into two categories, tears of the plaque shoulder and radial tears (Fig.4). We also evaluated the cross-sectional areas within the external elastic lamina, internal elastic lamina, and lumen area. The stenosis rate was calculated using the following formula: (1−[lumen area / external elastic lamina area])×100.
Immunohistochemistry
The primary antibodies used were mouse monoclonal antibodies against CD68 to detect macrophages (working dilution 1:1200, Agilent, Santa Clara, CA), and α-smooth muscle actin (α-SMA) to detect smooth muscle cells (working dilution 1:400, Agilent, Santa Clara, CA). We measured the CD68- or α-SMA-positive area in the intima to clarify the characteristic histological features of plaques. Positive areas of these markers were defined as the ratio of the immunoreactive area / vessel area (Fig.5).
Statistical Analysis
Results for continuous variables with normal distributions are presented using means±SD. The normality of the distribution was examined using the Shapiro-Wilk test. Variables with non-normal distributions are expressed as medians (interquartile range). For statistical analysis, differences between groups were evaluated using Pearson’s chi-square test for categorical variables. For the analysis of soft X-ray radiographic images, the differences between ATAs and PTAs were checked for normal distribution, and the paired t-test was used. The Wilcoxon test was used for histopathological and immunohistological analyses because the distributions were not normal. We considered p＜0.05 to indicate a statistically significant difference. JMP version 14.2 (SAS Institute, Cary, North Carolina) was used for statistical analyses.
Results
Clinical Characteristics of CLI Patients
The mean age of the 15 patients was 73±7.9 years and male accounted for 86.7% of the total subjects. The medical histories showed high prevalences of hypertension (n=12, 80.0%), chronic kidney disease (n=12, 80%) and smoking (n=13, 86.7%). There were 8 (53.3%), 11 (73.3%) and 6 (40.0%) patients with diabetes mellitus (DM), receiving hemodialysis (HD) and who had undergone EVT, respectively (Table 1).
Table 1.Patient Characteristics
| Patient characteristics |
n = 15 Mean±SD, %
|
| Age |
73±7.9 |
| Male |
13 (86.7) |
| Hypertension |
12 (80.0) |
| Diabetes mellitus |
8 (53.3) |
| Dyslipidemia |
6 (40.0) |
| Smoking history |
13 (86.7) |
| Chronic kidney disease |
12 (80.0) |
| Hemodialysis |
11 (73.3) |
| Atrial fibrillation |
3 (20.0) |
| Old myocardial infarction |
5 (33.3) |
| Post PCI |
3 (20.0) |
| Post EVT |
6 (40.0) |
The values are mean±standard deviation or n (%). EVT: endovascular treatment, PCI: percutaneous coronary intervention.
In total, 30 peripheral arteries (ATAs=15, PTAs=15) dissected from amputated lower limbs and were subjected to ex vivo soft X-ray radiography. This radiographic modality showed significant differences in the calcification area, represented as a percentage. The extents of calcification in PTAs was greater than that in ATAs (ATAs, 48.3%±19.2 versus PTAs, 61.6% ±23.9; p＜0.001, Fig.1).
Histological Assessment of Plaques
In total, 860 peripheral artery sections (461 ATA sections and 399 PTA sections) were evaluated histologically. Fibrous cap atheroma identified more often found in ATAs than in PTAs (ATAs, 11.3% versus PTAs, 3.8%; p＜0.0001). On the other hand, thromboembolic lesions were more common in PTAs (ATAs, 11.1% versus PTAs 15.8%; p=0.041). The prevalence of eccentric plaque was significantly higher in ATAs than in PTAs (ATAs, 63.7% versus PTAs, 49.1%; p＜0.0001).
Intimal calcification was greater in ATAs than in PTAs (ATAs, 0% [0 - 16.8%] versus PTAs, 0% [0 - 11.7%]; p=0.0046) and the prevalence of intimal sheet calcification was higher in ATAs than PTAs (ATAs, 25.4% versus PTAs, 17.5%; p=0.0055). However, medial calcification was more predominant in PTAs than in ATAs (PTAs, 38.2% [13.1 - 63.2%] versus ATAs, 15.7% [0 - 56.6%]; p＜0.0001), and the prevalence of medial sheet calcification was higher in PTAs than ATAs (PTAs, 78.2.% versus ATAs, 49.7%; p＜0.0001). Both ATAs and PTAs showed significant stenosis, which was more severe in ATAs than in PTAs (ATAs, 85.4% [68.1 - 98.2%] versus PTAs 77.0% [66.3 - 93.9%]; p=0.0043). The area of the external elastic lamina was larger in ATAs than in PTAs (ATAs, 6.52 mm2 [3.91-10.0 mm2] versus 5.77 mm2 [4.45-7.41 mm2], Table 2).
Table 2.Comparison of plaque lesions and calcification between below-the-knee artery
|
ATA (461 sections) (%), Median [25-75%] |
PTA (399 sections) (%), Median [25-75%] |
p Value
|
| Pathological intimal thickening |
230 (49.9) |
114 (28.6) |
＜ 0.0001
|
| Fibrous cap atheroma |
52 (11.3) |
15 (3.8) |
＜ 0.0001
|
| Fibrocalcific plaque |
45 (9.8) |
20 (5.0) |
0.0086
|
| Thromboembolic lesion |
51 (11.1) |
63 (15.8) |
0.041
|
| Percentage of intimal calcification area (%) |
0 [0 - 16.8] |
0 [0 - 11.7] |
0.0046
|
| Intimal speckled calcification |
27 (5.9) |
20 (5.0) |
0.59 |
| Intimal fragment calcification |
35 (7.6) |
26 (6.5) |
0.54 |
| Intimal sheet calcification |
117 (25.4) |
70 (17.5) |
0.0055
|
| Percentage of medial calcification area (%) |
15.7 [0 - 56.6] |
38.2 [13.1 - 63.2] |
＜ 0.0001
|
| Medial speckled calcification |
1 (0.25) |
7 (1.52) |
0.06 |
| Medial fragment calcification |
56 (12.2) |
25 (6.3) |
0.003
|
| Medial sheet calcification |
229 (49.7) |
312 (78.2) |
＜ 0.0001
|
| Stenosis rate (%) |
85.5 [68.1 - 98.2] |
77.0 [66.3 - 93.9] |
0.0043
|
| EEL (mm2)
|
6.52 [3.91 - 10.0] |
5.77 [4.45 - 7.41] |
0.0028
|
Values are median (interquartile range) or n (%). ATA: anterior tibial artery, EEL: external elastic lamina, PTA: posterior tibial artery.
Transverse arterial sections showing the most severe stenosis among 5 serial sections, examined by employing immunohistochemistry. Eighty-one ATA and 73 PTA sections excluding thromboembolic lesions were morphometrically analyzed. Anti-CD68 and anti-α-SMA antibodies were used to detect macrophages and smooth muscle cells, respectively. Positive areas of staining with each antibody were calculated morphometrically using an image analysis system. The ratio of the CD68- positive area per vessel area was significantly higher in ATAs than in PTAs (ATAs, 0.29% [0.095 - 1.1%] versus PTAs, 0.12% [0.029 - 0.36%]; p=0.0007). The ratio of the α-SMA-positive area per vessel area was higher in ATAs than in PTAs (ATAs, 2.3% [1.2 - 4.8%] versus PTAs, 1.5% [0.84 - 3.7%]; p=0.021, Fig.5).
Subgroup Plaque Analysis of Patient Clinical Backgrounds
Comparison of Histopathological Features of Atherosclerotic Lesions in Peripheral Arteries with versus without DM
Nine patients with DM (DM, 443 sections) and 6 without DM (non-DM, 417 sections) were compared. The prevalence of fibrous cap atheroma was higher in the DM group than in the non-DM group (DM, 9.9% versus non-DM, 8.0%; p=0.015). The ratio of intimal calcification area per intimal area was greater in the DM group than in the non-DM group (DM, 0% [0 - 21.3%] versus non-DM, 0% [0 - 9.0%]; p=0.011), and the prevalence of intimal sheet calcification was higher in the DM group than non-DM group (DM, 24.6% versus non-DM, 18.7%; p=0.036). While, the ratio of the medial calcification area per medial area was larger in the non-DM group than in the DM group (DM, 21.3% [0.00049 - 54.9%] versus non-DM, 38.6% [0.00021 - 64.5%]; p=0.0018), and the prevalence of medial sheet calcification was higher in the non-DM group than in the DM group (DM, 53.7% versus non-DM, 72.3%; p＜0.0001, Table 3)
Table 3.Subgroup plaque analysis of patient’s clinical background Comparison of plaque lesions and calcification between characteristics (DM, HD)
|
DM versus non-DM |
HD versus non-HD |
| DM (443sections) |
Non-DM (417sections) |
p Value
|
HD (615sections) |
Non-HD (245sections) |
p Value
|
| Pathological intimal thickening |
193 (43.8) |
150 (36.0) |
0.0019
|
260 (42.3) |
84 (34.3) |
0.031
|
| Fibrous cap atheroma |
44 (9.9) |
23 (8.0) |
0.015
|
60 (9.8) |
7 (2.9) |
0.0007
|
| Fibrocalcific plaque |
48 (10.8) |
17 (4.1) |
0.0002
|
50 (8.1) |
15 (6.1) |
0.31 |
| Thromboembolic lesion |
61 (13.8) |
55 (13.2) |
0.80 |
68 (11.1) |
46 (18.8) |
0.0030
|
| Percentage of intimal calcification area (%) |
0 [0 - 21.3] |
0 [0 - 9.0] |
0.0011
|
0 [0 - 21.9] |
0 [0 - 0] |
＜ 0.0001
|
| Intimal speckled calcification |
39 (8.8) |
8 (1.9) |
＜ 0.0001
|
44 (7.2) |
3 (1.2) |
＜ 0.0001
|
| Intimal fragment calcification |
27 (6.1) |
27 (8.2) |
0.24 |
55 (8.9) |
6 (2.5) |
0.0008
|
| Intimal sheet calcification |
109 (24.6) |
78 (18.7) |
0.036
|
157 (25.3) |
30 (12.2) |
＜ 0.0001
|
| Percentage of medial calcification area (%) |
21.3 |
38.6 |
0.0018
|
39.9 |
0 |
＜ 0.0001
|
|
[0.00049 - 54.9] |
[0.00021 - 64.5] |
|
[9.1 - 64.2] |
[0 - 35.5] |
|
| Medial speckled calcification |
8 (1.8) |
0 (0) |
0.006
|
7 (1.1) |
1 (0.4) |
0.31 |
| Medial fragment calcification |
66 (14.9) |
15 (3.6) |
＜ 0.0001
|
63 (10.2) |
18 (7.4) |
0.19 |
| Medial sheet calcification |
238 (53.7) |
303 (72.3) |
＜ 0.0001
|
432 (70.2) |
109 (44.5) |
＜ 0.0001
|
| Stenosis rate (%) |
91.0 |
72.9 |
＜ 0.0001
|
82.5 |
80.0 |
0.061 |
|
[75.7 - 99.4] |
[63.0 - 88.8] |
|
[69.5 - 95.9] |
[60.1 - 98.5] |
|
Values are median (interquartile range) or n (%). DM: diabetes mellitus, HD: hemodialysis.
Eleven patients receiving HD (HD, 615 sections) and 4 patients not receiving HD (non-HD, 245 sections) were compared. The prevalence of fibrous cap atheroma was higher in the HD group than in the non-HD group (HD, 9.8% versus non-HD, 2.9%; p＜0.0007). The ratios of the intimal calcification area per intimal area and the medial calcification area per medial area were greater in the HD group than in the non-HD group (HD, 0% [0 - 21.9%] versus non-HD, 0% [0 - 0%]: HD, 39.9% [0 - 64.2%] versus non-HD 0% [0 - 35.5%]; p＜0.0001, respectively), and the prevalence of intimal sheet calcification and medial sheet calcification were higher in the HD group than in the non-HD group (HD, 25.3% versus non-HD, 12.2%: HD, 70.2% versus non-HD, 44.5%; p＜0.0001, respectively, Table 3).
Pathological Examination of Vascular Injury Caused by EVT
The vascular injury associated with balloon angioplasty was observed as tears within the vessel wall in four ATAs (19 sections) and three PTAs (34 sections). Tears of ATAs were frequently observed along the plaque shoulder: around the boundary between an eccentric plaque and a normal vessel wall. In contrast, PTAs demonstrated radial tears, which were strikingly different from those affecting ATAs (Fig.4). The number of sections showing recognizable tears within the intima was higher in ATAs that in PTAs (ATAs, 65.0% versus PTAs, 26.5%; p＜0.01). On the other hand, tears reaching the adventitia were more common in PTAs than in ATAs (ATAs, 15.0% versus PTAs, 52.9%; p＜0.01). The number of tears observed in each section was higher in PTAs than in ATAs (ATAs, 2 [1 - 2] versus PTAs, 2 [2 - 3]; p＜0.05, Table 4).
Table 4.Histological evaluation of tears by endovascular therapy
|
ATA (19 sections) (%), Median [25-75%] |
PTA (34 sections) (%), Median [25-75%] |
p Value
|
| Tear up to the intima |
12 (63.2) |
9 (26.5) |
＜ 0.001
|
| Tear up to the media |
8 (42.1) |
13 (38.2) |
0.78 |
| Tear up to the adventitia |
3 (15.8) |
18 (52.9) |
0.0059
|
| The number of tears in each section |
2 (1-2) |
2 (2-3) |
0.031
|
| Tears in the shoulder region of the plaque |
13 (68.4) |
2 (5.9) |
＜ 0.0001
|
| Radial tears all around the circumference |
6 (31.6) |
32 (94.1) |
＜ 0.0001
|
| α-SMA-positive area along the tear/EEL area |
2.64 % [0.71 - 4.1%] |
4.47% [3.26 - 6.79 %] |
0.0087
|
| Days from EVT to amputation |
39 [20 - 60] |
39 [39 - 45] |
0.93 |
Values are median (interquartile range) or n (%). ATA: anterior tibial artery, PTA: posterior tibial artery, SMA: smooth muscle actin, EEL: external elastic lamina, EVT: endovascular therapy.
To evaluate the extent of neointima induced by the proliferation of smooth muscle cells after balloon angioplasty, we measured the α-SMA-positive area along the tears caused by EVT employing immunohistochemistry. The ratio of the α-SMA-positive area throughout the length of the tears per external elastic lamina area was higher in PTAs than in ATAs (ATAs, 2.64 % [0.71 - 4.1%] versus PTAs, 4.47% [3.26 - 6.79 %]; p＜0.01, Table 4). None of the post-EVT sections showed restenosis, i.e., more than 50% of the lumen area stenosed by neointima.
Discussion
Differences in Plaque Morphology between ATAs and PTAs
The present study revealed that ATAs had the eccentric plaque type, namely pathological intimal thickening and fibrous cap atheroma, according to the modified AHA classification of atherosclerotic lesions, and higher stenosis rates than PTAs. We speculated that there are three main anatomical factors possibly explaining the high prevalence of advanced eccentric plaques in the ATAs. First, the ATA passes between the tibia and the fibula through the interosseous membrane after branching off from the popliteal artery13). Then, the ATA descends into the anterior compartment of the leg along the shin near the peroneal nerve and the distal segment runs into the subcutaneous portion of the lower leg14, 15). Therefore, the ATA branches from the popliteal artery with an acute margin. It is known that atherosclerotic lesions tend to develop in curved and bifurcated areas due to low shear stress against the vessel wall16). This atherogenic effect may have resulted in more lipid rich lesions in the ATAs. Secondly, due to the anatomical distribution of tibial arteries, the peak systolic velocity is known to be lower in ATAs than in PTAs17). ATAs with slow blood flow experience low shear stress and are prone to developing atherosclerosis. Thirdly, the distal portion of the ATA is more vulnerable to the bruising because it runs on the subcutaneous surface layer, i.e., is more superficial than other lower extremity arteries14). Therefore, it is thought that such stimuli can produce endothelial damage18), leading to the formation of atherosclerotic lesions. In contrast to the ATA, the PTA branches straight from the popliteal artery13). It is expected that thromboembolic materials in the blood stream reach PTAs more frequently than ATAs. Due to the anatomy of the PTA, there may be a higher risk for embolization in PTAs than in ATAs. The area of the external elastic lamina was larger in ATAs than in PTAs, which is consistent with the higher incidence of fibrous cap atheroma in ATAs and diffuse medial calcification in PTAs, respectively. It is well known that necrotic core development accelerates while medial calcification suppressed beneficial remodeling. Differences in the external elastic lamina area between ATAs and PTAs reflect the plaque phenotypes of these peripheral arteries.
Difference in Calcified Patterns between the Intima and the Media
Arterial calcification is classified into intimal and medial calcification. Histologically, intimal calcification is observed close to the necrotic core and inflammatory debris. Medial calcification, on the other hand, is identified along the medial smooth muscle cell layers without lipid accumulation or inflammation19). On soft X-ray radiographs, the percentage of calcification of PTAs was higher than that of ATAs. Since the images cannot distinguish between intimal and medial calcification, the sum of intimal and medial calcification areas was compared employing radiography. Interestingly, radiographic and pathological analyses revealed diffuse medial calcification to be more marked in PTAs than in ATAs. In PTAs, the peak systolic velocity is higher than in ATAs, which would presumably result in higher shear stress17). A previous study demonstrated that nitric oxide released from vascular endothelial cells increases under high shear stress20). Increased nitric oxide reportedly promotes osteochondrocytic differentiation of smooth muscle cells21). High shear stress in PTAs might be one of the factors accelerating calcification in smooth muscle cell-rich media.
In contrast, intimal calcification was more common in ATAs than in PTAs. According to the histological features of intimal calcification, this might be attributable to be due to the high prevalence of fibrous cap atheroma with necrotic core in ATAs. We assumed that fibrous cap atheroma develops focally; therefore, the total calcified area was smaller in ATAs than in PTAs, as demonstrated by radiography. Recognizing the pathological differences among BK-arteries is crucial for devising the optimal EVT strategy. Severely calcified lesions can disturb balloon and stent expansion and carry a risk of slow flow after EVT22).
Distribution of Cell Types in Atherosclerotic BK-Arteries
We examined immunohistochemical evidence indicative of the distributions of macrophages and smooth muscle cells in the BK arteries. Macrophages play a key role in vascular inflammation and contribute to the development of lipid-rich atherosclerotic plaques23). Phenotypic modulation, migration, and proliferation of smooth muscle cells are also important factor in the pathogenesis of atherosclerosis24). Immunohistochemistry indicated the CD68-positive and the α-SMA-positive areas to both be larger in ATAs than in PTAs. The current study revealed that the histopathological features of atherosclerotic lesions not only differ between tibial arteries, but also show differing distributions of cell types in the atherosclerotic plaques of peripheral arteries. Lesions rich in macrophages and smooth muscle cells are consistent with fibrous cap atheroma, which shows inflammation and fibrous cap formation with a necrotic core.
Sub-Analysis by Patient Background
Metabolic abnormalities such as hyperglycemia and insulin resistance exacerbate atherothrombosis due to endothelial dysfunction and increased inflammation25). Although medial calcification was observed in one-fifth of the DM group, fibrous cap atheroma and intimal calcification were more common in DM than in non-DM patients in our present study. These data indicate DM to be critical for the plaque phenotype, which affects the distribution of calcification within the intima. On the other hand, medial calcification was prominent in HD patients. Even though both risk factors are important for vascular calcification, DM and HD are essential for intimal and medial calcification, respectively, at least in the tibial arteries26).
Post-EVT Pathology in BK-Arteries
Few reports on pathological studies have described post-EVT BK arteries. Interestingly, post-EVT ATAs and PTAs apparently differed pathologically. Tears at the shoulder of an eccentric plaque and radial tears of a concentric plaque were frequently identified in ATAs and PTAs, respectively. The cracks caused by balloon angioplasty were more numerous and showed deeper invasion in PTAs than in ATAs. The plaque shoulder is mechanically weak and balloon angioplasty triggers tears at the boundary between the eccentric plaque and the normal vessel wall in ATAs. In contrast, PTAs showed concentric plaque with diffusely distributed medial calcification, such that we expected PTAs would be more susceptible to balloon pressure circumferentially as well as more broadly than ATAs. This phenomenon was also indicated by the larger area of neointima immunohistochemically stained employing anti-α-SMA antibodies. A clinical study revealed that EVT for BK-arteries requires aggressive and early postoperative re-treatment27). Although restenosis was not identified in any of our specimens, possibly due to the short period from EVT to amputation, the pathology of PTAs suggested balloon angioplasty to be ineffective. There are reports showing the efficacy of drug-coated balloons compered to plain old balloon angioplasty in BK-arteries28). As a strategy for preventing neointima formation, as illustrated by the α-SMA-positive area along the tears, we speculate that drug-coated balloons might be more beneficial than plain old balloon angioplasty for PTAs.
Limitation
This study has several limitations.
The subjects were limited to CLI patients, which may have led to selection bias. However, this is the first study to compare histopathological features between ATAs and PTAs in CLI patients, and we consider the number of sections to have been sufficient to elucidate the pathogenesis of CLI. Six of 15 patients underwent EVT. Because the vessel wall is damaged by balloon dilation, EVT may have further exacerbated histopathological changes in the vessel wall. Only ATAs and PTAs were evaluated, not AK-arteries, because most patients were treated by amputation of the affected lower limbs below the knee. To investigate the pathology of AK-arteries in CLI patients, additional specimens from AK amputations need to be evaluated.
Conclusion
Histological features such as atherosclerotic and thromboembolic lesions differed markedly between ATAs and PTAs. Understanding the vascular calcification distribution and pathology of post-EVT vessels may contribute to the establishment of optimal treatment strategies for PAD.
Acknowledgments and Notice of Grant Support
This work was supported by JSPS KAKENHI Grant Number JP22K06970.
Conflicts of Interest
The authors have no conflicts of interest directly relevant to the content of this article.
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