Pathological Analysis of Medial and Intimal Calcification in Lower Extremity Artery Disease

Background The prevalence and degree of lower extremity artery disease in hemodialysis (HD) patients is higher than in the general population. However, the pathological features have not yet been evaluated. Objectives The aim of the study was: 1) to compare lesion characteristics of lower extremity artery disease in HD vs non-HD patients; and 2) to determine factors associated with severe medial calcification. Methods Seventy-seven lower limb arteries were assessed from 36 patients (median age 77 years; 23 men; 21 HD and 15 non-HD) who underwent autopsy or lower limb amputation. Arteries were serially cut at 3- to 4-mm intervals creating 2,319 histological sections. Morphometric analysis and calcification measurements were performed using ZEN software. Calcification with a circumferential angle (arc) ≥180° was defined as severe calcification. Multivariable logistic regression was used to identify risk factors for severe medial calcification. Results The degree of the medial calcification arc was significantly higher in the HD group compared to the non-HD group (P < 0.0001). In the multivariable analysis, HD was associated with severe medial calcification in below-the-knee lesions (OR: 17.1; P = 0.02). The degree of intimal calcification in above-the-knee lesions was also significantly higher in HD patients with a higher prevalence of advanced atherosclerotic plaque (P = 0.02). The prevalence of severe bone formation was more common in the HD patients (P = 0.01). Conclusions Hemodialysis patients demonstrated a higher degree of medial and intimal calcification compared with non-HD patients. The difference was more prominent in the medial calcification of below-the-knee lesions.

T he latest systematic review by Fowkes et al 1 reports that there are more than 200 million people worldwide with lower extremity artery disease (LEAD).The LEAD prevalence in the general population is reported to be between 1.4% and 8.3%, 1,2 whereas in hemodialysis (HD) patients it is significantly higher at 25.3%. 35][6] The reasons for the poor clinical outcome in patients undergoing HD have not been well evaluated.Losurdo et al 7 reported that medial calcification of below-the-knee (BK) arteries is strongly associated with the risk of lower extremity amputation in patients with diabetes.Another study also demonstrated higher LEAD prevalence and severity in HD patients with higher calcification scores in lower extremity arteries.However, previous studies, have based the diagnosis of medial or intimal calcification on X-rays 8,9 or computed tomography (CT), 10,11 which may not be precise enough for calcification localization.
3][14][15] Previous pathological LEAD analysis has been done in patients with abundant risk factors 16 and chronic limb-threatening ischemia (CLTI). 17However, there are no systematic evaluations of the pathologic features of lower extremity arteries in HD patients with LEAD.Such analysis could reveal the characteristics of the diseased peripheral arteries, facilitating better strategies to prevent atherosclerosis progression in HD patients, for example in improving the quality of dialysis, which depends upon dialysis membranes and fluids.Additionally, a better understanding of the pathological features would promote the development of newer devices for endovascular treatment.This study aimed to compare the pathological characteristics of lower extremity arteries in HD and non-HD patients with LEAD by evaluating specimens collected from autopsy and amputation cases.

METHODS STUDY POPULATION AND PATIENT CHARACTERISTICS.
A total of 36 patients with lower extremity amputation due to CLTI or acute limb ischemia (ALI) and autopsy subjects who died from various causes were enrolled in the current study.ALI was defined as an ischemic limb with acute onset to progressive exacerbation, 18 excluding acute progression of CLTI.The patients were listed in the "CV Hills pathology registry of LEAD patients" from November 2019 to December 2021.The registry involved 14 centers in Japan and included patients with symptomatic LEAD in Rutherford Categories 3 (severe claudication) to 6 (ulceration or gangrene).We histologically evaluated all 36 patients (39 limbs) who were divided into 2 patient groups: HD (n ¼ 21) and non-HD (n ¼ 15), (Figure 1).

This study was approved by the Ethics Review
Committee of Tokai University (No. 19R289).
PATHOLOGICAL EVALUATION OF THE LOWER EXTREMITY ARTERY.Lower extremity arteries were trimmed from the legs and classified into 2 groups based on X-ray and gross findings: femoropopliteal arteries were classified as above-the-knee (AK) arteries, while anterior tibial arteries, posterior tibial arteries, and peroneal arteries were classified as BK arteries.In all, 32 patients had undergone previous revascularization (31 endovascular therapy and 1 bypass).Sections with stents and artificial grafts were excluded from the evaluation.We fixed 77 arteries (19 AK and 58 BK) in 10% formalin, and anatomic location and calcification severity were determined using radiographs.Vessels were further decalcified as necessary.All arteries were cut sequentially in 3-to 4-mm intervals and tissue sections were prepared and stained with hematoxylin and eosin, and Movat pentachrome as previously reported. 19Morphometric analysis and calcification measurements were performed in the scanned sections using ZEN software (ZEISS) as previously demonstrated. 16In brief, the percent stenosis and circumferential angle (arc) of intimal and medial calcification were measured.The sections stained with hematoxylin and eosin were used to evaluate calcification severity, and the Movat pentachrome stain was used to determine the exact media location (internal and external elastic laminae).Medial calcification was defined as primary calcification within the media and did not include intimal calcification extending to the medial wall.The internal and external laminae, the inner and outer border of the media, respectively, were carefully examined especially in sections stained with Movat pentachrome stain.

CLASSIFICATION OF ATHEROSCLEROTIC PLAQUE
AND CALCIFICATION.Intimal plaques were classified following the modified American Heart Association classification. 12,16,17,20,21In brief, adaptive intimal thickening was defined as the spontaneous Fibroatheroma was characterized by the presence of macrophages around a necrotic core, with a dense fibrous cap.Thin-cap fibroatheroma was identified as a necrotic core covered by a thin fibrous cap.Plaque rupture consists of a necrotic core and an overlying disrupted thin fibrous cap.Fibrous plaque was characterized by collagen-rich neointimal tissue with a few SMCs but no lipid pool or necrotic core.Fibrocalcific plaque was defined as neointimal growth with calcified lesions, and calcified nodule was defined as calcified lesions erupting into the vessel lumen with thrombi on the surface.
Adaptive intimal thickening, fibrous plaque and PIT were classified as nonadvanced atherosclerotic lesions, whereas fibroatheroma, plaque rupture, thincap fibroatheroma, fibrocalcific plaque, and calcified nodule were classified as advanced atherosclerotic lesions.Calcification with a circumferential angle (arc) $180 was defined as severe calcification.Bone formation was also evaluated with the arc measurement in the media and intima similar to calcification.
Bone formation with a circumferential angle $90 was defined as severe bone formation.4.8% vs 33.3%; P ¼ 0.01).More than 90% of patients had CLTI with Rutherford category $ IV, resulting in majority of lower limb amputations in both groups.

Kato et al
In AK lesions, in the HD group, fibrocalcific plaque was the most common plaque type (47.0%), followed by fibrous plaque (16.5%) and calcified nodule (15.5%); and in the non-HD group, fibrous plaque (30.9%) was the most common followed by fibrocalcific plaque (27.9%).
In BK lesions of both the HD and non-HD groups, fibrous plaque was the most common plaque type (51.3% vs 39.0%) (Figure 5C).
THROMBOTIC LESIONS.All the thrombotic lesions (calcified nodule or plaque rupture) were observed in  the AK group, except for 1 lesion that showed a calcified nodule in anterior tibial artery.The prevalence of vessels with calcified nodules in AK lesions was more common in the HD group than the non-HD group (77.8% vs 20.0%, respectively; P ¼ 0.009).The prevalence of lesions with plaque rupture was similar in both groups (22.2% vs 10.0%, respectively; 6).

DISCUSSION
The increasing number of patients undergoing HD with LEAD has resulted in more amputations and deaths due to CLTI; however, the reasons for poor clinical outcomes in patients on HD have not been well evaluated.This study aimed to investigate the LEAD pathologic features in HD patients collected at autopsy and from amputated legs.The main findings were as follows: 1) the arc of intimal and medial calcification were significantly higher in the HD patients than the non-HD patients; 2) HD was an important factor associated with severe medial calcification in BK lesions; 3) severe bone formation was more common in the HD patients; 4) the HD patients had more advanced intimal atherosclerotic AK lesions than the non-HD patients but the groups had diabetes, and chronic kidney disease with arterial calcification. 23,24However, reports on calcification of lower extremity arteries did not differentiate medial and intimal calcification.Ohtake et al, 10 for example, used multidetector-row CT to investigate the degree of lower limb arterial calcification in HD patients.However, they did not distinguish between medial and intimal calcification. 10Another study on HD patients identified the linear rail track type as medial calcification, and irregular and patchy distribution type as intimal calcification based on X-rays, which may be not precise enough for identifying the calcification distribution.In addition, arterial calcification was only analyzed in limited locations (pelvis and thigh), not including BK arteries. 9e current study evaluated all the histopathological specimens collected from the lower limb arteries, resulting in the most accurate characterization to date in HD patients with LEAD.The results showed that the degree of medial and intimal calcification was significantly higher in HD patients than in non-HD patients.The prevalence and arc of medial calcification in BK lesions were particularly striking, and, surprisingly, a median circumferential calcification of more than 300 was observed in HD patients.Medial calcification was more pronounced in the HD group even though the Rutherford class was comparable in both groups.The study suggests that intimal and medial calcification would be a likely cause of worse clinical outcomes in HD patients.
5][26] This suggests that medial calcification could affect the prognosis of CLTI patients.Reports suggest that medial calcification is a result of various processes (eg, inflammation, apoptosis, Ca/P homeostasis, and extracellular matrix organization); however, the mechanisms of its progression are still unclear. 22,27other study also demonstrated significant medial calcification in patients without common cardiovascular risk factors. 28e present study supports findings of HD as an important factor associated with severe medial calcification in BK lesions.Furthermore, significantly higher serum phosphorus levels in the HD group suggest HD-induced electrolyte abnormalities may have contributed to medial calcification.These results highlight the importance of dialysis quality, which is dependent upon the dialysis membrane and fluids, to maintain optimal serum phosphorus levels.Further studies are therefore needed to reveal the mechanisms of the medial calcification progression.

BONE FORMATION IN HD PATIENTS.
Previous pathologic evaluation demonstrated that bone formation was observed in 6% of the lower extremity arteries of amputated limbs. 24Age and diabetes mellitus were associated with bone formation, whereas renal failure was not.However, the prevalence of bone formation may have been underreported because not all the serial sections were evaluated in the study.There were few sections with bone formation (5.7%; 132 in 2,319 sections), whereas the current study demonstrated bone formation in 20 lower extremity arteries (26%).Evaluating all the serial sections of the artery is believed to be the most accurate method to determine the prevalence of bone formation.The higher prevalence of bone formation in lower extremity arteries could be a cause of poor prognosis in patients undergoing HD.
Recent histological studies have shown the effectiveness of optical coherence tomography in detecting calcification and bone formation in the lower extremities. 29Further observational studies are needed to evaluate the association of bone formation and prognosis, such as amputation.Kato et al   The prevalence of intimal atherosclerosis was common in the HD group when restricted to AK lesions (A), whereas no significant difference in the prevalence of intimal atherosclerosis was found between the HD and non-HD groups when restricted to BK lesions (B).(C) Comparison of intimal plaque types by site in HD and non-HD groups.In the HD group, the fibrocalcific plaque was the most common plaque type, followed by fibrous plaque and calcified nodule.In the NonHD group fibrous plaques were the most common AK lesion.Fibrous plaques were the most common plaque type of BK lesion in both groups, and advanced atherosclerosis was less common.AIT ¼ adaptive intimal thickening; AK ¼ above-the-knee; BK ¼ below-the-knee; HD ¼ hemodialysis; PIT ¼ pathological intimal thickening; TCFA ¼ thin-cap fibroatheroma.

A
B B R E V I A T I O N S A N D A C R O N Y M S AK = above-the-knee ALI = acute limb ischemia BK = below-the-knee CLTI = chronic limbthreatening ischemia GEE = generalized estimating equation HD = hemodialysis LEAD = lower extremity artery disease SMC = smooth muscle cell Kato et al J A C C : A D V A N C E S , V O L . 2 , N O .9 , 2 0 2 3 LEAD Pathology in Patients Undergoing HD N O V E M B E R 2 0 2 3 : 1 0 0 6 5 6 accumulation of smooth muscle cells (SMCs) within the proteoglycan collagen matrix, with the absence of lipid or macrophage foam cell infiltration.Pathological intimal thickening (PIT) was defined as the lipid pool that indicates the presence of apoptotic SMCs.

FIGURE 1
FIGURE 1 Study Flow of the Manuscript HD and non-HD groups showed extremely rare intimal calcification in BK lesions.Medial calcification was found in almost all the sections in BK lesions of HD patients, with severe medial calcification in 73.3% of the sections.Representative histopathological AK to BK findings in HD patients presenting with CLTI leading to lower limb amputation are shown in Figure 3 (Central Illustration).

FIGURE 2
FIGURE 2 Comparison of Calcified Lesions and Bone Formation in the HD and Non-HD Groups

FIGURE 3
FIGURE 3 Histopathological Findings of Above-the-Knee to Below-the-Knee in Patients Undergoing HD With Chronic Limb-Threatening Ischemia Several studies have investigated the pathological characteristics of intimal plaque in lower limb arteries.Torii et al16 and Narula et al17 performed a detailed pathological evaluation of LEAD patients.However, studies have not focused on HD patients.In the current study, the intimal plaque type of all tissue specimens from HD and non-HD patients was assessed following the Modified American Heart Association classification.Intimal calcification was significantly more common in the HD patients than the nonHD patients in AK lesions.Furthermore, advanced atherosclerotic changes (eg, fibrocalcific plaque and calcified nodule in AK lesions) were more frequent in the HD group.Moreover, fibrous plaques were most frequently observed in the BK lesions of HD patients compared to non-HD patients with fewer advanced atherosclerotic changes.Atherosclerotic changes in lower extremity arteries were more pronounced in AK lesions, which was in line with previous pathological studies. 16,17Ablation with debulking devices such as the Jetstream atherectomy device (Boston Scientific), orbital atherectomy (Diamondback 360, Cardiovascular Systems Inc) or intravascular lithotripsy (Shockwave medical) might be effective for intimal calcification such as fibrocalcific plaques and calcified nodules, which were relatively common in the HD group.Conversely, as the degree of atherosclerosis in BK lesions was not as advanced as expected, newer treatment devices for endovascular treatment of BK lesions in HD patients will be needed to improve clinical outcomes.STUDY LIMITATIONS.First, the number of subjects evaluated was relatively small, and the number and length of vessels trimmed varied.However, the number of subjects may be enough to characterize the pathological differences between medial and intimal calcification in HD and non-HD patients because all the serial sections of artery were evaluated in the study with detailed clinical data (eg, serum P/Ca levels).Second, the prevalence of coronary artery disease in the current study may be underdiagnosed as coronary angiography or coronary CT was not performed in all the cases.Third, detailed laboratory parameters including patient coagulation parameters were not available.CONCLUSIONSHD patients had a higher degree of medial and intimal calcification compared with non-HD

FIGURE 4
FIGURE 4 Comparison of the Prevalence of Severe Bone Formation in HD and Non-HD Groups

FIGURE 5
FIGURE 5 Comparison of Atherosclerotic Lesions in HD and Non-HD Groups

J
A C C : A D V A N C E S , V O L . 2 , N O .9 , 2 0 2 3 LEAD Pathology in Patients Undergoing HD N O V E M B E R 2 0 2 3 : 1 0 0 6 5 6 Hospital), Dr Haruya Yamane (National Hospital Organization Osaka National Hospital), Dr Amane Kozuki (Osaka Saiseikai Nakatsu Hospital), Dr Akinori Sumiyoshi (Sakurabashi Watanabe Hospital), Dr Eiji Karashima (Shimonoseki City Hospital), and the patients and their families for sending their valuable specimens.

FIGURE 6
FIGURE 6 Comparison of the Incidence of Calcified Nodules and Plaque Rupture Resulting in Acute Thrombotic Events in Above-the-Knee Lesions in the HD and Non-HD Groups

J
A C C : A D V A N C E S , V O L . 2 , N O .9 , 2 0 2 3 Kato et al N O V E M B E R 2 0 2 3 : 1 0 0 6 5 6 LEAD Pathology in Patients Undergoing HD R E F E R E N C E S

TABLE 2
Comparison of Vessel Characteristics Between HD and Non-HD Groups Values are n (%) or median (IQR).

TABLE 4
Uni/Multivariable Analyses of Predictors for Severe Medial Calcification

TABLE 5
Comparison of Bone Formation by Site Between HD and Values are n (%).HD ¼ hemodialysis.