The Increase of The Relative Amount of Nodular Calci cation In Femoral Plaques is Associated With Milder Lower Extremity Arterial Disease

Mae Azeez University of Helsinki Mirjami Laivuori Helsinki University Hospital and University of Helsinki Johanna Tolva University of Helsinki Nina Linder University of Helsinki Johan Lundin University of Helsinki Anders Albäck Helsinki University Hospital and University of Helsinki Maarit Venermo Helsinki University Hospital and University of Helsinki Mikko I Mäyränpää University of Helsinki and Helsinki University Hospital Marja-Liisa Lokki University of Helsinki A. Inkeri Lokki (  inkeri.lokki@helsinki. ) University of Helsinki Juha Sinisalo Helsinki University Hospital and University of Helsinki


Introduction
The amount of calci cation in lower limb arteries has been associated to increased degree of stenosis, and higher cardiovascular morbidity and mortality [1], [2].
Vascular calci cation is a bone-like formation continuum that presents different morphologies, which in turn impact differently on the plaque histology and the clinical presentation [3], [4]. It initiates as microcalci cation, which predestines the plaque to an unstable state [5]. The propagation of microcalci cation into macrocalci cation is believed to have a stabilizing effect on the plaque, despite its potential stenosing impact [6]. Macrocalci cation can occur in the form of a calci ed plate, called sheet calci cation (ShCa) or the form of osteoid metaplasia [4], [7]. Nodular calci cation (NodCa) may be caused by a fracture of ShCa and is accompanied by surrounding brin deposits [7], [8]. The latter may also suggest an additional thrombotic origin of the nodular calci cation [9]. NodCa may erupt through the protective cap of the plaque with an overlying thrombus to form a calci ed nodule [10].
Semi-quanti cation of calci cation in lower limb arteries and its clinical implication has been analyzed using imaging scoring [1], [2], [11]. Histological quanti cation is a valuable instructive tool for understanding the clinical signi cance and mechanistic of different plaque structures. To our knowledge, no study has analyzed the quantity of NodCa and ShCa in femoral atherosclerosis and their impact on LEAD presentation.
Interestingly, despite its dense brocalci c character, atherosclerosis in super cial femoral artery is reputable for its milder clinical presentation compared to other vascular territories [12]. This is partly by the virtue of the compensatory vascular remodeling observed in this artery, which compensates stenosis Page 4/16 even in sizable atherosclerotic lesions [12], [13]. Thus far, studies have not addressed the impact of different calci ed morphologies on vascular remodeling. Therefore, in this study, we quantify NodCa and ShCa in femoral plaque sections and analyze and compare the contribution of each category area proportion to the LEAD severity. We also analyze the effect of each quanti ed calci cation category on regional vascular remodeling.
We applied a deep learning algorithm to obtain a consistent measuring method in all the studied sections and to facilitate quanti cation of the co-existing categories to be studied.

Results
Patients' characteristics are shown in Table 1.  Table S2).

Discussion
We have, for the rst time, quanti ed the amount of NodCa and ShCa on femoral artery plaque sections. The results revealed that the increase of the relative amount of NodCa is associated with reduced vascular ischemia of the lower limb, even at over 90% level of stenosis. Additionally, higher relative amount of NodCa is associated with reduced risk of semi-urgent operation. The latter may implicate a slower obstruction and eventually a milder LEAD presentation.
NodCa and ShCa typically coexist in the same plaque, according to the continuum of vascular calci cation.To understand their clinical relevance, the quanti cation of NodCa and ShCa lesions is needed. Quantifying of more than one feature accurately and consistently is di cult to attain using the conventional subjective scoring method. Therefore, we applied deep learning algorithm analysis for the localization and quanti cation of these calci ed structures. The quanti cation was optimized and validated by the high sensitivity and speci city, with around 2% area error.
NodCa is believed to fracture from sheet calci cation [15]. This possibly explains the highest prevalence of NodCa in artery with more dynamic activity like super cial femoral artery compared to coronary and carotid arteries [10]. Furthermore, smooth muscle cells of femoral artery have an inherently different molecular makeup from other vascular beds; their higher expression of TGFβ promotes mineralizing activity [16]. This may explain the prevailing dense calci cation observed in this study sections.
All study samples were obtained from operated patients, and they had over 90% including occlusion, identi ed histologically, and con rmed from preoperative magnetic resonance angiography. Interestingly, distal limb perfusion in these patients, quanti ed by measurements of toe pressure and ankle brachial index, varied in their severity. This clinical variation is re ected in a variable presentation and relative quantity of the calci ed structures, ShCa and NodCa in the patients' lesions. The observed clinical variation, however, was mainly associated to the quanti ed NodCa according to this study analysis.
In the current study, NodCa was encountered in 96% of sections with over 90% level of stenosis. Such a high prevalence can be attributed to the good exposure of the longitudinally sectioned plaques. Despite the high prevalence of NodCa in the studied obstructed/semi-obstructed lesions, quanti cation of NodCa in these samples has revealed that its relative amount contributed to a milder presentation of LEAD. The milder presentation was assessed by the negative association of the quanti ed NodCa with severely lowered TP and ABI. Furthermore, increase of the relative amount of NodCa was associated with decreased risk of semi-urgent intervention. The association of the higher relative amount NodCa with milder LEAD in obstructed/semi-obstructed samples may be implicated by a slow rate of stenosis progression [17]. No association was observed between the quanti ed ShCa and the clinical presentation of LEAD.
The applied quantifying approach has demonstrated that the increase of the relative amount of NodCa contributed signi cantly to the increase of the measured vessel diameter in samples with over 90% stenosis. Larger arteries at the obstructive/semi-obstructive level may incubate larger plaques and likely more NodCa. However, the quanti cation in this study is made relative to the plaque area. Furthermore, the analysed association, after adjustment to the demographic determinants of vessel size: gender, age and BMI; and modi ers of vascular remodeling: smoking, hypertension, diabetes, GFR, and hs-CRP, remained signi cant [18], [19]. This indicates that NodCa may have some contribution to an expansive vascular remodeling. No association was noted between the amount of ShCa and the measured vessel diameter in these samples.
We postulate, that the loss of the compact calci ed structure by the fragmentation of NodCa, and the separation of NodCa fragments by brin that collects from the leaky capillaries may enhance the expansive vascular remodeling [15]. Expansive vascular remodeling is proved to delay stenosis progression [20]. This possibly indicates a slower obstruction of vessels that incubate abundant NodCa in their lesions, presented by reduced propensity to semi-urgent intervention [20].
Previous studies on the clinical association of the quanti ed NodCa in LEAD have not, to our knowledge been published. However, the presence of calci ed nodules on femoral plaque samples has been also observed to protect against post-operative major amputation and/or re-intervention of the re-vascularized limb [21]. In the current study, the patients were not analyzed post-operatively.
The amount of ShCa was signi cantly reduced in normal BMI patients compared to overweight/obese patients. In line with this nding, the amount of ShCa inversely correlated with serum LDL cholesterol and triglyceride, and positively correlated with serum HDL cholesterol in the studied patients. Level of lipids in serum is believed to increase mainly the deposition of early calci cation in the form of microcalci cation and has been demonstrated histologically [22]- [24].
The strength of the study is the rigorous clinical examination results obtained pre-operatively. We developed a reliable neural network method to quantify different forms of arterial calci cation. The trained algorithm attained high sensitivity and speci city. The study material consisted of plaques harvested during surgical intervention; therefore, they represent a later stage of the disease. No conclusion could be drawn from this material about less severe stage of disease. Our ndings about NodCa and disease severity and vessel diameter represent a cross-sectional study and to our knowledge the rst study to quantify the relative amount of NodCa and ShCa.

Conclusion
Femoral plaques with over 90% stenosis demonstrate variable clinical severity of LEAD. The applied deep learning analyzing tool allows for categorical quanti cation of calci cation types in the plaque's sections.
The quantifying approach is more reliable than simply indicating the presence of calci cation category in terms of clinical signi cance assessment.This quantifying approach for calci cation categories has revealed that the relative predominance of NodCa indicated a slowly progressing obstruction diagnosed by alleviated ABI and TP readings and reduced semi-urgent surgical intervention and inferred by a possible expansive vascular remodeling contribution. These results improve our understanding of the complex pathophysiology of LEAD and pave way for personalized treatment options targeted at the speci c pathological ndings of endarterectomy patients.

Cohort description
Femoral plaque samples were collected during endarterectomy of the femoral artery bifurcation between October 2014 and January 2017 [4]. The severity of LEAD of the patients (N=90) was determined by ankle brachial index (ABI), toe pressure (TP), Fontaine classi cation of LEAD symptoms, and urgency of the operation. Preoperative magnetic resonance angiography images were used to con rm the severity of the stenosis (Fig. 1).
A and B are preoperative magnetic resonance images of two male patients that present obstructive level of stenosis of femoral artery (blue arrow) caused by the atherosclerotic lesions C and D respectively. Obstruction in these lesions is demonstrated histologically (arrowhead). Lesion C is dominated by NodCa, closely seen in the magni ed image (thin arrow) surrounded by brin (white asterisk), while lesion D has mainly ShCa (black asterisk) in the magni ed image. The trained algorithm (E) and (F) recognizes and calculates area proportion of NodCa (blue color) and ShCa (red color) to the sectioned plaque (green color) area. The calculated area proportion in E is 0.26 for NodCa and 0.10 for ShCa, while in F is 0.06 for NodCa and 0.40 for ShCa.
We compared the area proportion of NodCa and ShCa observed in the plaque tissue to the clinical characteristics listed above. We analyzed endarterectomy samples, which ful lled two criteria:1) more than 90% stenosis including obstruction, was observed both histologically and by magnetic resonance angiography, and 2) internal elastic lamina for vessel diameter measurement was identi able in the plaques' histological section.
Plaques were formalin-xed, decalci ed, and longitudinally sectioned into two halves. Sections were stained with Hematoxylin and eosin stain for histomorphometry analysis. Sample processing and laboratory analyses are described in detail elsewhere [4].
The patients signed a written informed consent to the study before endarterectomy operation. To quantify each of the prede ned calci cation categories, two sequential algorithms were developed; the rst algorithm, the plaque tissue algorithm recognized and quanti ed the area of plaque tissue from slide background. The algorithm was set to region context size of 50 µM, the complexity level of Complex and the default speci cations. Upon this algorithm, a strati ed second algorithm, the calci cation algorithm, was built for quanti cation of the calci cation categories, NodCa and ShCa. While developing the algorithms, accuracy was assessed through 1) Veri cation of each annotation 2) Analysis of the untrained regions and whole section slides. Calci cation algorithm was ne adjusted on the following parameters: iterations = 7000, eld of view = 100 x, image augmentation range (-10 to 10), aspect ratio=1, maximum sheer=1, luminance range (-30 to 30), contrast range (-30 to 30), maximum white balance change= 10, and noise= 2. The algorithm quanti ed ShCa and NodCa as the area of every recognized structure of the category in mm2, the collective area of each category, and the area proportion of the calci cation category to that of the plaque section tissue area (Fig. 1). To optimize the analysis, visual validation of the nal algorithm analysis was conducted, whereafter, tiny lesions that were part of the continuum yet did not contribute to an actual nodule, were excluded. This was done after visually determining the cut-off size limit of the lesion per each slide. The precision of the nalized algorithm was 97.53%, sensitivity was 97.66% and total area error (false positive and negative) was 2.06%.
The maximum width of the vessel diameter in the most stenosed part was measured to assess the vascular remodeling in relation to the area proportion of NodCa and ShCa. This was done using the measurement tool in the Aiforia platform (supplementary gure S1).

Data analysis
Area proportions of NodCa and ShCa was analyzed in relation to patients' continuous and categorized binomial variables. Continuous data analysis of the patients is presented as the mean (± standard deviation). Data were analyzed for normal distribution by Shapiro-Wilk test. Normally distributed data were analyzed by two-tailed t-test and Pearson correlation test, while non-normally distributed data were analyzed by Mann-Whitney U and Spearman rank analysis. Analysis of covariance was set to assess vessel diameter association with area proportion of NodCa and ShCa along with other confounding factors; age, gender, body mass index (BMI), smoking, hypertension, glomerular ltration rate (GFR) and in ammatory state (high sensitivity C-reactive protein (hs-CRP)). Our data t the assumption of logistic regression analysis for the association of area proportion of NodCa and ShCa with the clinical parameters of LEAD, adjusted by hypertension, diabetes and dyslipidaemia. P value < 0.05 was considered statistically signi cant. Data were analyzed using SPSS 25 (Armonk, NY: IBM Corp).
The age was categorized by the median into patients older or younger than 70.5 years. was categorized by the cutoff point of normal and overweight (25 kg/m2) into normal, and overweight or obese (combined). Laboratory measurements were categorized according to the standardized age/genderrelevant reference values that are adopted in the analyzing facility, Helsinki University Hospital Laboratory Services (HUSLAB). Hs-CRP was considered increased for females when levels exceeded 2.5 mg/L and for males when levels exceeded 3 mg/L. GFR was considered impaired if it was less than the following values measured in ml/min /1.73 m2: 77 for patients aged 50-59 years, 69 for patients aged 60-69 and 59 for patients aged 70 years and older. Leukocytosis was labelled for readings higher than 8.2 E9/L, while anaemia was deduced from females' haemoglobin readings < 117 g/L and from males' readings < 134 g/L. The categorization of ABI and TP parameters was based on the clinical reference [14]. ABI less than 0.4 and TP less than 30 mmHg were deemed as severe disease indicators. Fontaine class was categorized into two groups, patients with claudication were deemed to have mild symptoms and patients with rest pain, ischaemic ulcer or gangrene were considered to have severe symptoms [4].
Surgical interventions were classi ed into elective operations, semi-urgent operations were determined if an exceeded intervention was mandated within 4 weeks of their clinical evaluation.
A and B are preoperative magnetic resonance images of two male patients that present obstructive level of stenosis of femoral artery (blue arrow) caused by the atherosclerotic lesions C and D respectively.
Obstruction in these lesions is demonstrated histologically (arrowhead). Lesion C is dominated by NodCa, closely seen in the magni ed image (thin arrow) surrounded by brin (white asterisk), while lesion D has mainly ShCa (black asterisk) in the magni ed image. The trained algorithm (E) and (F) recognizes and calculates area proportion of NodCa (blue color) and ShCa (red color) to the sectioned plaque (green color) area. The calculated area proportion in E is 0.26 for NodCa and 0.10 for ShCa, while in F is 0.06 for NodCa and 0.40 for ShCa.

Figure 2
The collective area proportion of nodular calci cation (NodCa) and sheet calci cation (ShCa) of the 51 patients with > 90% stenosis included in the study

Supplementary Files
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