Evidence of woven bone formation in carotid artery plaques

Objective: Plaque morphology plays an important prognostic role in the occurrence of cerebrovascular events. Echolucent and heterogeneous plaques, in particular, carry an increased risk of subsequent stroke. Depending on the quality of the plaque echogenicity based on B-mode ultrasound examination, carotid plaques divide into a soft lipid-rich plaque and a hard plaque with calcifi cation. The aim of this study was to investigate structural changes in the basement membrane of diff erent carotid artery plaque types.


Introduction
In addition to the degree of stenosis, analysis of the internal morphological structure of the plaques is becoming an increasingly important, contributory factor in stroke risk. Compared with calcium-containing "hard plaques", plaques with a high lipid content are associated with an increased risk of ipsilateral cerebral ischemia [1,2]. Most of the trials emphasizing the relationship between plaque echo structure and stroke risk are based on high-resolution ultrasound (US) using a visual method of classi ication [3,4]. To date, the plaques of carotid arteries are divided into friable, ibroatheromatous and atheromatous forms [5]. In a previous study on explanted human aortic valves, calci ied areas showed characteristic morphological features of woven bone formation [6]. In this report, we describe the morphology of plaques of carotid arteries using polarised light microscopy in addition to conventional electron microscopical techniques. Especially, we were interested in the identi ication of bony structures mimicking valve calci ication.

Tissue biopsies
Tissue samples from the carotid artery were taken from patients undergoing routine endarteriectomy of the carotid artery in operations at the Department of Thoracic, Heart and Vascular Surgery. 17 biopsies were taken from 10 male patients (average age; 75 + 1 years) and 7 females (68 + 3 years). The study population included patients suffering from iliform stenosis of the carotid artery. In 9 cases, stenosis of the carotid artery occurred without any symptoms, in 8 patients signs of circulatory disturbance of the vertebrobasila system or the carotid artery were observed. Table 1 summarizes the basic data of the study patients.

Ethics
The Ethics Committee of the University of Göttingen has approved the investigations on expanted carotid artery plaques on patients to determine of woven bone formation in carotid artery (reference: 25/6/2010).

CT-Angiography fi ndings
The CT-angiography of a patient with soft plaque shows bilateral high-grade internal carotid stenosis due to artheromatous plaque. Compared to the vascular environment, hypodense concentric plaque is shown intraluminally. The lumen itself contrasts hyperdensely calci ied plaque is not detectable (Figure 1a).
The CT angiography of a patient with hard plaque also shows high-grade internal carotid stenosis due to calcifying plaques. There is hardly any evidence of hypodense internal plaques. A small hyperdensely contrasted lumen with extensive, bizarre, issured calci ications, sometimes with adjacent hardening artefacts, is shown ( Figure 1b).  Figure 2a). The endothelial layer was divided by small islands. At higher magni ication, multiple initial tearings were observed and the matrix was uncovered. In these areas,

CT-Angiography
All patients underwent preoperative CT angiography in a 64-row CT scan. For this purpose, the bolus-triggered examination technique was used with a bolus of 80 -100 ml KM at low rates of 3-5 ml/s with scan velocities up to almost 10 cm/s after reaching an enhancement of 100 HU with a delay of 6-12 s. The source recordings were then reconstructed in 3-D technique.

Scanning electron microscopy
In order to reveal the surface morphology of the explanted valves, scanning electron microscopy was performed. Specimens from valve lea lets were ixed for 6h in a solution containing 2.5% glutaraldehyde and 0.2 M cacodylate. Afterwards, samples were dehydrated in a series of increasing concentrations of alcohol. After critical point drying, all samples were sputtered with gold-palladium. Samples were visualized using the digital scanning microscope Zeiss DSM 960.

Polarised light microscopy
To evaluate the presence of woven bone tissue in calci ied areas of pathologically altered aortic and mitral valves, polarized light microscopy was performed. To prepare thin ground sections from non-decalci ied materials for polarised light microscopy, a special technique was established based upon the method of plastination developed by Hagens, et al. and modi ied for histological purposes by Schultz and Drommer [7,8] incorporation of erythrocytes into the extracellular matrix was observed. Some large plaques were covered with a thin layer of endothelial cells (Figure 2b,c).

Polarised light microscopical fi ndings
Recent techniques employing polarised light microscopy as described by Bloebaum, et al. are potentially useful to evaluate the presence of woven bone tissue [10]. Tissue samples from normal artery walls revealed no pathological alterations.
Polarised light microscopy enabled) a distinction between two different types of plaques. In transmitted plane light, the collagenous ibers as well as intravascular deposits were transparent. Using a hilfsobject red 1 st order, deposits looked lucent (Figure 3a,b). When unstained thin ground sections of pathologically altered samples of the second group were viewed in transmitted plane light, the intravalvular localized inhomogeneous inclusions appeared as a secondary substance of yellowish or blue color (Figure 3c). In regular light, the character of this substance could be described as ibrous resembling natural woven bone. At high magni ication (620 x), bundles of collagen ibers were detected (Figure 3d).
We detemine paque group 1 with evidence of woven bone formation in 4 patients with symptoms and 4 asymptomatic patients. The palque group without evidence of woven bone formations was found predominantly in symptomatic patients (5 symptomatic versus 3 asymptomatic patients). Nevertheless, a clinical correlation between plaque form and symptomatology cannot be deduced.

Discussion
Thromboembolism from extra-cranial atherosclerosis accounts for the neurological de icit in approximately half of the patients with ischaemic stroke [9]. Selection for eradicating interventions is conventionally determined by measuring luminal stenosis that results from in situ atheromatous plaques [7][8][9]. Apart from the degree of stenosis, plaque morphology has emerged in recent years as an important contributory factor in stroke risk [3]. During onset of thrombembolic events in arteresclerosis, vulnerable plaques play an important role [10]. Histological studies of coronary atherosclerosis suggest that plaques can be identi ied by thin ibrous caps that overlie large, often necrotic lipid cores [11,12]. A multimodal assessment of plaque vulnerability involving the combination of systemic markers, new imaging methods, for example, ultrasonic investigation, ct-imaging and MRI that target in lammatory and thrombotic components, and the potential of emerging therapies may lead to a new strati ication system for atherothrombotic risk and to a better prevention of atherothrombotic stroke [3][4][5][13][14][15][16]. Mechanisms of plaque rupture have been extensively studied and several parameters have been found to interact: extracellular matrix, in lammatory cells, gelatinases, stromelysins, matrilysin and MMp expression induced by oxidised lipids etc. [17][18][19][20][21][22].
In a previous study on degenerative explanted human aortic valves, calci ied areas were detected with characteristic morphological features of woven bone formation. Pathologically altered heart valves appear to exhibit distinct stages of desmal osteogenesis [6]. Formation of woven bone formation indicates an inappropriate biomechanical stress of collagenous ibers, potentially due to malsynthesis.
In this paper we report on our systematic SEM and polarised light microscopical investigations on explanted human carotid plaques. In all the samples, we detected uniform changes in the endothelium and the basement membrane. The endothelial cells often showed hyperplasia with loose binding to each other. Rarely, an endothelial layer was complete. The loss of endothelial cells may expose the extracellular matrix, which obviously sets various pathological processes in motion [23][24][25][26]. The increased activation of matrix metalloproteinases in pathologically altered human cardiac valves emphasizes the crucial role of the extracellular matrix in the development of this disease [26]. Similar mechanisms are held responsible for rupture of atherosclerotic plaques [21].
Polarised light microscopy identi ied two different types of plaques, a soft one with a transparent structure and without the presence of bone tissue, called soft plaques, and hard plaques with woven bone structures. These indings are probably identical with echolucent (predominant lipid core) und echo-opaque plaques (predominantly ibrous tissue/ calcium) as identi ied by sonography and magnetic resonance tomography [27][28][29][30][31]. The major inding of our study is the presence of woven bone tissue in hard carotid plaques. Dystrophic calci ication irst described by Mönckeberg is the most common pathological inding in surgically explanted valves [32]. Virchow recognized already that the mineralization of the walls of arteries in atherosclerosis is a process of ossi ication and not only a process of calci ication [33]. Several case reports and clinical studies have identi ied bone proteins in ossi ied areas. The studies on the pathophysiology of heterotopic enchondral ossi ication in atherosclerotic plaque of arterial walls showed that osteoprogenitor cells resemble microvascular pericytes [34,35]. Myo ibroblast-like cells, situated throughout the ibroid layer of cardiac valves and cultured in vitro, are capable of phenotypic differentiation into osteoblast-like cells [36,37]. Many authors, therefore, suggest the existence of a population of ossifying cells in both aorta and cardiac valves [34][35][36][37][38]. Atherosclerosis is a multifactorial, multistep disease that involves chronic in lammation as well as oxidation [34]. Detection of woven bone structure in pathologically altered human aortic valves and in carotid plaques indicates an additional pathogenic factor in the onset of atherosclerosis. In accordance with indings on human aortic valves, malsynthesis of collagenous ibers might contribute to pathogenesis of atherosclerosis of carotid arteries. Dystrophic calci ication is a passive process in degenerating connective tissue, whereas heterotrophic ossi ication is an active process of abnormal tisssue repair. During the process of desmal ossi ication, collagen is produced which can easily be diagnosed by light microscopic examination using polarised light. The newly formed primitive woven bone separates intravalvular inclusions from surrounding collagen ibers [6].
The detection of woven bony tissue suggests that inadequate strain favours the mineralization of carotid plaques. The altered mechanical environment and the a b d c

Conclusion
The submitted study demonstrated 2 fundamentally different carotid plaques. Whereas the irst group shows a distinct of woven bone tissue, the second group completely lacks this tissue. In the past, we could demonstrate the woven bone tissue in degenerated aortic and mitral valves. we assume that, in addition to low physiocomechanical changes in blood low, collagen metabolism disorders play a central role in the pathogenesis of carotid stenosis.