ORGINAL ARTICLE
Sneddon‘s syndrome as a disorder of small arteries with endothelial cells proliferation: ultrastructural and neuroimaging study

Introduction
Sneddon’s syndrome is characterized by association of livedo reticularis (typical skin lesions) and cerebrovascular diseases: stroke or transient ischemic attack [1,7,23]. This disease is a rare progressive disorder of unknown aetiology, affecting small and medium-sized arteries of the skin and the brain [23,32]. The pathological change is noninflammatory occlusive arteropathy [21,25]. The disease usually starts with vascular pathology in the epidermis but neurological defects become evident later [31]. For this reason, examination of skin biopsy has a particular importance for the early diagnosis [31]. Sneddon’s syndrome mainly affects women before or during middle age [10]. The clinical differentiation of Sneddon’s syndrome from other phenomenologically similar disorders may be difficult [12,16,19,26] and requires supplementary examinations.
We present histological, immunohistochemical, ultrastructural, and neuroimaging methods used commonly for an early diagnosis of Sneddon’s syndrome. Special attention was focused on ultrastructural examinations of the skin and muscles, since, to our knowledge, there are only a few data concerning ultrastructural examination of the blood vessels in diagnosis of Sneddon’s syndrome to date [18,24].

Case report
A 18-year-old woman had been well until 2 years earlier when the non-specific prodromal symptoms (headache, vertigo) and livedo reticularis developed. Dermatological examination revealed livedo reticularis on both lower legs and the trunk (Fig. 1). These skin lesions were exacerbated by cold. Additionally, a diagnosis of labile hypertension was made. Two years later she was first seen at our ward. On the neurological examination, she was well oriented and cognition was intact. There was mild dysarthria. The right pupil was 8 mm, and the left 6 mm; both constricted normally. A prominent peripheral left facial weakness was evident. The remaining cranial-nerve functions were intact. The patient barely lifted her left arm from a bed; the distal muscles of the left upper extremity were graded 3-4/5 in motor power; she lifted her left leg 10 cm off the bed against minimal resistance; the distal leg muscles were graded 4/5; the right extremities were graded 5/5. The deep tendon reflexes were ++ in the right extremities and +++ in the left; left Babinski, Oppenheim and Chadock signs were present. The cerebrospinal fluid (CSF) was normal and its protein electrophoresis did not show any presence of discrete bands in the immunoglobulin G (IgG) region.
Complete blood count and differential count (CBC & DC), prothrombin time, activated partial thromboplastin time, electrolytes (Na, K, Cl) were all within a normal limit.
Tests for lupus anticoagulant, antinuclear antibody (ANA), and antiphospholipid antibodies, cryoglobulin, human immune deficiency virus (HIV), Coxackie and ECHO viruses, tuberculosis (PCR) were all negative.

Material and methods
The samples of skin and skeletal muscles were fixed in buffered formalin, and embedded in paraffin. Sections were stained with hematoxylin and were immunostained with the following antibodies: smooth-muscle actin (SMA) DAKO 1:75, CD31 DAKO 1:20, and CD34 Novocastra 1:25.
For the ultrastructural studies, the small samples were fixed by immersion in 2.5% glutaraldehyde with post-fixation in osmium tetroxide, and routinely processed into epoxy resin. The arteries were identified on semithin sections stained with toluidyne blue, and ultrathin sections were cut from the selected area. The ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Opton DPS 109).

Results
The light microscopy revealed a significant reduction of a vascular lumen in the capillaries, small and medium arteries of the skin, and in muscle samples (Fig. 2, 3). Proliferation of endothelial cells, frequently with multilayer arrangement with their nuclei placed perpendicularly to the vascular lumen and/or basement membrane, was visible (Fig. 2). The basement membrane was often thickened (Fig. 3). The proliferating cells were positive with antibody CD34, CD31 and sporadically with SMA (Fig. 2c ).
In the skin biopsy, the electron microscopy showed that the capillaries, like small and medium-sized arteries had a different range of lumen reduction and often resembled a narrow crevice (Fig. 4-7). In the cytoplasm of the cells surrounding the lumen of the vessels the abundant filaments (about 10 nm in diameter – a diameter of the indirect filaments) were present. They were usually organized in bundles frequently filling a substantial part of the cytoplasm (Fig. 8, 9). Their nuclei were often folded or invaginated (Fig. 7, 8). In these cells, Weibel-Palade bodies and numerous pinocytotic vesicles under plasmalemma, were seen (Fig. 8, 9). In some of the capillaries and the small arteries junctions between the endothelial cells were partially detached, sometimes forming prominent extensions (Fig. 10, 11). Outside the endothelia, the smooth muscle cells characterized by attachment plaques, were located (Fig. 12-14). Some of them were electron denser than normal ones and their cytoplasmic protrusions penetrated to the endothelial cells (Fig. 14). The capillaries with ultrastructural picture typical of proliferating vessels were also observed (Fig. 11). Their endothelial cells produced numerous cytoplasmic protrusions, both at the lumen and abluminal surface (Fig. 12, 14).
Vessels with the different range of the lumen reduction were also observed in the skeletal muscle. Contrary to the skin, in the muscle biopsy, the cells surrounding the vessels lumen were characterized by a large number of pinocytotic vesicles as well as under plasmalemma and in the all cytoplasm, but proliferation of filaments was not to seen (Fig. 15).
CT scans showed only two big ischemic focuses in the pons (Fig. 16a,b). Additionally, MRI picture revealed some small disseminated ischemic focuses in the pons (Fig. 17) and in the deep structure of both brain hemispheres, the biggest one was placed in the right thalamus (Fig. 18). MRA showed no changes in big extracranial and intracranial arteries (Fig. 19a,b).
Discussion
The relationship between livedo reticularis and cerebrovascular lesions was first reported by Sneddon in 1965 [25] as a clinical entity. The clinical findings of our patient, including the presence of livedo reticularis and particular neurological symptoms, suggested Sneddon’s syndrome. To confirm Sneddon’s syndrome diagnosis neuroradiological, immunohistochemical and ultrastructural investigations were performed.
MRI picture usually shows lacunar infarct and leukoaraidosis; bilateral patchy of diffuse hyperintensive areas of signal alterations in the white matter, observed particularly on T2-weighted images, suggesting chronic ischemia and cortical atrophy. Progression of MRI changes indicates the correlation with a worsening clinical condition in the follow up [6,28]. Our CT scan and MRI demonstrated abnormalities that agree with earlier observations of other authors [4,6]. CT has shown ischemic focuses in the pons and MRI picture confirmed the CT findings and revealed the additional small disseminated ischemic focuses in the deep structures of both brain hemispheres. Severity of the disease seems to be correlated with MR imaging aspects [28]. In our patient, MRA of the big extracranial and intracranial arteries appeared normal, which does not rule out that the pathology is still concerned around the small and medium-sized arteries.
Our data are in agreement with Tourbach et al. [28] that neuroimaging like CT, MR and MRA contribute to better understanding of the pathophysiology, and natural course of cerebral lesions in Sneddon’s syndrome.
Our histological and ultrastructural examination of the skin and muscle biopsy revealed a significant reduction of vascular lumen in the capillaries, and in the small and medium-sized arteries. Their basement membrane was often thickened. The cells surrounding the blood lumen were CD31 positive and contained Weibel-Palade bodies. It is commonly accepted that, besides the Weibel-Palade bodies, the immunoreactivity for factor VIII and CD31 are of the most specific to identify the endothelial cells [2,11,14]. On this basis, we believe that the proliferating cells surrounding the vascular lumen displayed the phenotype characteristic of endothelial cells. The most of the endothelial cells showed abundant intermediate filaments, frequently filling a substantial part of the cytoplasm and numerous pinocytotic vesicles. Such a fibrillary appearance of the cytoplasm is probably an exaggerated intermediate filaments content in the cells [13]. A large number of intermediate filaments was observed in aging human lens epithelial cells and in many other degenerative, and aging cells [8,20] The identity of the cells engaged in proliferation remained controversial, which has been already noted by Seep et al.[24]. Generally, our findings are in contrast to most observations of other authors, who claim that the lumen of vessels is occluded by subendothelial proliferating smooth muscle cells or myofibroblastic cells and noninflammatory thrombotic occlusions [15,18,22,31,32]. It was also observed that in the skin small to medium-sized arteries of the dermis-subcutis boundary are affected in a stage-specific sequence from stage I to stage IV, and that the cells involved in subendothelial proliferation displayed the phenotype characteristic of smooth muscle cells and were vimentin and actin positive but desmin negative [24]. However, in our immunohistochemical and ultrastructural investigations neither proliferation of the medial smooth muscle cells nor their presence in the lumen of the vessels was observed. However, the presence of endothelial cells, characterized by Weibel-Palade bodies among the smooth muscle cells in the occluded vessel lumen, was noted [24]. An endothelial origin of proliferating cells, which were factor VIII positive, was postulated earlier [9].
Between the endothelial cells with numerous intermediate filaments the junctions were often detached to a different degree. In our opinion, this picture may be comparable with the initial stage of changes described by Zelger et al. [32]. However, it is worth remembering that, in some cases, the mature vessels must be first destabilized to allow angiogenesis [29].
Our ultrastructural pictures indicate the formation of new vessels. Angiogenesis is a formation of new capillaries from the pre-existing vessels by migration and proliferation of the endothelial cells. It plays a key role in normal and pathological conditions, including diseases [5,27,30]. The ultrastructural picture of the endothelial cells during angiogenesis is comparable to endothelial activation [17]). The endothelial activation is characterized by the presence of lumininal and abluminal protrusions, an increase of cytoplasmic organelles and basement membrane degradation [17].
The skin has a rich blood supply, but when stenoses or even occlusions occur in several neighbouring arterioles, tissue hypoxia develops [31] and this, in our opinion, can lead to the proliferation of vessels.
Additionally, cerebral angiography is abnormal in approximately 75% of Sneddon’s syndrome cases. The common findings are an obliterating noninflammatory arteriopathy with stenosis or occlusion of the intracranial vessels and the so called pseudo-angiomatosis presented by vessels malformations like transdural anastomoses, a wide network of fine collateral branches or granulomatous leptomeningeal infiltration. The name pseudoangiomatosis comes from the term angiomatosis, but in this case there is no evidence for neoplastic vascular proliferation. We support the view that in the course of Sneddon’s syndrome the process of leptomeningeal vessels proliferation (pseudoangiomatosis) may be due to the repeated episodes of ischemia [3].
Most cerebrovascular events in Sneddon’s syndrome are ischemic lesions in the brain, but there are some case reports about intracranial haemorrhage. The suspected mechanism seems to be rupture of an abnormal vessel. [3].
To sum up, we believe that the picture of the vessels in our skin and muscle samples results from the area selected for biopsy. The histological changes in the arterioles of the skin within an area of racemose livedo are irregularly distributed, so a place selected for biopsy would be therefore of decisive importance [31].
Our findings, on the basis of immunohistochemical and electron microscopy examination, suggest that the occlusions of the capillaries and the small arteries are composed of the endothelial cells. Clinical data and neuroimaging pictures support the Sneddon’s syndrome diagnosis. Further investigations in this field are necessary for a better understanding of the pathogenesis and the course of the illness.

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