Neo-linfogenesi nelle lesioni aterosclerotiche della biforcazione carotidea

INTRODUCTION: The indications for surgical or pharmacologic treatment of the patients with carotid stenosis are based on the macroscopic characteristics of the atherosclerotic carotid plaque (obtained by diagnostic investigations: ECD/CT). The stability of the plaque depends on inflammatory cells, inflammatory cytokines and on the neoangiogenesis. In the literature there are many studies about atherosclerotic plaques but the mechanism of development, progression and instability of the plaque are not known. A lot of studies show how the progression and instability of the plaques would depend on the formation of new blood vessels that are responsible for the migration of inflammatory plaques and intraplaque haemorrhage. The new “actors” in the atherosclerotic plaques are lymphatic vessels but their role is unknown. AIM: The aim of this project is to evaluate the inflammatory cells in atherosclerotic carotid plaque, the lymphatic and blood vessels, their location and their relationship with inflammatory cells in the plaque. MATERIAL AND METHOD: In this project, 31 patients with atherosclerotic carotid plaques were enrolled (10 plaques of patients were used to develop the method) and undergone to carotid endarterectomy (CEA) following ESVS guidelines at the Vascular Surgery Unit of the Ospedale di Circolo in Varese. For each clinical and biological data were collected. The carotid plaques were classified independently of clinical symptoms by their morphology (stenosis and plaque composition) by computed-tomography angiography into fatty/soft (< 60 Hounsfield units (HU), mixed 60>HU<129) and calcified (> 130 HU). The calcified plaques were excluded from this study. Carotid endarterectomy tissue samples, during CEA, were removed with minimal trauma and immediately placed in paraformaldehyde for histological evaluations researching inflammatory cells, lymphatic and blood vessels in the plaques. The Immunohistochemestry slides were processed with MIAquant that is a novel system for automatic segmentation, measurement and localization comparison of different biomarkers from serialized histological slices. This software would allow to analyze, with objective criteria, a large number of images but, especially, the entire surface of the section. RESULTS: In soft plaques there are a lot of inflammatory cells and vessels (lymphatic and blood) compared to mix plaques. Working on serial plaque sections, I focused on the localization of neovascularization, using Ulex to detect blood vessels and Podoplanin (D2-40) as marker of lymphatic vessels. In the soft plaques, the expression of D2-40 (lymphatic vessels) is near the lipidic core, unlike the blood vessels are localized in soft and mixed plaques, predominantly near the cap of the plaques and there isn’t any relationship with the lipid core. For a better understanding of the origin and function of lymphatic and blood vessels inside atherosclerotic carotid plaques, some components of the inflammatory infiltrate were examined, particularly, I have analysed T lymphocytes (CD-3) and two different macrophage populations (CD-68, CD-163). In addition to this, their position was also evaluated against lymphatic and blood vessels. In atherosclerotic carotid plaques Lymphocytes (CD-3) are near blood vessels and are spread homogenously in the plaques. T lymphocytes and hematic vessels are not localized in the same territory of lymphatic vessels marked with D2-40. The macrophages cells (CD-68, CD-163) are located near the lipid core, in the same layer of lymphatic vessels. The colocation of lymphocytes with blood vessels and macrophages with lymphatic vessels were confirmed by double coloration with confocal microscopy. To achieve comparable and reproducible data, all soft plaque sections have been examined automatically, by MIAquant software, to obtain a localization image for each segmented marker (map), which shows the locations of all markers inside the atherosclerotic carotid plaques. Furthermore, the software allows to create an histogram with united density value of markers of all plaques. This software allow to confirm that macrophages cells and lymphatic vessels in all plaques are located near the lipid core, in particular at a distance of 0-400 pixels. Instead blood vessels and lymphocytes are spread homogenously. The co-location analysis of lymphatic vessels and macrophage cells was further scrutinized by way of immunofluorescence in confocal microscopy. Taking advantage of the resolution power of confocal microscopy, it observed that only rarely we can find tubular structures attributable to one or collapsed lymphatic vessels, this is frequently seen in structures that could also be isolated cells near macrophage cells CD68. In some cases, cells seem that they express both markers. To characterise macrophage cells and to better understand how they could influence new lymph genesis, an analysis of macrophage cells CD-68 was done on the confocal microscope with doublemarkings and I observed the presence in the plaques of cells that show positive labelling for CD-68 and they don’t express HLA-DR. Such features are characteristic for myeloid-derived suppressor cells (MDSC) of the monocyte-macrophage line. DISCUSSION: These data show that the lymphatic and blood vessels are located in different place in the carotid atherosclerotic plaque. The blood vessels co-locate with lymphocyte CD-3 cells, these cells are important to determinate the development of the lesions, in fact they can cause apoptosys and they produce cytokines that promote the endothelial proliferation and develop of neo-vessels. Furthermore they reduce macrophages proliferation and so they cause plaque instability. The lymphatic vessels, near the lipid core in atherosclerotic plaque, could be the attempt to absorb and metabolize lipids and cholesterol. In fact in physiological conditions they have a key role in the reverse transport of lipid and cholesterol in peripheral blood. These data are further support by the co-location of lymphatic vessels and macrophage cells, in fact it is known that the macrophage cells in atherosclerosis are important in the process of phagocytosis of lipid and cholesterol performing the role of “scavenger”. Macrophages localize in the proximity of the lymphatic vessels and co-localize with themselves, probably because they represent inflammatory cells mainly involved in the lymphogenesis process. They determine lymphogenesis via two methods: the production of paracrine signals represents the first one on behalf of macrophages; the second one is symbolized by the differentiation of macrophages in lymphatic endothelial cells. The first mechanism of macrophages that supports lymphogenesis, is determined by the up-regulation of VEGF-C during the inflammatory process inside the atherosclerotic plaque. In fact, macrophages produce TNF-α that activates the TNFR1 receptor facilitating the creating of the VEGF-C, which activates VEGFR-3 promoting the development and the activation of lymphatic endothelial cells. In addition to the paracrine mechanism, macrophages contribute to lymphogenesis using transdifferentiation in lymphatic endothelial cells. Through this investigation, I have also recognized the presence of myeloid-derived suppressor cells of the monocyte-macrophage line inside the atherosclerotic carotid plaques. These cells inside neoplasms have a dual function: trying to inhibit T lymphocytes activity from avoiding self-inflicted damage, although, at the same time, they facilitate neo-angiogenesis and neo-lymphogenesis, and thus tumor’s growth and its dissemination. In atherosclerotic carotid plaques, these cells will probably inhibit T cells’ activity, as they do not localize near such cells, and they foster lymphogenesis more extensively that angiogenesis, given their localization close to lymphatic vessels and not near blood vessels. CONCLUSIONS: Lymphatic vessels are new actors in atherosclerotic carotid plaques but it is unknown if they determine the progression, stability or instability of the plaques. This project might be an optimal start point to continue the study trying to identify the functions of myeloid-derived suppressor cells in particular, by pinpointing the cytokines that they produce, to understand that type of activity they possess inside the atherosclerotic plaque. Therefore, this investigation, regardless of the riveting and not yet identified data in the literature, it represents the preliminary phase of other possible examinations of atherosclerotic carotid plaques. The inspection of new plaques and additional markers will allow us to acquire significant results for an overall assessment of the plaque, and hopefully, to the identification of areas at a higher atheroembolic risk inside the atherosclerotic carotid plaque.