SCIENZE CHIRURGICHE The vulnerable carotid plaque. Identification of endothelial markers predictive of plaque weakness, rupture predisposition and neoangiogenesis

Ciclo Xxvi, Tesi Presentata, Dott Francesco, Vasuri Coordinatore, Dottorato Relatore, Chiar Mo, Stella Andrea, Chiar, Mo, Gianandrea Pasquinelli
A Al lm ma a M Ma at te er r S St tu ud di io or ru um m --U Un ni iv ve er rs si it tà à d di i B Bo ol lo og gn na a DOTTORATO DI RICERCA IN Esame finale anno 2014 II Ancora una volta, a Debbie, con tanto amore... ...e alla mia famiglia, che mi ha sempre sostenuto in questo lunghissimo percorso! III ABSTRACT Background. Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group demonstrated that intra-plaque neovessels show both a Nestin+/WT+ and a
more » ... /WT+ and a Nestin+/WT1-phenotype, the latter being correlated with complications and plaque instability. Aims. The aims of the present thesis are: (i) to confirm our previous results on Nestin/WT1 phenotype in a larger series of carotid atheromatous plaques, (ii) to evaluate the relationship between the Nestin+/WT1-neoangiogenesis phenotype and plaque morphology, (iii) to evaluate the relationship between the immunohistochemical and histopathological characteristics and the clinical instability of the plaques. Materials and Methods. Seventy-three patients (53 males, 20 females, mean age 71 years) were consecutively enrolled. Symptoms, brain CT scan, 14 histological variables, including intraplaque hemorrhage and diffuse calcifications, were collected. Immunohistochemistry for CD34, Nestin and WT1 was performed. RT-PCR was performed to evaluate Nestin and WT1 mRNA (including 5 healthy arteries as controls). Results. Diffusely calcified plaques (13 out of 73) were found predominantly in females (P=0.017), with a significantly lower incidence of symptoms (TIA/stroke) and brain focal lesions (P=0.019 and P=0.013 respectively) than not-calcified plaques, but with the same incidence of intraplaque complications (P=0.156). Accordingly, both calcified and not calcified plaques showed similar mean densities of positivity for CD34, Nestin and WT1. The density of Nestin and WT1 correlated with the occurrence of intra-plaque hemorrhage in all cases, while the density of CD34 correlated only in not-calcified plaques. Conclusions. We confirmed that the Nestin+/WT1-phenotype characterizes the neovessels of instable plaques, regardless the real amount of CD34-positive IV neoangiogenesis. The calcified plaques show the same incidence of histological complications, albeit they do not influence symptomatology and plaque vulnerability. Female patients show a much higher incidence of not-complicated or calcified plaques, receiving de facto a sort of protection compared to male patients. V OVERVIEW OF THE PHD PROJECT The principal aims of the current PhD program is the evaluation of the neoangiogenesis in carotid atheromatous plaques, in term both of neovessel density and phenotype, and its correlation with plaque morphology and instability. We performed a morphological and immunohistochemical study on 73 carotid plaques, in order to evaluate the phenotype of the intra-plaque neovessels, and its relationship with plaque morphology and clinical presentation. In previous studies conducted during the PhD program, we found that specific vasa vasorum of normal arteries co-express Nestin and WT1, two markers of progenitor endothelia and vascular proliferative potential. Those results were published in 2012 [Vasuri F, Fittipaldi S, Buzzi M, Degiovanni A, Stella A, D'Errico-Grigioni A, Pasquinelli G. Nestin and WT1 expression in small-sized vasa vasorum from human normal arteries. Histol Histopathol 2012;27:1195-1202], and gave us a theoretical basis for the evaluation of Nestin and WT1 in plaque neovessels. During the second year of the PhD program, we evaluated CD34, Nestin and WT1 in 49 specimens, and we found that intra-plaque neovessels express both the Nestin+/WT1+ phenotype and the Nestin+/WT1-phenotype, i.e. the WT1/Nestin ratio was inferior than 1. Moreover, the Nestin+/WT1-phenotype correlated with the occurrence of intra-plaque histological complications (and plaque vulnerability) regardless the density of CD34-positive vessels. These results are under revision [Fittipaldi S, Vasuri F, Degiovanni A, Pini R, Mauro R, Faggioli G, D'Errico-Grigioni A, Stella A, Pasquinelli G. Nestin and WT1 expression in atheromatous plaque neovessels: association with vulnerability. Histol Histopathol]. The results exposed in the present thesis are the progression of our study, with an enlargement of the carotid specimen series (n=73), and special focus on the different neoangiogenetic profiles in neovessel of plaques with different morphology, and on the correlations with clinical data. VI SUMMARY 1.3.2 AHA classification of intimal lesions: advanced lesions pag. 14 1.3.3 Modifications to the original classification pag. 18 1.4 PREVIOUS RESEARCH OF OUR GROUP pag. 21 1.4.1 Nestin and WT1 as markers of active neoangiogenesis pag. 21 1.4.2 Physiology of vasa vasorum and neoangiogenesis VII in diseased arteries pag. 22 1.4.3 Nestin and WT1 expression in healthy vasa vasorum pag. 23 1.4.4 Nestin and WT1 in neoangiogenesis of atheromatous plaques pag. 25