Atherosclerosis and its devastating clinical complications such as arterial thrombosis, myocardial infarction, ruptured aortic aneurysms and stroke, are the leading cause of illness and death in Western countries. Carotid atherosclerotic plaque is causative of cerebrovascular insufficiency and represents the most frequent lesion associated to ischemic stroke (Wein and Bornstein 2000). Carotid endarterectomy (CEA), the most frequently performed intervention in vascular surgery, and still the gold standard procedure for hemodynamically significative carotid atherosclerosis(Mayberg et al, 1991), constitutes an injury to the vascular wall, which is followed by arterial wall healing response. Only in a small percentage of cases (4-7%) exaggeration of the normal reparative process ensues, which leads to a hemodinamically significant re-narrowing (restenosis) of the vessel lumen. Restenosis is the main cause of failure in vascular surgery and represents the most frequent long-term complication of carotid artery surgery. The restenotic process occurs more frequently between 3 to 12 months after surgery, less frequently thereafter. It represents an important clinical problem, in as much as patients with restenosis require a tight follow-up and eventually new interventions. The mechanisms underlying the restenotic process have been studied mainly in the coronary arteries, where this complication after percutaneous transluminal coronary angioplasty (PTCA) revascularization is much more frequent (30-50%). This difference in frequency can be ascribed to the different injury caused by revascularization procedures: during CEA the plaque is completely removed, while it is crushed and disrupted during PTCA. Cell migration and proliferation, matrix remodeling and thrombus deposition at injury site are believed to be important factors in the pathogenesis of the restenotic process (Schwartz, 1998). However, the biochemical and cellular components implicated, and the molecular mechanisms of the vascular response to injury are only partially known. Moreover, animal models of restenosis have many and important limitations. Taking into account the research expertise of our group, the study will particularly focus on issues related to the hemostatic process, or others components that can interact with this pathway and among them: a) regulation of endothelial function, that includes production of hemostatic molecules (i.e. vonWillebrand factor) or receptors (TFPI, thrombomodulin, protein C receptor), negative regulators of the coagulation process; b) inflammation, influencing the expression of several coagulation factors, among them tissue factor and fibrinogen; c) receptors directing plasma molecules to the intracellular degradation and thus potential regulators of levels of coagulation factors and matrix proteases; d) proteolytic components that could interact with the main proteolytic system able to degrade fibrin, the fibrinolytic system. Although most of the markers proposed for this study have been suggested to contribute to restenosis, conflicting results and sporadic findings have been produced. The components of the primary carotid atherosclerotic plaque (endarterectomy specimens) and of the restenotic lesion have been poorly investigated in humans. This phase of the project is designed to compare the primary lesions with the restenotic ones for morphology and specific markers to improve the knowledge on the origin of cells responsible for the restenotic tissue.This part of the project will be developed in the frame of a recently established collaboration with the Department of Pathology and Immunology, Geneva (dr. ML Bochaton-Piallat, prof.G. Gabbiani). Expression of candidate genes will be investigated in tissue specimens by in situ RNA hybridisation to reinforce findings from the association studies performed in the first part of the project. It's worth noting that, whereas protein studies are limited by availability of proper functional or antigenic assays, probes to investigate the expression at the mRNA level can be designed virtually for all genes. Associations with a weak statistical significance could be validated by these approaches (immunohistochemistry and in situ hybridisation). Vascular Smooth Muscle Cells (V-SMC) are the main contributor to the atherosclerotic lesion and are strong candidates for the restenotic process.Cultured V-SMC will be characterized for differentiation markers used to discriminate between SMCs and myofibroblasts. In the human V-SMC model we will investigate: - TF expression (ELISA, Western Blotting) - TF activity by FXa generation fluorogenic assay in cell lysates and medium. - TF bearing microparticles from V-SMC, after Fas-Ligand induced apoptosis, will be quantified by flow cytometry after labelling with the annexin V-FITC.

Progetto PRIN "Traditional and non traditional risk predictors of cardiovascolar disease and restenosis" Sottoprogetto: Genetic, plasmatic and cellular markers of restenosis after carotid endarterectomy

MARCHETTI, Giovanna;
2005

Abstract

Atherosclerosis and its devastating clinical complications such as arterial thrombosis, myocardial infarction, ruptured aortic aneurysms and stroke, are the leading cause of illness and death in Western countries. Carotid atherosclerotic plaque is causative of cerebrovascular insufficiency and represents the most frequent lesion associated to ischemic stroke (Wein and Bornstein 2000). Carotid endarterectomy (CEA), the most frequently performed intervention in vascular surgery, and still the gold standard procedure for hemodynamically significative carotid atherosclerosis(Mayberg et al, 1991), constitutes an injury to the vascular wall, which is followed by arterial wall healing response. Only in a small percentage of cases (4-7%) exaggeration of the normal reparative process ensues, which leads to a hemodinamically significant re-narrowing (restenosis) of the vessel lumen. Restenosis is the main cause of failure in vascular surgery and represents the most frequent long-term complication of carotid artery surgery. The restenotic process occurs more frequently between 3 to 12 months after surgery, less frequently thereafter. It represents an important clinical problem, in as much as patients with restenosis require a tight follow-up and eventually new interventions. The mechanisms underlying the restenotic process have been studied mainly in the coronary arteries, where this complication after percutaneous transluminal coronary angioplasty (PTCA) revascularization is much more frequent (30-50%). This difference in frequency can be ascribed to the different injury caused by revascularization procedures: during CEA the plaque is completely removed, while it is crushed and disrupted during PTCA. Cell migration and proliferation, matrix remodeling and thrombus deposition at injury site are believed to be important factors in the pathogenesis of the restenotic process (Schwartz, 1998). However, the biochemical and cellular components implicated, and the molecular mechanisms of the vascular response to injury are only partially known. Moreover, animal models of restenosis have many and important limitations. Taking into account the research expertise of our group, the study will particularly focus on issues related to the hemostatic process, or others components that can interact with this pathway and among them: a) regulation of endothelial function, that includes production of hemostatic molecules (i.e. vonWillebrand factor) or receptors (TFPI, thrombomodulin, protein C receptor), negative regulators of the coagulation process; b) inflammation, influencing the expression of several coagulation factors, among them tissue factor and fibrinogen; c) receptors directing plasma molecules to the intracellular degradation and thus potential regulators of levels of coagulation factors and matrix proteases; d) proteolytic components that could interact with the main proteolytic system able to degrade fibrin, the fibrinolytic system. Although most of the markers proposed for this study have been suggested to contribute to restenosis, conflicting results and sporadic findings have been produced. The components of the primary carotid atherosclerotic plaque (endarterectomy specimens) and of the restenotic lesion have been poorly investigated in humans. This phase of the project is designed to compare the primary lesions with the restenotic ones for morphology and specific markers to improve the knowledge on the origin of cells responsible for the restenotic tissue.This part of the project will be developed in the frame of a recently established collaboration with the Department of Pathology and Immunology, Geneva (dr. ML Bochaton-Piallat, prof.G. Gabbiani). Expression of candidate genes will be investigated in tissue specimens by in situ RNA hybridisation to reinforce findings from the association studies performed in the first part of the project. It's worth noting that, whereas protein studies are limited by availability of proper functional or antigenic assays, probes to investigate the expression at the mRNA level can be designed virtually for all genes. Associations with a weak statistical significance could be validated by these approaches (immunohistochemistry and in situ hybridisation). Vascular Smooth Muscle Cells (V-SMC) are the main contributor to the atherosclerotic lesion and are strong candidates for the restenotic process.Cultured V-SMC will be characterized for differentiation markers used to discriminate between SMCs and myofibroblasts. In the human V-SMC model we will investigate: - TF expression (ELISA, Western Blotting) - TF activity by FXa generation fluorogenic assay in cell lysates and medium. - TF bearing microparticles from V-SMC, after Fas-Ligand induced apoptosis, will be quantified by flow cytometry after labelling with the annexin V-FITC.
2005
M., Anselmi; Marchetti, Giovanna; E., Bonora; D., Girelli; I., Sheiban
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1683428
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