¿ROLE OF ENDOTHELIAL DYSFUNCTION IN THE PATHOGENESIS OF UNPROVOKED VENOUS THROMBOEMBOLISM¿
Tesi di Dottorato
Data di Pubblicazione:
2023
Citazione:
¿ROLE OF ENDOTHELIAL DYSFUNCTION IN THE PATHOGENESIS OF UNPROVOKED VENOUS THROMBOEMBOLISM¿ / A. Cancellara ; tutor: S. Della Bella ; supervisore: F. Calcaterra ; phd course coordinator: N. Landsberger. Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, 2023 Jul 25. 35. ciclo, Anno Accademico 2022.
Abstract:
Background and Aim
Venous thromboembolism (VTE) is a multifactorial disease that includes deep vein thrombosis (DVT) and pulmonary embolism (PE). VTE patients can be classified on the basis of the risk factor(s) that trigger the thromboembolic event. Indeed, up to 50% of VTE events occur in absence of predisposing conditions and are therefore classified as unprovoked VTE (uVTE). Being uVTE pathogenesis still unknown, uVTE patients are characterised by a high risk of recurrence, and the therapeutic management of these patients to prevent VTE recurrence is still controversial. A better comprehension of uVTE pathogenesis and the identification of the molecular mechanisms underlying VTE triggering in these patients are needed to guide patient treatment through the definition of new clinicopathological entities and the definition of molecular targets for the development of novel therapeutic strategies. Endothelial dysfunction (ED) plays a crucial role in promoting thrombus formation, yet its presence in uVTE patients has been poorly investigated, so far. Therefore, in this study we investigated ED in uVTE, using patient-derived endothelial colony-forming cells (ECFCs) that represent an optimal non-invasive model to assess the functionality of the endothelial compartment. In particular, in this project we aimed to:
• set-up and optimize ECFC-based functional assays aimed at assessing thrombogenic endothelial properties;
• investigate the presence of constitutive ED in uVTE patients and the involved underlying molecular mechanisms;
• investigate whether ED in uVTE patients is further promoted by pro-inflammatory stimuli;
• evaluate whether ED contribute to VTE recurrence risk in uVTE patients;
• assess whether ECFC features of ED are restricted to uVTE or shared with secondary VTE.
Methods
Optimization of functional assays. ECFC-based TGA and thrombogenesis assay were preliminarily set up on commercially-available human umbilical venous endothelial cells (HUVECs), then tested on ECFCs isolated and expanded from 8 healthy donors (HDs). ECFCs were used at passages 4-6, in basal conditions or after TNF-stimulated activation. TNFinduced EC activation was assessed as upregulation of the adhesion molecule VCAM-1 and the pro-coagulant molecule Tissue Factor (TF) by flow cytometry. EC function was assessed by analyzing: a) thrombin generation assessed by TGA on ECs - a modification of Hemker’s method; b) platelet deposition and fibrin formation under flow conditions, assessed in thrombogenesis assay.
Assessment of ED role in uVTE pathogenesis. 78 VTE patients and 27 HDs were enrolled in the study. VTE patients were classified as: i) provoked VTE (pVTE) (n=28) when the VTE event was associated with major risk factors, ii) weakly provoked VTE (wpVTE) (n=28) when the VTE event was associated with minor risk factors; iii) unprovoked VTE (uVTE) (n=22) when the VTE event occurred in the absence of any provoking risk factors. A peripheral blood sample was collected at a single timepoint from all the enrolled subjects for ECFC isolation and plasma collection. In VTE patients, blood withdrawal was executed 3 months after the index VTE event, along with simultaneous assessment of validated predictors of VTE recurrence (namely, post thrombotic syndrome, D-dimer levels, and presence of residual venous obstruction). To investigate the presence of constitutive ED, ECFCs obtained from the different subgroups of VTE patients and HDs were characterized by assessing: i) their immunophenotype; ii) their ability to promote thrombosis in ECFC-based TGA and thrombogenesis assay; iii) their secretion of soluble ED markers (soluble adhesion molecules including the soluble forms of ICAM-1, VCAM-1, PECAM-1 and E-Selectin) in culture superna
Tipologia IRIS:
Tesi di dottorato
Keywords:
endothelial dysfunction (ED) ; endothelial colony-forming cells (ECFCs) ; thrombogenesis assay ;
thrombin generation assay (TGA) ; unprovoked thromboembolism (uVTE).
Elenco autori:
A. Cancellara
Link alla scheda completa: