The role of endothelial colony-forming cells (ECFCs) in acute deep vein thrombosis patients

Author(s)

Letícia Queiroz da Silva

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2022-04-26

Examining board members

Joyce Maria Annichino Bizzacchi; Bruno Kosa Lino Duarte; Erich Vinicius De Paula; Luciana Pereira Ruas; Carolina Caliari Oliveira

• Advisor: Joyce Maria Annichino Bizzacchi

Abstract

Deep Vein Thrombosis (DVT) is defined as a pathological process resulting from changes in the vessel walls, blood flow, and/or blood composition, which leads to the formation of a thrombus within the vessel. Despite current knowledge, much remains to be understood about the risk factors associated with DVT. When the vascular endothelium actively responds to pathophysiological stimuli, the antithrombotic pattern of this endothelium can change; and with the loss of integrity, occurs a release of endothelial cells (CECs), which have been considered possible markers for several diseases. In addition, endothelial colony-forming cells (ECFCs) are a subtype of endothelial progenitor cells with remarkable clonogenic potential and in vivo vascularization capacity, and due to these characteristics of endothelial nature, are being used as an ex vivo model in disease investigation. However, despite the endothelium playing a key role in hemostasis, no study has evaluated the ECFCs in acute DVT. In addition, the impossibility of a direct analysis of the endothelium makes the actual role of the endothelium in the disease difficult to understand. Therefore, the main objective of this work was to characterize and evaluate the cellular function of the ECFCs of patients with acute DVT compared to healthy individuals (controls, CN). Patients were selected after confirmation of the diagnosis of DVT of the lower limbs from July 2019 to September 2021. The groups consisted of 25 patients with acute DVT (mean age 45 ± 13 years), with 20 follow-ups (DVT>6 months) and 38 HI (mean age 36 ±10 years). We divided the assessments into 2 groups: (1) descriptive and laboratory assessment of patients with acute DVT, DVT >6 months, and HI; and (2) evaluation of endothelial function and biological parameters, with the characterization of cells by immunophenotyping and percentage of senescent cells, proliferation, apoptosis, cell migration, and thrombin generation capacity (TGT), compared to HI. In addition, we performed a sub analysis for the identification of biological markers involved in the success of obtaining ECFCs previously isolated from peripheral blood and expanded in culture. As a result, in group 1 we demonstrated a plasmatic increase of the pro-inflammatory interleukins (IL) IL-6, IL-8, platelet-derived growth factor (PDGF-AA/AB), and P-selectin adhesion molecule, and a higher frequency of ECs in patients with acute DVT compared to HI. Thus, we observed that in DVT >6 months IL-6, IL-8, P-selectin, and CECs were decreased compared to acute DVT, however, we observed an increase in Soluble Intercellular Adhesion Molecule 1 (sICAM-1) and Vascular Adhesion Molecule soluble 1 (sVCAM-1). In group 2, success in obtaining CEFCs was 37% in the HI group, 20% in the acute DVT, and 25% in the DVT >6 months. Cell function tests of ECFCs in patients with acute DVT vs. HI revealed cells with lower proliferative and migratory capacity, and more cells in apoptosis. TGT showed cells with similar properties between groups. Cell supernatant analyzes showed an increase in IL-6 expression in the ECFCs of patients with acute DVT. We have yet to complete these tests at DVT >6 months to compare DVT at two different times. Finally, some factors appear to be involved in the emergence and maintenance of cell culture, such as the increase in sICAM-1 and sVCAM-1 both in plasma and in the supernatant of cells that were isolated. In conclusion, we defined that it is possible to establish the isolation of ECFCs in patients with acute DVT, however with low success rates, and this can be linked to the findings of this work regarding the change in functional properties, especially in the replicative and regenerative capacity. Furthermore, we believe that cells can also be affected by the extracellular environment in the acute phase in the face of pro-inflammatory stimul (AU)

• FAPESP's process: 18/15618-3 - Evaluation of the activity of circulating colony forming endothelial cells (ECFCs) in patients with spontaneous and induced Venous Thrombosis
• Grantee: Letícia Queiroz da Silva Justo
• Support Opportunities: Scholarships in Brazil - Doctorate