Evaluation of circulating nucleosomes and influence of simvastatin IN VITRO in the adhesive and chemotactic properties of neutrophils in patients with venous thromboembolism

Author(s)

Kiara Cristina Senger Zapponi

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2018-02-26

Examining board members

Joyce Maria Annichino Bizzacchi; Erich Vinicius De Paula; Marina Pereira Colella; Suely Meireles Rezende; Luciane Alarcão Dias Melício

• Advisor: Fernanda Loureiro de Andrade Orsi; Joyce Maria Annichino Bizzacchi

Abstract

Recently, animal models and clinical studies have explored the participation of neutrophils in the pathophysiology of VTE. However, it remains unclear whether the activation of neutrophils is persistent after the acute phase of the disease. Furthermore, there are clinical evidences supporting that simvastatin may prevent recurrent VTE, since the drug has pleiotropic anti¬inflammatory effects. The aim of this study was to evaluate the occurrence of neutrophil activation in patients with VTE compared to healthy controls and to determine the effect of simvastatin in the adhesive properties and chemotaxis of the neutrophils. Neutrophils activation was determined by the expression of activated adhesive molecules (CD11a/ Mac-¬1), reactive oxygen species (ROS), chemotaxis assays, and serum nucleosome and DNA-MPO complex, as markers of neutrophil extracellular traps (NETs). The major endothelial ligands of neutrophils, circulating endothelial adhesive molecules (s-ICAM-1 / s-VCAM-1) as well as serum high sensitive C-reactive protein (CRP) levels as a marker of inflammation were also evaluated. For CD11a and CD11b integrins expression and migration assays, neutrophils were evaluated under basal conditions and after inflammatory stimulus, pretreated, or not, with simvastatin. The study group consisted of thirty¬seven patients with personal history of VTE and thirty¬seven controls matched with patients according to age, gender and ethnicity. Median time since VTE occurred was 25 months, the event was spontaneous in 51.35% of the cases, 62,16 % of patients presented proximal VTE and 24,32% with pulmonary embolism (PE). According to Villalta Scale, 64.28% of the patients presented post-thrombotic syndrome (PTS). Activated CD11a adhesive molecule was higher in VTE patient neutrophils, both in basal conditions and after TNF? stimulus. Higher of CD11b was observed in patient neutrophils, compared with controls, only after TNF? stimulus, and the stimulus was reverted by pre¬treatment with simvastatin. Neutrophils from VTE patients also presented increased basal chemotaxis and IL-¬8¬ stimulated chemotaxis. We also observed increased DNA-MPO complexes in VTE patients, as well as in the s-ICAM-1 and s-VCAM-1 endothelial adhesive molecules. The amount of ROS and circulating nucleosomes were similar in VTE patients and controls. However, when patients were divided into subgroups according to factors associated with recurrence, such as: high D-dimer (> 500ng / ml) and spontaneous VTE, or by the presence of SPT and the time period after the acute event, we could observe a significant increase of circulating nucleosomes in patients with SPT. There were no differences in the other subgroups. In addition, hs-CRP levels were significantly higher in VTE patients. We demonstrated that patients with VTE presented neutrophil activation for a long time after the acute thrombotic episode. In addition, these results suggest that there is an increased interaction of the neutrophils adhesive molecules with their endothelial ligands, consequentely favoring the process of neutrophil migration, as well as suggesting an increase in the endothelial activation. The increase of NETs in VTE patients may be involved in the activation of other cells, especially endothelial cells, and other pathways, which are back-stimulated. These results may support the hypothesis that increased neutrophils activation is part of the chronic inflammatory condition associated with VTE and may be downregulated, in parts, by the effects of simvastatin (AU)

• FAPESP's process: 13/22981-3 - Evaluation of circulating nucleosomes and influence of Simvastatin in vitro in the adhesive and chemotactic properties of neutrophils in patients with venous thromboembolism
• Grantee: Kiara Cristina Senger Zapponi Cerri
• Support Opportunities: Scholarships in Brazil - Doctorate