Dottorato in FISIOPATOLOGIA ED IMAGING CARDIO-TORACO-VASCOLARE

Titolo della tesi: “Role of Intestinal Permeability in Patients at Risk of Cardiovascular Diseases”

Introduction: Platelet toll-like receptor 4 (TLR4) is overexpressed in patients with atrial fibrillation (AF) and myocardial infarction (MI), but its activation and potential trigger are not understood. Methods: Serum levels of lipopolysaccharide (LPS) and platelet aggregation (PA) to collagen alone or in combination with a TLR4 inhibitor (TLR4i) were studied ex vivo in platelets from 50 patients with AF, 50 with MI and 50 controls matched for age, sex and atherosclerotic risk factors. The TIR domain-containing platelet adaptor protein (TIRAP) and the TIRAP-MyD88 interaction were investigated by Western blot and co-immunoprecipitation, respectively. In addition, the role of LPS in patients and its association with markers of gut permeability (zonulin), platelet aggregation, oxidative stress (sNOX2-dp, H2O2) and endothelial dysfunction (NO bioavailability, ET-1) were evaluated ex vivo by enzyme-linked immunosorbent or colorimetric assays. Furthermore, two different in vitro experiments were performed: 1) to test whether LPS induces activation of TLR4 pathway by evaluation of platelet TIR domain-containing adaptor protein (TIRAP) and TIRAP-MyD88 interaction, investigated by western blot and co-immunoprecipitation, respectively. 2) to test, on human intestinal mucosa cell line (Caco-2), whether LPS treatment induces direct damage to tight junction proteins (occludin) by Western blot analysis and co-immunoprecipitation and indirect damage by oxidative stress production. Results: Serum LPS was significantly higher in both patient groups, AF an MI, compared to controls. Collagen-stimulated platelets from AF and MI pre-treated with TLR4i showed a significant decrease in PA compared to collagen-stimulated platelets. The ex vivo study showed that TIRAP phosphorylation and TIRAP-MyD88 immunocomplex formation were higher in both patient groups compared to controls. Also, serum zonulin, a biomarker of impaired gut barrier function, was significantly higher in both patient groups, AF an MI, compared to controls and positively correlated with serum LPS levels. The in vitro studies showed that: 1) LPS, at concentrations similar to those found in AF and MI patients circulation, dose-dependently activated TLR4 pathway as highlighted by increase of TIRAP phosphorylation, TIRAP-MyD88 immunocomplex formation and platelet response to the agonist, all these effect were attenuated by TLR4i. 2) the stimulation with LPS of Caco-2 cells was able to induce an increase in oxidative stress and damage to TJs proteins, reducing NOX2 activation and the expression of occluding, respectively. Conclusion: The study proves that platelet TLR4 is activated in the AF and MI patient groups, suggesting circulating LPS as a potential trigger for platelet aggregation and intestinal barrier damage.