Digital Theses Archive


Tesi etd-09202019-123124

Type of thesis
Study on the role of sevoflurane in promoting the release and uptake of endothelial exosomes protecting cardiomyocytes against perioperative ischemia/reperfusion injury.
Scientific disciplinary sector
SCIENZE MEDICHE - Translational Medicine
Membro Dott.ssa FORINI, SIMONA
  • Exosome
  • Cardioprotection
  • Sevoflurane
Exam session start date
Background<br><br>To date, the perioperative myocardial infarction is the leading cause of morbidity and mortality after cardiac surgery. Hence, an improvement of the clinical outcome of patients exposed to myocardial ischemia/reperfusion (I/R) is still a desirable achievement. Several studies have demonstrated that volatile anesthetics prevent cardiac I/R injury. Moreover, it has been demonstrated that cardioprotection by sevoflurane is not mediated by inflammatory cytokines. To best of our knowledge, halogenated ethers including sevoflurane exert cardioprotection activating pro-survival kinases similar to coronary ischemic preconditioning. In 2014, it was demonstrated that remote ischemic preconditioning increases the release of cardiac exosomes and that exosomes recapitulate cardioprotection due to ischemic preconditioning. Exosomes are 40–100 nm-sized particles stored in endosomal compartments, which are secreted when these intracellular bodies fuse with the plasma membrane. Conventional surface markers of exosomes include the tetraspanin family members CD63 and CD81, among others. Exosomes carry proteins, lipids, and nucleic acids including DNA, mRNAs, long non-coding RNAs and microRNAs (miRNAs), which they can shuttle to recipient cells, even at a distance. The hypothesis of my study is that Sevoflurane induces the endothelial release of cardioprotective exosomes during cardiac surgery with extracorporeal cardiopulmonary bypass (CPB).<br><br>Aim 1<br>-To evaluate the effects of Sevoflurane or Propofol in modulating the profile of circulating biomarkers of myocardial injury in patients undergoing on-pump minimally invasive mitral valve surgery.-<br><br>For this purpose, I have performed a single-center randomized controlled trial named Trial MINI-SEVO (NCT02551328) at FTGM/OPA Hospital (Massa, Italy) in accordance with the Declaration of Helsinki. The study was approved by the Area Vasta Toscana Ethics Committee (CEAVNO no. 452/14). <br>Patients signing informed consent and undergoing to minimally invasive mitral valve repair or replacement (MIMV) via right mini thoracotomy were recruited to one of two groups: Sevo-group (31 patients) and Propofol group (31 patients). Between March 2015 and December 2016, 225 patients were assessed for study inclusion; of these, 160 did not meet the inclusion/ exclusion criteria and were excluded. During CPB, sevoflurane was administered in the oxygenator circuit through a calibrated vaporizer. In both groups, depth of anesthesia was monitored with bispectral index. The primary outcome was myocardial injury, assessed by measuring myocardial Troponin I from venous blood samples collected pre-operatively (baseline), before CPB, before cardiac reperfusion and 6, 12, 24, 48 and 72h after cardiac reperfusion. Secondary outcomes were systemic metabolic stress as assessed by: blood pH, lactate, serum creatinine level, mechanical ventilation time, length of ICU stay, left ventricular function. <br>Concentrations of cTnI were increased postoperatively and peaked at 6hours after surgery in both groups. Postoperative blood pH, lactate, and serum creatinine were lower in the sevoflurane group compared to the propofol group. Mechanical ventilation time was lower in the sevoflurane group compared to the propofol group (p = 0.05), whereas ICU stay and total length of stay were similar in both groups. <br>There were neither in-hospital deaths nor unexpected serious adverse events. All patients were discharged with no or negligible residual mitral regurgitation. <br><br>Aim 2<br>-To evaluate the effects of Sevoflurane or Propofol in modulating the profile of circulating exosomes in patients undergoing on-pump minimally invasive mitral valve surgery.-<br><br>For this purpose, the exosome-rich fraction was isolated from blood samples of 14 patients using a standard protocol of serial, differential centrifugation and ultracentrifugation steps. I have measured levels of CD63+ and CD81+, established exosome biomarkers, by Western Blotting. CD81 is a marker of endothelial exosomes, whereas CD63+ is a generic exosomal surface marker. CD81+/CD63+ ratio, an index of endothelial exosomes, in patients treated with propofol was higher than patients receiving Sevoflurane. I found an inverse relationship between cardiac troponin I values and endothelial exosomes levels in the propofol group, but not in the Sevoflurane patients. <br><br>Aim 3<br>-To evaluate the cardioprotective effects of endothelial-derived exosomes in vitro.-<br><br>Murine coronary endothelial cells were exposed to increasing dose of Sevoflurane or PBS for 6h. Then, cells were stressed with transient exposure to 1.5mM hydrogen peroxide for 1h in order to simulate I/R injury. Dihydroethidium staining was used for detection of superoxide anion mage. The previous exposure of endothelial cells to Sevoflurane caused a statistical significant dose-dependent reduction of superoxide anion, apoptotic index revealed by TUNEL assay and higher release of exosomes rich of HSP70. In order to verify if endothelial exosomes protect cardiomyocytes, murine cardiomyocytes HL1 were pre-treated for 6h with medium of Sevoflurane-treated or PBS-treated cells. Medium exosome depleted of Sevoflurane-treated cells was also used as control. Finally, HL-1 cells were exposed to 1.5mM hydrogen peroxide.<br>When cardiomyocytes were exposed to hydrogen peroxide 1mM for 1h, cell survival was reduced to 31.76%, (P &lt;0.05). Never the less Conditioned medium of Sevoflurane-treated cells increased cell survival (P &lt;0.05). Conversely, exosome depleted conditioned medium failed in evoking cardioprotection (p &lt;0.05). <br>