DTA

Digital Theses Archive

 

Tesi etd-12062018-190846

Type of thesis
Perfezionamento
Author
VANNINI, FEDERICA
URN
etd-12062018-190846
Title
Identification of circulating low molecular weight endothelium-protective factors
Scientific disciplinary sector
BIO/15
Course
SCIENZE MEDICHE - Translational Medicine
Committee
Membro Prof. RECCHIA, FABIO ANASTASIO
Membro Prof. LIONETTI, VINCENZO
Membro Dott. ARMIROTTI, ANDREA
Membro Prof. DE CATERINA, RAFFAELE
Membro Prof. REGGIANI, ANGELO
Keywords
  • Atherosclerosis
  • endothelial dysfunction
  • endothelium-protective factors
  • plasma ultrafiltrates
  • dog plasma fractionation
  • omic analysis on plasma
Exam session start date
;
Availability
parziale
Abstract
In literature, many sources report that dog is not a good model for atherosclerosis because it is not susceptible to atherogenesis, even under a high-cholesterol diet. Therefore, given the pivotal role of the endothelium in atherogenesis, we have hypothesized the presence of protective factors in the plasma of dog that may reduce the risk of endothelial dysfunction under stress condition. Identification and isolation of the circulating endothelium-protective factors can provide the basis for the development of new drugs against atherosclerosis.<br>The composition of plasma from pigs, dogs and human subjects were analyzed. Human plasma samples were separated in two groups: elder human plasma from healthy subjects over 65 years old and young human plasma from healthy subjects under 30 years old. 3K plasma samples from human atherosclerotic patients were included in the analysis to search for metabolites related to the pathology.<br>Human aortic endothelial cells (HAEC) were treated with α-tumor necrosis factor or oxidized low density lipoproteins for 24 hours in the presence of 50K or 3K plasma in order to test the potential endothelium-protective effect of the samples. The different cellular response to the treatment has been evaluated by the analysis of VCAM-1 or HO-1 protein expression and TNFα, IL-6 and PAI-1 mRNA expression. The antioxidant ability of plasma was evaluated by adding plasma samples to low density lipoproteins (LDL) in the presence of copper sulfate and by measuring the conjugates dienes formation. Liquid Chromatography-Mass Spectrometry (LC-MS) was used to identify the endothelium-protective factors in 3K plasma by lipidomic, peptidomic and metabolomic analysis. <br>Among the studied groups, 50K and 3K dogs present the highest endothelium-protective effect in the in vitro analysis as well as young human plasma. Results showed the same protective effect of 50K and 3K plasma samples on HAEC, suggesting that proteins are not involved in the investigation. The antioxidant activity of NAC on TNF-treated HAEC was comparable with 3K dog plasma, while IL-6 and PAI-1 were not modulated, suggesting that 3K dog plasma exerted endothelium-protection by antioxidant properties.<br>LC-MS/MS analysis revealed the presence of several metabolites and peptides markers significantly different from elder human compared to dog or young human plasma. These selected markers were tested in vitro, but none of them exhibited endothelium-protection by itself. <br>Fractionation of 3K dog plasma with C18 column revealed that the active part of the 3K sample was in the flow-through (unretained) fraction, meaning that the potential protective factors are polar molecules.<br>Metabolomic analysis suitable for polar compounds did not improve the investigation of the potential protective factors among species and further investigations are currently ongoing to search similarities between dogs and young humans.<br>By analyzing 3K human plasma from young, elder and atherosclerotic subjects with metabolomics, positive correlation was seen with endothelium-protective effect. In addition, the age factor was positively associated with our in vitro results for endothelium-protection.
Files