Platelets exhibit a unique secretory profile consisting of multiple growth factors, cytokines, microRNAs, small soluble molecules and proteins with a dual pro and antiangiogenic role. This balanced combination of mediators is mainly contained in extracellular vesicles (EV) , now conceived as biological signaling systems or vectors of which platelets represent the most abundant source of the human body. The EVs may be of heterogeneous size and composition (exosomes, microparticles, macrovesicle) and it has been reported that they reflect the hemostatic properties of the platelets. Consequently, the clinical preparation known as platelet lysate (LP) is also able to enhance the angiogenic properties of platelets. The plethora of highly concentrated factors, soluble molecules and EV in this blood product is considered to be the main responsible for its angiogenic capacity, but the way in which the LP-derived EV could regulate angiogenesis has yet to be fully addressed.
In this regard, the purpose of my study is to confirm that LP Mesengen®, (patent No.WO2013042095) represents a source of bioavailable EV and therefore validate the individual role of EV in relation to the ability to mediate angiogenesis. In addition, we want to demonstrate which molecular mechanisms and biological actions mediated by LP and LP-derived EVs alone can contribute to the restoration of the endothelium after insult and therefore open a new path on the use of LP as a tool for vascular regeneration in the context of cell-free therapy.
So, the main purpose of my work is characterization in terms of content, quantity and size of the Evs contained in the LP Mesengen. This information is necessary for a better understanding of the biological activities of LP in the molecular and consequently clinical context.
Given the presence in the literature of studies on platelet functionality mediated by microRNA and their transport related to vesicular functions, we analyzed the EVs of PL content.
For the first time the complete miRNoma of the LP profile was analyzed by means of RNA Seq.
The result was an RNA content, represented by miRNA (43%), followed by Y RNA (17%), antisense RNA (10%) and lincRNA (8%). Subsequent in silico analyzes allowed us to select highly expressed and coherent miRNAs among the 4 replicates of LP analyzed and corral, thanks to gene ontology databases, to known miRNAs involved in endothelial activation, vasculogenesis and NADPH oxidation. So to obtain a list of 5 miRNA: miR-320a; miR-148b-3p; miR-25-3p; miR-26b-5p; miR-152-3p. Furthermore, we discovered that miR126, also known as angiomiRNA and selectively expressed on endothelial cells15, was among the first 40 miRNA expressed in LP. Consequently, we validated its presence in our LP by finding miR 126 levels Real Time PCR which increase proportionally to the quantity of LP tested (p = 0.043).