LIPIDOMIC CHARACTERIZATION OF CELL SECRETOME COMBINED WITH THE STUDY OF SELECTED BIOACTIVE LIPIDS IN AN OSTEOARTHRITIS MODEL

Lipidomics is the large-scale identification and quantification of lipids and their variations in various physio-pathological conditions. This recent “-omic” area is rapidly developing as an emerging field, and it sustains the advancement of current knowledge in the realm of lipid biology. Indeed, if lipids are conventionally considered as structural components of cellular membranes, they have emerged recently as key players in a wide range of biological processes such as signaling events and trafficking. However, their precise physio- pathological function is still poorly understood and a comprehensive characterization of lipids in each cell type can provide pivotal information to better understand the roles exercised by these compounds in several biological phenomena. In this project, the attention was focused on the roles of specific bioactive lipids families, as polyunsaturated fatty acids (PUFA), PUFA-derived molecules (known as eicosanoids), endocannabinoids (EC) and EC- related compounds N-acylethanolamines (NAE), and their involvement in the mesenchymal stem/stromal cells (MSC)-related inflammatory context. Indeed, MSC have attracted much attention for their capacity in regulating inflammation and reparative roles. Since it is known that MSC therapeutic action largely depends on paracrine mechanisms, the scientific interest has shifted to the study of their secretome, namely conditioned medium (CM). The therapeutic potential of the CM, derived from MSC in disparate medical fields (from immunology to orthopedics), has been extensively endorsed by in vitro and in vivo evidence. However, lipidomics knowledge in the MSC field and their secretome remain rather limited and a broadly characterization of lipids is still missing. In recent years, our group have investigated and characterized the adipose-derived stem/stromal cells (ASC)-CM content, in terms of both soluble factors and vesicular components (extracellular vesicles – EV), through different approaches (i.e.Raman spectroscopy and proteomic analysis), highlighting substantial differences in the total lipid content and inflammatory factors between CM and EV. In this project, two advanced mass spectrometry (MS) analytical methods for the absolute quantification of 32 bioactive lipid molecules - belonging to PUFA, eicosanoids and EC/NAE families and highly implicated in the inflammatory scenario - were developed and validated according to the Food and Drug Administration guidelines. A double liquid-liquid extraction step was set up, starting from a single sample, in different mixture of organic solvents, to cover the broad polarity range of selected analytes. Linearity was observed in the range of 0.1-2.5ng/ml (1-25ng/ml for PUFA) with a r2>0.991. Regarding precision and accuracy, the methods showed good performance in terms of both repeatability and reproducibility, showing CV values below 15%, while lowest limits of quantitation (LLOQ) were <0.1ng/ml. Extraction recovery, matrix effect and post-extraction stability values were within acceptance limits. The analytical methods were applied to the secretome (in terms of CM and EV) derived from MSC, isolated either from human bone marrow (BMSC) or adipose tissue (ASC), and from dermal fibroblasts (DF), since they share common characteristics with MSC, and they have started to be considered a convenient alternative. A total of 9 lipid molecules in MSC and DF-derived CM and EV samples were quantified. In detail, the presence of 5 EC/NAE (including 2-arachidonoilglycerol -2AG- and N- palmitoylethanolamide -PEA) and 3 PUFA were reported in both preparations. Prostaglandin E2 (PGE2) was found only in CM samples. An enrichment in lipid content were displayed in almost all MSC-CM and DF-CM rather than coupled MSC- and DF-derived EV. Then, the biological function of 2 lipid compounds quantified in ASC-CM - 2AG and PEA – was assessed in a well-established in vitro model of osteoarthritis (OA), based on the administration of 10ng/ml tissue necrosis factor alfa (TNFα) to human primary articular chondrocytes (CH). The CH were isolated by femoral head of 14 patients and treated with TNFα alone or in association with 2AG and PEA at observed ASC-CM concentrations (0.05 and 0.02 ng/mL per million cells, respectively). The expression of both CB1 and CB2 on primary articular CH was confirmed by western blot (WB) analysis. TNFα increases the extracellular concentration of the inflammatory lipid prostaglandin E2 (PGE2) analyzed by MS and an additional increment was highlighted when CH were treated with the combination of TNFα and 2AG. In contrast, PEA showed a protective effect on the PGE2 release, providing a downmodulation up to the levels quantified in inactivated CH. Accordingly, TNFα increased the expression of cyclooxygenase 2 (COX2) especially when in association with 2AG, while PEA partly blunts TNFα-induced COX2. In parallel, also the nitric oxide (NO) production in CH cell media was significantly enhanced under TNFα as well as TNFα+2AG treatments, while PEA was able to blunt NO release. Finally, the targeted MS analysis, using previously described analytical methods, showed a significant decrease in PUFA induced by TNFα, suggesting a possible implication in PUFA-derived mediators effect. Our results allowed a first partial lipids characterization of MSC and DF secretome, demonstrating a specific lipid profile for CM and EV and supporting a possible implication of some bioactive lipids molecules in the OA scenario and in the future use of this cell-free products therapeutic approach.