Proteome Investigation of Rat Lungs Subjected to Ex Vivo Perfusion (EVLP)

Abstract

Ex vivo lung perfusion (EVLP) is an emerging procedure that allows organ preservation, assessment and reconditioning, increasing the number of marginal donor lungs for transplantation. However, physiological and airflow measurements are unable to unveil the molecular mechanisms responsible of EVLP beneficial effects on lung graft and monitor the proper course of the treatment. Thus, it is urgent to find specific biomarkers that possess these requirements but also accurate and reliable techniques that identify them. The purpose of this study is to give an overview on the potentiality of shotgun proteomic platforms in characterizing the status and the evolution of metabolic pathways during EVLP in order to find new potential EVLP-related biomarkers. A nanoLC-MS/MS system was applied to the proteome analysis of lung tissues from an optimized rat model in three experimental groups: native, pre- and post-EVLP. Technical and biological repeatability were evaluated and, together with clustering analysis, underlined the good quality of data produced. In-house software and bioinformatics tools allowed the label-free extraction of differentially expressed proteins among the three examined conditions and the network visualization of the pathways mainly involved. These promising findings encourage further proteomic investigations of the molecular mechanisms behind EVLP procedure.

Human Red Blood Cells as Oxygen Carriers to Improve Ex-Situ Liver Perfusion in a Rat Model

“…The perfusion system (Figure 2) consisted of a customized circuit derived from the isolated lung perfusion system (Hugo Sachs Elektronik, Harvard Apparatus, March-Hugstetten, Germany) implemented and described by our laboratory [21,22,23,24]. It consisted of a reusable heated glass reservoir, a heated bubble trap, a circulating tube derived by an infusion system, an octagonal peristaltic pump, a polystyrene lid, and a single-use artificial lung (Supplementary Tables S3 and S4).…”

“…The concentration of total HA in outflow perfusate was determined by ELISA using a commercially available kit (sensitivity, 0.068 ng/mL; low standard, 0.625 ng/mL; R&D Systems, Minneapolis, MN, USA). The procedure was validated in preliminary experiments by using HA preparations with a known concentration and known average molecular mass (Select-HA HiLadder and LoLadder; Hyalose, Oklahoma City, OK, USA) [21,22,23,24].…”

Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers

“…A spectral counts (SpCs) based quantitative approach, although sometimes it is less accurate that other methods, is more easy to apply, and it is enough for statistical analysis if it is applied to evaluate differential expression comparing the same proteins and its value [ 36 ], combined with statistical analysis, such as by t-test, too [ 37 ]; proteins selected by both LDA and MAProMA (Multidimensional Algorithm Protein Map) were evaluated by hierarchical clustering [ 38 , 39 ], applying Ward’s method and the Euclidean distance metric. LDA using common covariance matrix for all stratified groups and Mahalanobis distance and hierarchical clustering were performed by JMP 5.1 software (SAS Institute, Cary, NC, USA company, city, state abbrev if USA, country)).…”

Procurement and ex-situ perfusion of isolated slaughterhouse-derived livers as a model of donors after circulatory death

“…Based on our experience in clinical and preclinical ex-situ perfusions (Bassani et al, 2016;Lonati et al, 2018;Roffia et al, 2018;Dondossola et al, 2018), we designed a study aiming to improve and standardize an ethical and cost-effective procurement/perfusion model using slaughterhouse-derived liver grafts. This model could help characterization of liver metabolism and viability during ex-situ perfusion and provide data that could be translated effectively to clinical settings.…”