Title Proteomska analiza bioloških tekućina pacijenata s progresivnom osificirajućom fibrodisplazijom i pacijenata s koštanim prijelomima Title (english) Proteomic analysis of biological fluids of fibrodysplasia ossificans progressiva patients and patients with bone fractures Author Stela Hrkač Mentor Lovorka Grgurević (mentor)Committee member Fran Borovečki (predsjednik povjerenstva)Committee member Zdravko Petanjek (član povjerenstva)Committee member Lovorka Grgurević (član povjerenstva)Granter University of Zagreb Defense date and country 2021-07-16, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Scientific / art field, BIOMEDICINE AND HEALTHCARE Abstract UVOD: Progresivna osificirajuća fibrodisplazija (FOP) rijetka je genetska bolest koju karakteriziraju skeletalne malformacije i epizode egzacerbacije (engl. flare-ups) koje uzrokoju progresivnu heterotopnu osifikaciju (HO) mišića i vezivnog tkiva. Koštani prijelom je prekid kontinuiteta strukture kosti, nakon kojeg slijedi složeni proces koštanog cijeljenja u koji su uključeni brojni biološki procesi. Proteomska analiza bioloških tekućina pacijenata s FOP-om i koštanim prijelomima omogućuje njihovu usporedbu i potencijalno bolje razumijevanje bioloških procesa uključenih u HO i fiziološko koštano cijeljenje. ----- CILJ: Cilj ovog istraživanja bio je analizirati citokine iz krvne plazme i proteine izolirane iz ekstracelularnih vezikula (EV) kako bi se usporedila relativna ekspresija citokina i funkcija proteina iz EV-a koji su uključeni u fiziološko koštano cijeljenje (poslije prijeloma) i patološku HO u FOP-u. ----- ISPITANICI I METODE: U istraživanje su uključeni: ispitanici s FOP-om (N=3), s prijelomom duge kosti (N=20) i kontrolna skupina zdravih ispitanika (N=10). Krvni uzorci vađeni su u FOP i kontrolnoj skupini u jednoj vremenskoj točki, dok su ispitanicima s prijelomom vađeni u dvije vremenske točke – prva vremenska točka bila je između 1. i 6., a druga između 7. i 21. dana poslije prijeloma. Analizirana je relativna ekspresija 23 citokina plazme koristeći panel protutijela na ljudske citokine te su rezultati prikazani kao faktor promjene (engl. fold change) u usporedbi s kontrolnom skupinom. EV iz plazme izolirane su ultracentrifugiranjem i analizirane tekućinskom kromatografijom-spektrometrijom masa (engl. liquid chromatography-mass spectrometry, LC-MS) te bioinformatičkom analizom. ----- REZULTATI: Uočena je izražena razlika u relativnoj ekspresiji citokina između analiziranih skupina. Rezultati FOP pacijenta s flare-up-om pokazuju zamjetan porast relativne ekspresije 21 citokina, dok su relativne ekspresije IL-3 i IL-8 povišene kod svih FOP ispitanika. Citokini MCP-1 i RANTES pokazuju najveći porast u svim ispitivanim skupinama. Analiza EV proteina iz skupine s koštanim prijelomom pokazuje najveći relativni udio proteina uključenih u biološke procese i signalne puteve (BMP, TGF-β, Wnt, VEGF, PDGF, IGF-1 i citokinima posredovani putevi) koji su važni za koštano remodeliranje i cijeljenje, u usporedbi s ostalim skupinama. ----- ZAKLJUČAK: Nađene su izražene razlike u ekspresiji citokina između FOP pacijenata (s flare-up-om i bez) i pacijenata s koštanim prijelomom, kao i funkcionalne razlike EV proteina između ispitivanih skupina. Proteinski sadržaj EV izoliranih od ispitanika s FOP-om ne pokazuje odstupanja od ostalih skupina koja bi upućivala na njihovu važnost u patofizologiji FOP-a. Istraživanja poput ovog omogućuju bolje razumijevanje patofiziologije FOP-a i koštanog remodeliranja koje je ključno za razvoj novih terapijskih opcija za ovu izrazito tešku bolest.
Abstract (english) INTRODUCTION: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease which is characterized by skeletal malformations and episodic disease flare-ups which cause progressive heterotopic ossification (HO) of skeletal muscles and connective tissue. A bone fracture is a brake in the continuity of bone structure, which is followed by the compex process of bone healing, which involves numerous biological processes. Proteomic analysis of biological fluids from patients with FOP and bone fractures enables their comparison and a possible better understanding of biological processes involved in HO and physiological bone healing. ----- AIM: The aim of this study was to analyse plasma derived cytokines and proteins isolated from extracellular vesicles (EVs) in order to compare cytokine expression and functions of EV proteins which are involved in physiological bone healing (after fracture) and pathologic HO in FOP. ----- PARTICIPANTS AND METHODS: The study included participants with FOP (N=3), patients with long bone fractures (N=20) and a healthy control group (N=10). Plasma samples used were obtained from FOP patients and healthy participants at a single timepoint and at two timepoints in participants with bone fractures – the first timepoint was from day 1 to day 6 and the second was from day 7 do day 21 after fracture occurrence. Relative expression of 23 plasma cytokines was analysed using a human cytokine antibody array and results were expressed as fold changes compared to the control group. Plasma EVs where isolated by ultracentrifugation and their protein content was analysed using liquid chromatography-mass spectrometry (LC-MS) and bioinformatics. ----- RESULTS: There was a marked difference in relative cytokine expression between the studied groups. The FOP patient with a flare-up showed marked increase in 21 cytokines, whereas a change of IL-3 and IL-8 was observed in all FOP patients. Cytokines MCP-1 and RANTES show the biggest fold change in all studied groups. EV proteins from the fracture group showed the biggest relative ratio of proteins involved in biological processes and signaling pathways (BMP, TGF-β, Wnt, VEGF, PDGF, IGF-1 and cytokine mediated pathways) important for bone remodeling and healing compared to the other groups. ----- CONCLUSION: There are marked differences in cytokine expression between FOP patients (with and without flare-ups) and patients with bone fractures, as well as functional differences of EV proteins between the fracture and control group. The EV protein content of FOP patients did not show differences from the other groups which would indicate their importance in FOP pathophysiology. Studies like this grant further insight into FOP pathophysiology and bone remodeling which are needed to develop treatment options for this debilitating disease.
Keywords
ekstracelularne vezikule
citokini
FOP
cijeljenje koštanog prijeloma
Keywords (english)
extracellular vesicles
cytokines
FOP
fracture healing
Language croatian URN:NBN urn:nbn:hr:105:867071 Study programme Title: Medicine Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2022-02-09 11:38:57
