1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.
2Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of California San Diego, 3020 Children's Way, MC 5030, San Diego, CA, 92103-8450, USA.
3Genomics and Microarray Core, University of Colorado Denver, Aurora, CO, USA.
4Department of Biomedical Informatics, University of California San Diego, La Jolla, CA, USA.
5Gilead Sciences, Inc., Foster City, CA, USA.
6Inova Fairfax Hospital, Falls Church, VA, USA.
7Pinnacle Clinical Research, San Antonio, TX, USA.
8Virginia Commonwealth University, Richmond, VA, USA.
9Department of Visceral Surgery and Medicine and Department for Biomedical Research, Inselspital, University of Bern, Bern, Switzerland.
10Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS)-CIBERehd, University of Barcelona, Barcelona, Spain.
11Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of California San Diego, 3020 Children's Way, MC 5030, San Diego, CA, 92103-8450, USA. firstname.lastname@example.org.
Primary sclerosing cholangitis (PSC) is an idiopathic and heterogenous cholestatic liver disease characterized by chronic inflammation and fibrosis of the biliary tree. Currently, no effective therapies are available for this condition, whose incidence is rising. At present, specificity and sensitivity of current serum markers used to diagnose PSC are limited and often unreliable. In this study, we characterize circulating extracellular vesicles and provide supporting data on their potential use as novel surrogate biomarkers for PSC. EVs are membrane surrounded structures, 100-1000 nm in size, released by cells under various conditions and which carry a variety of bioactive molecules, including small non-coding RNAs, lipids and proteins. In recent years, a large body of evidence has pointed to diagnostic implications of EVs and relative cargo in various human diseases. We isolated EVs from serum of well-characterized patients with PSC or control subjects by differential centrifugation and size-exclusion chromatography. A complete characterization identified elevated levels of circulating EVs in PSC patients compared to healthy control subjects (2000 vs. 500 Calcein-FITC + EVs/μL). Tissue and cell specificity of circulating EVs was assessed by identification of liver-specific markers and cholangiocyte marker CK-19. Further molecular characterization identified 282 proteins that were differentially regulated in PSC-derived compared to healthy control-EVs. Among those, IL-13Ra1 was the most significantly and differentially expressed protein in PSC-derived EVs and correlated with the degree of liver fibrosis. In addition to protein profiling, we performed a miRNA-sequencing analysis which identified 11 among established, liver-specific (e.g., miR-122 and miR-192) and novel miRNAs. One of the newly identified miRNAs, miR-4645-3p, was significantly up-regulated fourfold in PSC-derived EVs compared to circulating EVs isolated from healthy controls. This study provides supporting evidence of the potential role of circulating EVs and associated protein and miRNA cargo as surrogate noninvasive and reliable biomarker for PSC.