Fibrogenic signals persist in DAA-treated HCV patients after sustained virological response
Authors: Claudia Montaldo, Michela Terri, Veronica Riccioni, Cecilia Battistelli, Veronica Bordoni, Gianpiero D’Offizi, Maria Giulia Prado, Flavia Trionfetti, Tiziana Vescovo, Eleonora Tartaglia, Raffaele Strippoli, Chiara Agrati, Marco Tripodi
Journal: Journal of Hepatology (2021)

•HCV T0/T6 EVs have been functionally and structurally compared to HD EVs.•HD EVs ameliorate LX2 cells' fibrogenic phenotype while HCV T0/T6 EVs do not.•HCV T0/T6 EVs contain lower levels of antifibrogenic miRNAs compared to HD EVs.•HCV T0/T6 EVs' cargo is enriched in profibrogenic proteins (e.g. DIAPH1).•SVR does not restore antifibrogenic cargo and activity in HCV EVs. Background & AimsPatients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary.MethodsExtracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot.ResultsOn the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p <0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (Log2 fold change of 4.4).ConclusionsTaken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR.Lay summaryDirect-acting antivirals lead to virological cure in the majority of patients with chronic hepatitis C virus infection. However, the risk of liver disease progression or complications in patients with fibrosis and cirrhosis remains in some patients even after virological cure. Herein, we show that extracellular vesicle modifications could be linked to long-term liver disease progression in patients who have achieved virological cure; these modifications could potentially be used as biomarkers or treatment targets in such patients. Patients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary. Extracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot. On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p <0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (Log2 fold change of 4.4). Taken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR.