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. 2022 Oct 30;5(1):1155.
doi: 10.1038/s42003-022-04123-z.

Tracking matricellular protein SPARC in extracellular vesicles as a non-destructive method to evaluate lipid-based antifibrotic treatments

Cristina Zivko  1   2   3 , Kathrin Fuhrmann  3 , Gregor Fuhrmann  4   5 , Paola Luciani  6   7
Affiliations

Tracking matricellular protein SPARC in extracellular vesicles as a non-destructive method to evaluate lipid-based antifibrotic treatments

Cristina Zivko et al. Commun Biol. .

Abstract

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis could expedite the development of effective treatments and noninvasive diagnosis for liver fibrosis. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers as potential alternative to liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2, and we investigated their biological effect onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. We mined our proteomic data for EV-associated proteins whose expression correlated with HSC treatment, choosing the matricellular protein SPARC as proof-of-concept for the feasibility of fluorescence nanoparticle-tracking analysis to determine an EV-based HSCs' fibrogenic phenotype. We thus used EVs to directly evaluate the efficacy of treatment with S80, a polyenylphosphatidylcholines-rich lipid, finding that S80 reduces the relative presence of SPARC-positive EVs. Here we correlated the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. Along with providing insights into polyenylphosphatidylcholines treatments, our findings pave the way for precise and less invasive diagnostic analyses of hepatic fibrogenesis.

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Conflict of interest statement

No private study sponsors had any involvement in the study design, data collection, or interpretation of data presented in this manuscript. P.L. declares the following competing interests: she has consulted Lipoid GmbH and Sanofi-Aventis Deutschland and received research grants from Lipoid, Sanofi-Aventis Deutschland and DSM Nutritional Products Ltd. C.Z., K.F., and G.F. declare no competing interests.

Figures

Fig. 1
Fig. 1. Workflow of EV isolation and purification protocols from LX-2 cells.
Schematic overview of the protocols to obtain highly purified EVs from LX-2 cells subjected to different treatments.
Fig. 2
Fig. 2. Isolation and characterization of EVs from LX-2 cells.
a Size distribution profiles of EVs isolated from differently treated cells (mean ± SD, n = 3). b Quantile subtraction of the yields. c, d SEM (c) and cryo-TEM (d) images of EVs isolated from untreated cells. e–g Protein content and vesicle number in the collected SEC-fractions obtained from untreated (e), quiescent (f), and perpetuated (g) LX-2 (mean ± SD, n = 3). h–j Representative EAF4 fractograms of EV collected from untreated (h), quiescent (i), and perpetuated (j).
Fig. 3
Fig. 3. Treatment of fresh LX-2 with EV-pellets from previously treated cells.
a Representative images of ORO staining in fluorescence (seen as red spots; nuclei stained with blue DAPI) of differently treated cells after thresholding (see Fig. S3). b Quantitative analysis of stained lipid droplets, whereby the fluorescent area (correlating to a quiescent-like status) was normalized to cell count, and bars corresponding to treatment using EV-pellets are in purple (mean ± SD, n = 3). For more results, see Figs. S4–7.
Fig. 4
Fig. 4. Main proteomic findings from SEC-purified EVs originating from differently treated LX-2.
a, b Principal component analysis and hierarchal clustering (also in Fig. S19) showing similarity degrees between biologically independent samples undergoing the same treatments, and differences between treatment groups. c Summary of the number of persistent proteins found in each treatment group. d Summary of the number of persistent proteins shared across treatment groups, excluding 1931 shared by all. e Panel resulting from cross-referencing the proteins consistently found in all treatment groups and the proteins that were under or over-expressed in the least one direct comparison after Welch’s t-test. For ease of comparison, a simple, normalized recovery score was developed by adding the LFQ values of every protein for each treatment condition and normalizing it to the sum of all of them, so that the panel could be visually inspected as a heat map. f Venn diagram depicting the number of identified proteins persistently found in S80 and ROL/PA groups as opposed to TGF and vice versa; numbers in parenthesis indicate proteins within those subsets which were either tissue-specific, membrane-bound, and/or secreted. (Supplementary Data 1).
Fig. 5
Fig. 5. Main proteomic findings from AF4-purified EVs originating from differently treated LX-2.
a–c Principal component analysis for samples originating from AF4-peak 1 (a), peak 2 (b), and for the two peaks combined (c). d Hierarchal clustering for biologically independent AF4-peak 2 samples undergoing the same treatments. e Summary of the number of persistent proteins found in each treatment group. f Summary of the number of persistent proteins shared across treatment groups. g Venn diagram comparisons of SEC and AF4-purified samples: for every treatment group, the proteins found in the SEC fraction are compared to those found in AF4-peak 1 (AF4 p1) and AF4-peak 2 (AF4 p2).
Fig. 6
Fig. 6. Feasibility of f-NTA for the detection of physiologically relevant proteins.
a SEC-purified EVs stained with unspecific membrane dye PKH67 by systematically increasing dye concentrations. b SEC-purified EVs incubation with varying amounts of AF488-CD81 and for different times at 24 °C. c, d EV-containing pellets incubated with AF488-CD81 prior to SEC and detected after purification (c) and comparison with additional incubation with AF488-CD9 (d). e Direct comparison of CD9 detection by f-NTA and FACS using EVs from differently treated LX-2, including isotype control (IC). f Detection of SPARC and CD81 on EVs isolated from differently treated LX-2 cells following the optimized f-NTA protocol (mean ± SD, n = 3). P values (p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****)) were determined by one-way ANOVA on ranks and Tukey’s multiple comparison.
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References

    1. Trefts E, Gannon M, Wasserman DH. The liver. Curr. Biol. 2017;27:R1147–R1151. doi: 10.1016/j.cub.2017.09.019. - DOI - PMC - PubMed
    1. Zivko C, Fuhrmann G, Luciani P. Liver-derived extracellular vesicles: a cell by cell overview to isolation and characterization practices. Biochim. Biophys. Acta Gen. Subj. 2020;1865:129559. doi: 10.1016/j.bbagen.2020.129559. - DOI - PubMed
    1. Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Burden of liver diseases in the world. J. Hepatol. 2019;70:151–171. doi: 10.1016/j.jhep.2018.09.014. - DOI - PubMed
    1. Byass P. The global burden of liver disease: a challenge for methods and for public health. BMC Med. 2014;12:159. doi: 10.1186/s12916-014-0159-5. - DOI - PMC - PubMed
    1. Pimpin L, et al. Burden of liver disease in Europe: epidemiology and analysis of risk factors to identify prevention policies. J. Hepatol. 2018;69:718–735. doi: 10.1016/j.jhep.2018.05.011. - DOI - PubMed

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