Functional secretome analysis reveals Annexin-A1 as important paracrine factor derived from fetal mesenchymal stem cells in hepatic regeneration

Abstract

Background: Human mesenchymal stem/stromal cells (MSCs) and their secreted molecules exert beneficial effects in injured tissues by promoting tissue regeneration and angiogenesis and by inhibiting inflammation and fibrosis. We have previously demonstrated that the therapeutic activity of fetal MSCs derived from amniotic fluid (AF-MSCs) and their hepatic progenitor-like cells (HPL) is mediated by paracrine effects in a mouse model of acute hepatic failure (AHF).

Methods: Herein, we have combined proteomic profiling of the AF-MSCs and HPL cell secretome with ex vivo and in vivo functional studies to identify specific soluble factors, which underpin tissue regeneration in AHF.

Findings: The anti-inflammatory molecule Annexin-A1 (ANXA1) was detected at high levels in both AF-MSC and HPL cell secretome. Further functional analyses revealed that the shRNA-mediated knock-down of ANXA1 in MSCs (shANXA1-MSCs) decreased their proliferative, clonogenic and migratory potential, as well as their ability to differentiate into HPL cells. Liver progenitors (oval cells) from AHF mice displayed reduced proliferation when cultured ex vivo in the presence of conditioned media from shANXA1-MSCs compared to control MSCs secretome. Intra-hepatic delivery of conditioned media from control MSCs but not shANXA1-MSCs reduced liver damage and circulating levels of pro-inflammatory cytokines in AHF.

Interpretation: Collectively, our study uncovers secreted Annexin-A1 as a novel effector of MSCs in liver regeneration and further underscores the potential of cell-free therapeutic strategies for liver diseases. FUND: Fondation Santé, GILEAD Asklipeios Grant, Fellowships of Excellence - Siemens, IKY, Reinforcement of Postdoctoral Researchers, IKY.

Keywords: Acute hepatic failure; Annexin-A1; Hepatic progenitors; MSCs; Secretome.

Fig. 1

Analysis and classification of the identified proteins in AF-MSCs and HPL cells. (a) Venn diagrams showing the high percentage of overlapping proteins among the secretome of the two groups (AF-MSCs vs HPL). The information shown in the Venn diagram has been derived after the frequency threshold application (only peptides identified in 3/4 samples in at least one group were considered for further analysis) in the total number of identifications obtained in the two groups of samples. (b) Classification of the identified proteins. The high percentage (61%) of secreted proteins (classically and non-classically secreted) obtained in both groups, indicates the efficiency of the utilized protocol for the enrichment of secreted proteins.

Fig. 2

Comparative proteomic analysis between the HPL and AF-MSC secretome. (a) (i) Pie chart illustrates the percentage of the proteins that were common and differentially expressed (fold change<0.67 or > 1.5, p < 0.05) between HPL cells and AF-MSCs, as well as of the proteins that were uniquely expressed per category. (ii) Bar graph representing the percentage of the 219 differentially expressed proteins that were statistically significant upregulated or downregulated in the secretome of HPL cells versus AF-MSCs. (b) The heat map illustrates the expression level changes of the 219 differentially expressed proteins among the HPL and the AF-MSC CM samples. Green color indicates up-regulation whereas red color indicates down regulation of the reported proteins. (c) Bar graph representing the relative expression levels of selected secreted proteins in HPL cells vs AF-MSCs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Lentiviral mediated RNAi knockdown of ANXA1 reduces the molecule's expression in AF-MSCs and their secretome. (a) Western blot analysis of ANXA1 in AF-MSCs, AF-MSC shscramble and AF-MSC shANXA1. Quantification was performed using the Quantity One software and the results were normalized to GAPDH positive control and then to AF-MSCs. (b) ELISA analysis of ANXA1 expression levels in shscramble-CM and shANXA1-CM. Data presented as mean ± SD of at least three independent experiments and analyzed by Student's test (****p < 0.0001). (c) Western blot analyses of the secreted ANXA1 in conditioned media (CM) derived from AF-MSC-shANXA1 and AF-MSC-shscramble. The densitometry analysis of the results was performed using the Quantity One software. Data presented as mean ± SD of two independent experiments and analyzed by Student's test (*p < 0.05).

Fig. 4

Proliferation, apoptosis, clonogenic potential and migratory capacity of AF-MSCs, after knock down of ANXA1 expression. (a) Comparative analysis of the proliferation rate of AF-MSC-shANXA1 versus AF-MSC-shscramble and AF-MSC during 7 days in culture. The values presented are the mean ± SD of three independent experiments (*, p < 0.05; **, p < 0.01, Student's t-test). (b) Apoptosis in AF-MSCs, AF-MSC-shscramble and AF-MSC-shANXA1 was examined by FACS analysis of Annexin V staining and 7AAD was used to assess late apoptosis/necrosis. (c) The clonogenic potential of AF-MSCs, AF-MSC-shscramble and AF-MSC-shANXA1 was determined by CFU-F assay. (i): The mean numbers ± SD of CFU-F per 100 cells in a 14-day clonogenic assay are presented. The values represent the mean ± SD of three independent experiments (*, p < 0.05, ***, p < 0.001; Student's t-test). (ii): Representative images of the colonies formed. (d) (i) Representative diagram of the migratory potential of AF-MSCs, AF-MSC-shscramble and AF-MSC-shANXA1 to fibronectin. The values presented are the mean ± SD of three independent experiments (**, p < 0.01, Student's t-test). (ii) Representative microscopy images of the migrated AF-MSCs, AF-MSC-shscramble and AF-MSC-shANXA1. Original magnifications, (original magnification x20. scale bar: 100 μm). (e) The differentiation potential of AF-MSCs, AF-MSC-shscramble and AF-MSC-shANXA1 into HPL cells, was determined by periodic acid Schiff staining assay. (i): Quantification analysis of PAS staining. The values represent the mean ± SD of three independent experiments (*, p < 0.05; ***, p < 0.001; Student's t-test). (ii): Representative images of HPL, HPL-shscramble and HPL-shANXA1, after PAS staining (original magnification x20, scale bar: 100 μm).

Fig. 5

Ant-inflammatory efficacy of secreted ANXA1 in AHF mouse model. (a) H&E staining of liver sections from control and AHF (CCl₄-induced) mice, as well as AHF mice receiving CM from AF-MSC-shscramble and AF-MSC-shANXA1 cultures. (Original magnification x20, scale bar: 100 μm). Arrows show necrotic areas. (b) Serum AST and ALT levels were measured in heathy mice (control), AHF mice, AHF mice that were administered CM from AF-MSC-shscramble or CM from AF-MSC-ANXA1 cultures. Data presented as the mean ± SD of five mice per group and analyzed by Student's t-test. P-values were estimated compared to AHF mice (*, p < 0.05; **, p < 0.01, Student's t-test). (c) Analysis of mouse serum cytokine levels, after treatment with shANXA1- or shscramble-CM. Mouse serum levels for IFN-γ, TNF and IL-10 from healthy mice, AHF mice or AHF mice that received shANXA1- and shscramble-CM, 24 h after administration. Each assay was performed in duplicates. Data are presented as the mean ± SD for at least 3 independent experiments and were analyzed by Student's t-test method (*, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 6

Determination of the biological activity of ANXA1 on mouse oval cell proliferation after 3 days in culture. Comparative analysis of the growth rates of oval cells after treatment with DMEM supplemented with 0.5% FBS as negative control, DMEM supplemented with 20% FBS as positive control, CM from parental AF-MSCs, AF-MSC-shscramble or AF-MSC-shANXA1 cultures, during 3 days in culture. Data are presented as the mean ± SD of three independent experiments (**, p < 0.01; Student's t-test).

Supplementary Fig. S1

Differentiation of AF-MSCs into HPL cells in vitro. (a) Representative microscopy images of AF-MSCs and HPL cells. Original magnifications 10×. (b) Semi-quantitative PCR analysis for a-fetoprotein, albumin and tyrosine aminotransferase expression levels in AF-MSCs and HPL cells (*, p < 0.05, **, p < 0.01, Student's t-test).

Supplementary Fig. S2

Predicted impact on the canonical signaling pathways involving (a): ANXA1 (lipocortin) and (b): IFNγ and TNF. An overall impact in the immune response is depicted. The pathway was generated by Ingenuity pathway Analysis software (IPA).