SILAC‐based quantitative proteomic analysis of secretome between activated and reverted hepatic stellate cells

Abstract

Activated hepatic stellate cell (HSC) is the main myofibroblast cell in the liver fibrosis (LF). An important characteristic of the recovery of LF is not only the apoptosis of activated HSCs but also reversal of myofibroblast-like phenotype to a quiescent-like phenotype. Understanding the changes of secreted proteins in the reversion of activated HSCs may provide the broader view of cellular regulatory networks and discover candidate markers or targets for therapeutic strategies of LF. In this study, stable isotope labeling with amino acids (SILAC) combined with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT MS) was performed on in vitro activated HSCs and reverted HSCs to obtain a proteomic view of secretory proteins. In total, 330 proteins showed significant differences in reverted HSCs. Among these, 109 upregulated proteins were mainly involved in amino acid metabolism pathway and glucose metabolism pathway using GeneGO/MetaCore software, while 221 downregulated proteins are closely associated with HSCs activation, such as cytoskeleton remodeling, chemokines, and cell adhesion. Additionally, a set of novel proteins associated with HSCs activation and reversion were validated by Western blotting in the cell secretion and in the sera of LF, including vitronectin, laminin beta 1, and ubiquitin conjugation factor E4B. Our study provided the valuable insight into the mechanisms in the reversion of activated HSCs and identified some potential biomarkers of LF in clinical studies. All MS data have been deposited in the ProteomeXchange with identifier PXD000773 (http://proteomecentral.proteomexchange.org/dataset/PXD000773).

Reversion of in vivo fibrogenesis by novel chromone scaffolds

“…This sort of explicit dedifferentiation of myofibroblasts into a quiescent state, which can also be trans-differentiated into myofibroblasts by TGF-β, has not been previously described. However, intermediate quiescent cells are frequently observed during recovery from human liver fibrosis [[55], [56], [57], [58], [59]]. In the case of sequential stimulation, although reversion to a normal ONGHEPA1-like morphology was evident, 81% of the genes that were differentially regulated in cells treated with TGF-β and TGF-β plus eupatilin were the same.…”

SILAC-based quantitative proteomic analysis of secretome of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus

“…Recently, SILAC technology with its convenient, accurate, and inexpensive advantages has become popular and been widely used for secretome analysis . In our SILAC‐based PRRSV‐infected Marc‐145 cells secretome study, several caveats exist, especially the possibility of nondirect responses to viral infection.…”

A simple in silico strategy identifies candidate biomarkers for the diagnosis of liver fibrosis in morbidly obese subjects

“…Central nodes correspond to the proteins involved in the fibrotic process, and their relevant information is reported in Table (Supporting information). In summary, the selected proteins, used to obtain each biological network, can be categorized into three different groups based on their role in NAFLD: (a) those reported as putative markers for the progression of the disease, such as CK‐18, adipocyte fatty acid binding protein (AFABP), fibroblast growth factor 21 (FGF21), insulin‐like growth factor‐binding protein 3 (IBP‐3) and lymphocyte cytosolic protein 1 (LCP‐1); (b) those reported to be involved in phenotype modulation (activation/reversion) of hepatic stellate cells (HSC), such as galectin‐1 (GALS1), ubiquitin conjugation factor E4B (UBE4B), vitronectin (VTN) and alpha smooth muscle actin (α‐SMA), laminin subunit beta 1 (LAMB1) and (c) those involved in extracellular matrix remodelling such as osteopontin (SPP1), collagen alpha‐1 (III) chain (COL1A3), matrix‐metalloproteinase‐2 (MMP2) and tissue inhibitor of metalloproteinase‐2 (TIMP2) …”