Following the SPARC: tracking Secreted Protein Acidic and Rich in Cysteine associated with LX-2´s extracellular vesicles as a non-destructive modality to evaluate lipid-based antifibrotic treatments

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis, a highly dynamic and active process ultimately responsible for liver failure if left untreated, could expedite the development of effective treatments and noninvasive diagnostic modalities for this often silent pathology. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers that might become a viable alternative to invasive 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 in vitro, and we investigated the biological effect they exert onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. Electrical/asymmetric flow field-flow fractionation (EAF4) revealed EV subpopulations with different physicochemical behaviors. Proteomic data from our samples was mined for EV-associated proteins whose expression correlated with HSC treatment. Consequently, we chose the secreted protein acidic and cysteine rich (SPARC), a matricellular protein previously reported to be upregulated in activated HSCs, as a proof-of-concept protein to explore the feasibility of using fluorescence nanoparticle tracking analysis as a non-destructive tool for the determination of HSCs’ fibrogenic phenotype based on EVs. We could thus use EVs to directly evaluate the efficacy of treatment with S80, a lipid rich (>75 %) in polyenylphosphatidylcholines (PPC). We found that PPC-rich S80 reduces the relative presence of SPARC-positive EVs. For the first time, we could correlate the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. In addition to providing novel insights into PPC treatments, our findings pave the way for more precise and less invasive diagnostic analyses of hepatic fibrogenesis.

Perilipin 5 Ameliorates Hepatic Stellate Cell Activation via SMAD2/3 and SNAIL Signaling Pathways and Suppresses STAT3 Activation

Comprehending the molecular mechanisms underlying hepatic fibrogenesis is essential to the development of treatment. The hallmark of hepatic fibrosis is the development and deposition of excess fibrous connective tissue forcing tissue remodeling. Hepatic stellate cells (HSC) play a major role in the pathogenesis of liver fibrosis. Their activation via the transforming growth factor-β1 (TGF-β1) as a key mediator is considered the crucial event in the pathophysiology of hepatic fibrogenesis. It has been shown that Perilipin 5 (PLIN5), known as a lipid droplet structural protein that is highly expressed in oxidative tissue, can inhibit such activation through various mechanisms associated with lipid metabolism. This study aimed to investigate the possible influence of PLIN5 on TGF-β1 signaling. Our findings confirm the importance of PLIN5 in maintaining HSC quiescence in vivo and in vitro. PLIN5 overexpression suppresses the TGF-β1-SMAD2/3 and SNAIL signaling pathways as well as the activation of the signal transducers and activators of transcription 3 (STAT3). These findings derived from experiments in hepatic cell lines LX-2 and Col-GFP, in which overexpression of PLIN5 was able to downregulate the signaling pathways SMAD2/3 and SNAIL activated previously by TGF-β1 treatment. Furthermore, TGF-β1-mediatedinduction of extracellular matrix proteins, such as collagen type I (COL1), Fibronectin, and α-smooth muscle actin (α-SMA), was suppressed by PLIN5. Moreover, STAT3, which is interrelated with TGF-β1 was already basally activated in the cell lines and inhibited by PLIN5 overexpression, leading to a further reduction in HSC activity shown by lowered α-SMA expression. This extension of the intervening mechanisms presents PLIN5 as a potent and pleiotropic target in HSC activation.

Fibroblast Growth Factor 21 Reduces Cholesterol-Induced Hepatic Fibrogenesis by Inhibiting TGF-β/Smad3C Signaling Pathway in LX2 Cells

Background: Liver fibrosis is often attributed to the activation of hepatic stellate cells (HSCs) and excessive scar formation in the liver. Advanced stages of the disease often lead to liver cirrhosis and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) is a secreted protein, which has anti-diabetic and lipocaic effects. Objectives: In this study, we investigated the ability of FGF21 to reduce hepatic fibrogenesis due to the accumulation of free cholesterol in the LX2 cell line (a type of HSC-derived cell line) and its mechanism of action. Methods: Cells were treated with 25, 50, 75, and 100 μM concentrations of cholesterol for 24 and 48 h. The mRNA expression of genes of TGF-β, αSMA, and collagen1α and the level of Smad3C protein were measured to assess liver fibrosis. Next, the cells were treated with FGF21 for 24 h, and the expression levels of TGF-β, αSMA, collagen 1α, and Smad3C protein were measured. Results: The results showed that the expression of TGF-β, αSMA, collagen 1α genes, and also the level of Smad3C protein in the presence of cholesterol increased significantly compared to the control group. Treatment with FGF-21 also significantly reduced the expression of TGF-β, αSMA, and collagen 1α genes. Conclusions: Cholesterol by increasing the level of Smad3C protein and activating the TGF-β signaling pathway increases major proteins involved in the production of extracellular matrix, including collagen 1α. Besides, FGF21 inhibits the further activation of HSCs by inhibiting the TGF-β/Smad3C signaling pathway and thus can prevent the progression of liver fibrosis.

Targeting Myosin 1c Inhibits Murine Hepatic Fibrogenesis

Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, TGF-β stimulation to hepatic stellate cells (HSCs, the liver pericyte and key cell type responsible for the deposition of extracellular matrix upregulates Myo1c expression, while genetic depletion or pharmacological inhibition of Myo1c blunted TGF-β induced fibrogenic responses, resulting in repression of α-SMA and Col1α1 mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response and contractility when compared with vehicle treated HSCs. Importantly, phosphorylation of SMAD2 and SMAD3 were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c-KO MEFs upon TGF-β stimulation. Using the genetic Myo1c knockout (Myo1c-KO) mice, we confirmed that Myo1c is critical for fibrogenesis as Myo1c-KO mice were resistant to CCl4 induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c-mediates hepatic fibrogenesis by modulating TGF-β signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.