The main characteristics of liver fibrosis are abnormal collagen deposition and structural destruction caused by multiple diseases, which further leads to portal hypertension and Liver failure. The activation of hepatic stellate cells (HSCs) has been proven to be the main source of collagen-producing myofibroblasts in the liver.1 Transforming growth factor β (TGF-β) is considered a key factor in activating HSCs.2 In the traditional model, the activation of HSCs will simultaneously induce high expression of alpha smooth muscle actin (α-SMA) and type I collagen, alpha 1 (COL1A1), indicating that myofibroblasts derived from HSCs possess enhanced migration and collagen production abilities. Based on the existence of TGF-β, we evaluated two characteristics of HSCs-derived myofibroblasts and constructed a new activation model.
Based on the Homo sapiens fibrosis liver Single-cell sequencing data set provided by P Ramachandran, we analyzed the correlation between α-SMA and COL1A1 expression in the cell population with myofibroblast characteristics.3 With the help of the Omnibrowser database (https://omnibrowser.abiosciences.com/#/home), we obtained a total of 11 cell subpopulations, among which mesenchyme cells possess the characteristics of myofibroblast (Fig. 1a). The expression of myofibroblast markers in mesenchyme cells, including α-SMA, COL1A1, platelet-derived growth factor alpha polypeptide (PDGFA), and platelet-derived growth factor receptor beta polypeptide (PDGFRB), is higher than that of other subpopulations (Fig. 1b). In mesenchyme cells, the expression of α-SMA is negatively correlated with COL1A1 (Fig. 1c). Next, we further validated this conclusion through models in vitro and in vivo.
Myofibroblasts are the main executors of migration and collagen secretion in the fibrotic liver. We further promoted the myofibroblast characteristics (α-SMA expression) of rat HSC cell line HSC-T6 using 5% rat liver fibrosis serum (Fig. 1d). After further treatment with gradient concentration TGF-β1, the expression of α-SMA mRNA in myofibroblasts was significantly increased, accompanied by a significant decrease in COL1A1 mRNA expression (Fig. 1e). Western blot further validated this conclusion at the protein level (Fig. 1f). In addition, COL1A1 concentration in the culture medium was determined by Elisa, and the result showed that TGF-β1 induced a decrease in COL1A1 secretion in myofibroblasts (Fig. 1g). The negative correlation between α-SMA and COL1A1 expression in myofibroblasts is consistent with single-cell sequencing analysis.
Next, we used carbon tetrachloride to construct the rat liver fibrosis model and evaluated the distribution of TGF-β1, α-SMA, and COL1A1 in the tissue (Fig. 1h). Multicolor fluorescence showed a network-like distribution of TGF-β1, while COL1A1 showed a patchy distribution in the TGF-β1 net. COL1A1 expression was lower near the TGF-β1 loop and higher in the distant region, presenting a centripetal pattern. The difference is that α-SMA expression was mostly concentrated next to the TGF-β1 loop.
Based on these data, we considered the differences in the expression characteristics of HSC-derived myofibroblasts in α-SMA and COL1A1 compared to traditional cognition. In our opinion, the negative correlation between α-SMA and COL1A1 expression seems to be more consistent with objective phenomena. If we infer that both SMA and COL1A1 are highly expressed in myofibroblasts, then the collagen fibers in the fibrotic liver should be constructed in the way of patchy distribution (Fig. 2a). However, collagen fibers exhibit elongated cords in the fibrotic liver without large aggregation. The expression of α-SMA brings stronger migration ability to myofibroblasts, which is closely related to the gradient concentration of TGF-β1. In the high concentration area of TGF-β1, myofibroblasts expand to the surrounding area. In the area far from TGF-β1, myofibroblasts weaken migration and enhance collagen production. This model can be named the dual mode transition of myofibroblasts in fiber liver (DMTM model) (Fig. 2b). The model includes the mechanism of TGF-β1 gradient-induced myofibroblasts and collagen expansion and reasonably explains the formation of striped collagen bundles. We actively look forward to discussing with peers and further improving the disease model for the progression of liver fibrosis.