Mechanotransduction-Targeting Drugs Attenuate Stiffness-Induced Hepatic Stellate Cell Activation <i>in Vitro</i>

Abstract

In hepatitis, activated hepatic stellate cells (HSCs) produce collagens, causing liver fibrosis. Microenvironmental stiffness is a known trigger of HSC activation and is communicated through mechanotransduction. Cell proliferation, alpha smooth muscle actin (α-SMA) and collagen type Iα (Col1α) are indicative of activated HSCs. We hypothesized that certain compounds could interfere with the HSC's recognition of microenvironmental stiffness by blocking cell adhesion signaling. To verify the potential of mechanotransduction, and in particular of focal adhesion proteins, as liver fibrosis drug targets, we evaluated existing drugs. We examined the effects of the integrin antagonist, BS-1417; the focal adhesion kinase (FAK) inhibitor, defactinib; the cyclin-dependent kinase (CDK) inhibitor, roscovitine; and two microtubule modulators, paclitaxel and colchicine, on stiffness-induced HSC activation. To determine the extent of transforming growth factor β (TGF-β) participation in mechanotransduction, we measured gene expression levels of α-SMA and Col1α. We also measured ATP levels to determine cell number. Results revealed that interestingly, although TGF-β did not show additional HSC activation after stiffness stimulation, the TGF-β receptor inhibitor, SB525334, markedly suppressed stiffness-induced α-SMA and Col1α mRNA expression. BS-1417, roscovitine, defactinib and colchicine suppressed α-SMA and Col1α mRNA expression as well as the number of HSCs. Paclitaxel also suppressed stiffness-induced α-SMA mRNA expression and the number of HSCs, but mildly reduced that of Col1α mRNA. Together, these results show that an integrin antagonist and mechanotransductiontargeting drugs reduced stiffness-induced HSC activation in a dose-dependent fashion. The targeting of focal adhesion proteins involved in mechanotransduction is promising in liver fibrosis drug development.

GA&HA-Modified Liposomes for Co-Delivery of Aprepitant and Curcumin to Inhibit Drug-Resistance and Metastasis of Hepatocellular Carcinoma

“…Previous studies have reported that aHSCs can promote ECM formation by synthesizing collagen and fibronectin. 49 , 50 The result showed that the combination therapy based on HA&GA-LPs could inhibit aHSCs-induced ECM deposition. Furthermore, alpha smooth muscle actin (α-SMA) was a marker protein of aHSCs, 51 and the immunofluorescence staining of α-SMA was performed to examine the activation of HSCs.…”

Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture

“…However, the activation capacity of the scHSCs was lost on day 14 of the culture. These results suggest that PSC-derived scHSCs are affected by in vitro culture similarly to pHSCs, which are known to acquire an activated phenotype in 2D cultures ( Sancho-Bru et al, 2005 ; Sakai and Yoshimura, 2021 ). On the other hand, our data suggest that scHSCs may retain a more quiescent-like phenotype than pHSCs, evident by the comparably low expression of fibrosis-related genetic markers and high storage capacity of vitamin A.…”

“…The time-dependent decline in cell-specific phenotypic traits and functionality is a well-known limitation of in vitro cell culture models ( Sancho-Bru et al, 2005 ; Xie et al, 2013 ; Sakai and Yoshimura, 2021 ). It is, therefore, crucial to thoroughly analyze the stability of cell types if they are to be included in vitro disease platforms.…”

“…However, the use of primary HSCs (pHSCs) and LSECs (pLSECs) from human donors is limited by availability, batch variations, and phenotypical and functional changes during isolation and in vitro cultivation ( Zeilinger et al, 2016 ). Specifically, pHSCs are easily activated, e.g., by the stiffness of the culture substrate, making it challenging to model progressive conditions like fibrosis where the activation process of HSCs is central to the pathogenesis ( Sancho-Bru et al, 2005 ; Sakai and Yoshimura, 2021 ). For pLSECs, no in vitro model known to the authors allow the preservation of their fenestrated phenotype, hampering their functionality.…”

Effect of Extracellular Matrix Stiffness on Candesartan Efficacy in Anti-Fibrosis and Antioxidation

“…Phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and ERK1/2, the downstream of FAK, are the two classical signaling factors that directly cause myocardial fibrosis [ 51 , 52 ], and can result in the α-SMA-positive myofibroblast diversity and the formation of various types of collagens. FAK also directly regulates the expression of α-SMA, and thus participates in the process of liver fibrosis [ 53 ]. the expression of AT 1 R can be induced by ECM stiffness, and the expression level of AT 1 R increases with the increase of ECM stiffness, demonstrating the activation effect of ECM stiffness on AT 1 R. For the group with CAN treatment, CAN leads to the decrease of AT1R expression, which shows the opposite effect to the increase of AT 1 R expression induced by ECM stiffness.…”