University of Illinois at Urbana Champaign, Urbana, USA.
University of Illinois at Urbana Champaign, Urbana, USA.
Acta Biomater. 2023 Sep 1;167:278-292. doi: 10.1016/j.actbio.2023.06.018. Epub 2023 Jun 19.
Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Cell microarrays demonstrated the cooperative effects of stiffness and ECM composition on H3K4 and H3K9 methylation/acetylation. ATAC sequencing revealed higher chromatin accessibility in HSCs on 1kPa compared to 25kPa substrates for all ECM conditions. Gene set enrichment analysis using RNA sequencing data of HSCs in defined ECM microenvironments demonstrated higher enrichment of NAFLD and fibrosis-related genes in pre-activated HSCs on 1kPa relative to 25kPa. Overall, these findings are indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs. STATEMENT OF SIGNIFICANCE: Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Overall, these findings were indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs.
肝星状细胞（HSCs）是非酒精性脂肪性肝病中肝脏纤维化的主要驱动因素之一。尽管肝脏疾病中 HSC 的激活与细胞外基质（ECM）沉积和重塑的变化有关，但 ECM 如何调节 HSC 的表型状态转变仍不清楚。我们使用高通量细胞微阵列，结合工程 ECM 微环境中的全基因组 ATAC 和 RNA 测序，研究了 ECM 和基质硬度对激活的原代人 HSCs 染色质可及性和基因表达的影响。细胞微阵列显示了硬度和 ECM 组成对 H3K4 和 H3K9 甲基化/乙酰化的协同作用。ATAC 测序显示，对于所有 ECM 条件，在 1kPa 相比 25kPa 基质上 HSCs 的染色质可及性更高。使用定义 ECM 微环境中的 HSCs 的 RNA 测序数据进行基因集富集分析表明，在 1kPa 相比 25kPa 条件下，前激活的 HSCs 中与非酒精性脂肪性肝病和纤维化相关的基因富集度更高。总的来说，这些发现表明 HSCs 存在微环境适应反应，并获得了持续激活状态。结合 ATAC/RNA 测序分析，鉴定出候选调节因子，包括 HSD11B1 和 CEBPb。HSD11b1 和 CEBPb 的 siRNA 介导的敲低表明在 HSCs 中，纤维化标志物的减少受到微环境的控制。
