Borah Dibakar, Blacharczyk Oliwia, Szafranska Karolina, Czyzynska-Cichon Izabela, Metwally Sara, Szymanowski Konrad, Hübner Wolfgang, Kotlinowski Jerzy, Dobosz Ewelina, McCourt Peter, Huser Thomas, Lekka Malgorzata, Zapotoczny Bartlomiej
Department of Biophysical Microstructures, Institute of Nuclear Physics, Polish Academy of Sciences, ul. Radzikowskiego 142, 31-342 Krakow, Poland.
Vascular Biology Research Group, Department of Medical Biology University of Tromsø-The Arctic University of Norway, 9019 Tromsø, Norway.
Cells. 2025 Apr 21;14(8):621. doi: 10.3390/cells14080621.
Liver sinusoidal endothelial cells (LSECs) play a crucial role in hepatic homeostasis, clearance, and microcirculatory regulation. Their fenestrations-patent transcellular pores-are essential for proper liver function, yet disappear in pathological conditions such as liver fibrosis and inflammation through a process known as defenestration. Defenestrated sinusoids are often linked to the liver stiffening that occurs through mechanotransduction-regulated processes. We performed a detailed characterization of polyacrylamide (PAA) hydrogels using atomic force microscopy (AFM), rheometry, scanning electron microscopy, and fluorescence microscopy to assess their potential as biomimetic substrates for LSECs. We additionally implemented AFM; quantitative fluorescence microscopy, including high-resolution structured illumination microscopy (HR-SIM); and an endocytosis assay to characterize the morphology and function of LSECs. Our results revealed significant local variations in hydrogel stiffness and differences in pore sizes. The primary LSECs cultured on these substrates had a range of stiffnesses and were analyzed with regard to their number of fenestrations, cytoskeletal organization, and endocytic function. To explore mechanotransduction in inflammatory liver disease, we investigated LSECs from a genetic model of systemic inflammation triggered by the deletion of Mcpip1 in myeloid leukocytes and examined their ability to restore their fenestrations on soft substrates. Our study demonstrates the beneficial effect of soft hydrogels on LSECs. Control cells exhibited a similar fenestrated morphology and function compared to cells cultured on plastic substrates. However, the pathological LSECs from the genetic model of systemic inflammation regained their fenestrations when cultured on soft hydrogels. This observation supports previous findings on the beneficial effects of soft substrates on LSEC fenestration status.
肝窦内皮细胞（LSECs）在肝脏内环境稳定、清除及微循环调节中发挥着关键作用。它们的窗孔——开放的跨细胞孔——对肝脏正常功能至关重要，但在诸如肝纤维化和炎症等病理状态下，会通过一种称为去窗孔化的过程而消失。去窗孔化的肝窦通常与通过机械转导调节过程发生的肝脏硬化有关。我们使用原子力显微镜（AFM）、流变学、扫描电子显微镜和荧光显微镜对聚丙烯酰胺（PAA）水凝胶进行了详细表征，以评估其作为LSECs仿生基质的潜力。我们还实施了AFM；定量荧光显微镜，包括高分辨率结构光照显微镜（HR-SIM）；以及内吞作用测定，以表征LSECs的形态和功能。我们的结果揭示了水凝胶硬度的显著局部差异和孔径差异。在这些基质上培养的原代LSECs具有一系列硬度，并就其窗孔数量、细胞骨架组织和内吞功能进行了分析。为了探索炎症性肝病中的机械转导，我们研究了来自髓系白细胞中Mcpip1缺失引发的全身炎症遗传模型的LSECs，并检查了它们在软基质上恢复窗孔的能力。我们的研究证明了软水凝胶对LSECs的有益作用。与在塑料基质上培养的细胞相比，对照细胞表现出相似的有窗孔形态和功能。然而，来自全身炎症遗传模型的病理LSECs在软水凝胶上培养时恢复了它们的窗孔。这一观察结果支持了先前关于软基质对LSEC窗孔状态有益作用的研究发现。
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