Three-dimensional (3D) cell culture conditions are often used to promote the differentiation of human cells as a prerequisite for the study of organotypic functions and environment-specific cellular responses. Here, we assessed the molecular and functional phenotype of vascular smooth muscle cells (VSMCs) cultured as 3D multilayered aggregates. Microarray studies revealed that these conditions decrease the expression of genes associated with cell cycle control and DNA replication and cease proliferation of VSMCs. This was accompanied by a lower activity level of the mitogen-activated protein kinase ERK1/2 and an increase in autocrine TGFβ/SMAD2/3-mediated signaling - a determinant of VSMC differentiation. However, inhibition of TGFβ signaling did not affect markers of VSMC differentiation such as smooth muscle myosin heavy chain (MYH11) but stimulated pro-inflammatory NFκB-associated gene expression in the first place while decreasing the protein level of NFKB1/p105 and NFKB2/p100 - inhibitors of NFκB transcriptional activity. Moreover, loss of TGFβ signaling also revived VSMC proliferation in 3D aggregates. In conclusion, assembly of VSMCs in multilayered aggregates alters their transcriptome to translate the cellular organization into a resting phenotype. In this context, TGFβ signaling appears to attenuate cell growth and NFκB-controlled gene expression representing important aspects of VSMC quiescence.

中文翻译：

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3D聚集体中血管平滑肌细胞的组装引起细胞静止。

三维（3D）细胞培养条件通常用于促进人类细胞的分化，这是研究器官型功能和特定于环境的细胞反应的先决条件。在这里，我们评估了培养为3D多层聚集体的血管平滑肌细胞（VSMC）的分子和功能表型。微阵列研究表明，这些条件会降低与细胞周期控制和DNA复制相关的基因的表达，并停止VSMC的增殖。这伴随着丝裂原活化蛋白激酶ERK1 / 2的活性水平降低和自分泌TGFβ/ SMAD2 / 3介导的信号传导增加-VSMC分化的决定因素。然而，抑制TGFβ信号传导不会影响VSMC分化的标志物，例如平滑肌肌球蛋白重链（MYH11），但首先会刺激促炎性NFκB相关基因的表达，同时降低NFKB1 / p105和NFKB2 / p100抑制剂的蛋白水平NFκB转录活性。此外，TGFβ信号传导的丧失也使3D聚集体中的VSMC增殖恢复了活力。总之，VSMC在多层聚集体中的组装会改变其转录组，从而将细胞组织转化为静止的表型。在这种情况下，TGFβ信号似乎减弱了细胞生长和NFκB控制的基因表达，这代表了VSMC静止的重要方面。