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.
Keywords: 3D culture; Differentiation; TGF; Vascular smooth muscle cells.
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