RESUMEN
Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.
Asunto(s)
Colesterol/metabolismo , Células Endoteliales/fisiología , Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Metabolismo de los Lípidos/genética , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismoRESUMEN
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 TGFß/SMAD2/3-mediated signaling - a determinant of VSMC differentiation. However, inhibition of TGFß 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 TGFß 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, TGFß signaling appears to attenuate cell growth and NFκB-controlled gene expression representing important aspects of VSMC quiescence.