Next Generation Sequencing on HMEC-1 or HUVEC residing on SOFT or STIFF matrices

Purpose: Endothelial cells respond to changes in subendothelial stiffness altering their proliferation, migration and barrier integrity but whether that is due to transcriptional reprogramming was largely unknown. Using RNA-Sequencing, we performed gene expression profiling for two endothelial cell types grown on soft or stiff matrices: primary human umbilical vein endothelial cells (HUVEC) and immortalized human microvascular endothelial cells (HMEC-1), to understand whether subendothelial stiffness-dependent changes in endothelial cell mechanics are due to transcriptional regulation. Methods and Results: By analyzing the differentially expressed genes between all samples we found that endothelial cell type rather that subendothelial stiffness is the primary determinant of the endothelial cell transcriptome. Both cell types respond to changes in their subendothelial stiffness by increasing the traction stresses they exert on stiffer as opposed to softer matrices, however it is apparently not the endothelial cell transcriptome that regulates this universal biomechanical response to subendothelial stiffness. Only a handful of genes were differentially expressed in each cell type in a stiffness-dependent manner, and none were shared between the two cell types examined. In contrast, thousands of genes were differentially regulated in HUVEC as compared to HMEC-1. HUVEC (but not HMEC-1) upregulate expression of TGF-2 on stiffer matrices, and also enhance their endogenous TGF-2 expression and their cell-matrix traction stresses in response to application of exogenous TGF-2. Conclusions: Altogether, these findings provide insights into the relationship between subendothelial stiffness, endothelial mechanics and variation of the transcriptome between distinct endothelial cell types, and reveal that subendothelial stiffness while critically impacting endothelial cell mechanics is minimally altering their transcriptome. Confluent endothelial cell mRNA profiles for cells residing for 24 h on soft 3 kPa or stiff 70 kPa polyacrylamide hydrogels coated with 0.25 mg/mL collagen I were generated in sextuplicate, using Illumina NextSeq 500 System using the High-Output Kit with 2x75 read length and 30 million reads per sample.

研究背景与目的：内皮细胞可响应内皮下刚度的变化，进而改变其增殖、迁移及屏障完整性，但上述效应是否由转录重编程介导，目前尚不清楚。本研究采用RNA测序（RNA-Sequencing）技术，对培养于软基质或硬基质的两种内皮细胞——原代人脐静脉内皮细胞（HUVEC）与永生化人微血管内皮细胞（HMEC-1）进行基因表达谱分析，旨在明确内皮细胞力学特性随内皮下刚度变化的现象是否由转录调控所介导。

方法与结果：通过分析所有样本间的差异表达基因，我们发现内皮细胞类型而非内皮下刚度，是内皮细胞转录组的主要决定因素。两种内皮细胞均可响应内皮下刚度变化：在硬基质上施加的牵引力相较于软基质更高，但调控这一针对内皮下刚度的通用生物力学响应的机制，显然并非内皮细胞转录组。仅少量基因在每种细胞中呈刚度依赖性差异表达，且两种受试细胞间无共有此类基因。与之形成对比的是，HUVEC与HMEC-1相比，存在数千个差异调控基因。HUVEC（而非HMEC-1）在硬基质上会上调转化生长因子β2（TGF-β2）的表达，且在添加外源性TGF-β2时，可增强其内源TGF-β2的表达水平与细胞-基质牵引力。

结论：综上，本研究结果阐明了内皮下刚度、内皮细胞力学特性与不同内皮细胞类型间转录组差异之间的关联，并揭示：尽管内皮下刚度可显著影响内皮细胞力学特性，但仅对其转录组产生极微小的改变。本研究通过Illumina NextSeq 500测序系统（采用高产出试剂盒，测序读长为2×75 bp，单样本测序量为3000万条reads），对铺覆0.25 mg/mL I型胶原（collagen I）的3 kPa软基质与70 kPa硬基质聚丙烯酰胺水凝胶上培养24 h的汇合态内皮细胞完成了6次生物学重复的mRNA谱分析。