Next Generation Sequencing on HMEC-1 or HUVEC residing on SOFT or STIFF matrices. 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. Overall design: 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.