Matrix stiffness-induced transcriptome alterations and regulatory mechanisms revealed by RNA-seq in Endothelial cells

Abstract

Abstract Background Changes in vascular stiffness are associated with the development and progression of many diseases, especially in cardiovascular disease. However, the effect of vascular stiffness on the endothelial cells (ECs) is not fully understood. This study focused on the transcriptional profiles of ECs cultured on the matrices with different stiffness (1kPa and 40kPa, respectively) to broaden the knowledge of mechanotransduction. Methods We first prepared hydrogel materials with different stiffness to simulate the vascular stiffness in normal and pathological states, respectively. RNA sequencing was then conducted on endothelial cells cultured on matrix for 24 hours. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia for Genes and Genomes (KEGG) pathway analysis, protein–protein interaction (PPI) network analysis and transcription factors (TFs) and their target genes analysis were performed on differentially expressed genes (DEGs). qRT-PCR was finally utilized to validate the hub dysregulated genes. Results We obtained 1775 differentially expressed genes (DEGs) by RNA-seq, with 450 up-regulated and 1325 down-regulated DEGs in ECs cultured on soft matrix (1kPa) compared to those cultured on stiff matrix (40kPa). According to GO and KEGG analysis, DEGs were mainly enriched in cell junctions and cell-matrix interaction related signaling pathways. In order to confirm the RNA-seq results, we performed real-time qPCR analysis on the genes of interest, including cadherin 5, F11 receptor (also known as junctional adhesion molecule-A), fibronectin 1, collagen α2(IV) chain, matrix metalloproteinase-14 and integrin α5, and found that the expression level of all these genes were down-regulated on soft matrix, suggesting that soft matrix caused by pathological conditions may directly attenuate vascular barrier function. Conclusions In summary, the results of RNA-Seq suggested that matrix stiffness may affect extracellular matrix (ECM) integrity, cell-ECM interactions, and cell-cell junctions. This study offers the insights about the effects of physical stimulation on cells, paving a way for vascular tissue engineering, regenerative medicine, disease modeling and therapies.