Bulk RNA Sequencing Analysis Reveals Signature Differences Between Porcine Venous and Arterial Smooth Muscle Cells

Background: One of our previous studies identified significant differences between porcine arterial and venous smooth muscle cells (ApSMCs and VpSMCs) in the expression of numerous genes and in the activity of several signaling pathways, using a candidate gene approach based on known protein function. In order to expand on our earlier findings especially with regard to differences in novel proteins and cell activation pathways, we describe herein a genome-wide comparison of VpSMCs and ApSMCs, using bulk RNA sequencing. Methods: ApSMCs and VpSMCs were isolated from pig carotid artery and jugular vein using standard explant culture protocols. The cells were cultured in DMEM containing 10% FBS and 1% P/S. When cells reached 100% confluency, total RNA was isolated using RNeasy plus mini kit (Qiagen) for bulk RNA sequencing (AZENTA life science). Results: PCA plots and heatmaps revealed a good separation within the 2 groups (ApSMCs and VpSMCs). Using standard cutoff (>=2 fold change, FDR<=0.05), 466 genes were upregulated in ApSMCs and 358 genes were upregulated in VpSMCs. Functional pathway analysis were conducted using the Gene Set Enrichment tool. The top 15 enriched pathways of the GSEA and ORA results were detected. Both GSEA and ORA results reveal the top regulated pathways are mostly involved in the cell cycle, cell structure and cell differentiation. In addition, deep analysis of specific genes changes in a specific pathway, we identified that differences of genes expression change in the same pathway between ApSMCs and VpSMCs. For example, in cell cycle pathway, TGFB1, GADD45A and P53 were expressed highly in ApSMCs while SKP2, PCK1, CDK1 and PPP2CA were expressed highly in VpSMCs. Furthermore, in PI3K/AKT signal pathway, TLR2, ITGB1, ATF4 and BCL2L11 were highly expressed in ApSMCs whereas ITGA6, PI3KCA, SGK1 and CCND1 were expressed highly in VpSMCs. Conclusion: The bulk RNA data clearly demonstrated the differences between two subsets of VSMCs in DEGs and genes that are utilized in a specific signal pathway. The top regulated pathways turned out to be involved in biological processes like cell cycle, cell structure formation and cell differentiation, which strongly support our previous observations and clinical manifestations. Funding: NIDDK Support