Differential Gene Expression and Signal Pathways Activation Lead to Differences in Cell Proliferation and Migration of Venous and Arterial Pig Smooth Muscle Cells

Background: Arteriovenous fistulae (AVF) are the preferred mode of dialysis vascular access but have a maturation failure rate of over 50% due to a venous segment stenosis, which is characterized by smooth muscle cells (SMCs) proliferation and migration, resulting in neointimal hyperplasia. Despite the clinical significance of venous segment AVF stenosis, the exact gene expression profile and activation pathways responsible for venous SMC proliferation and migration remain unclear. Methods: Pig arterial SMCs (ApSMCs) and venous SMCs (VpSMC) were cultured in DMEM containing 10% FBS and 1% P/S . Total RNA was isolated for bulk RNA sequencing. Cell lysates were collected for analyzing protein expression levels. MTT assays were used to measure cell proliferation, and cell scratch assays were used to assess cell migration ability. Results: Bulk RNA sequencing results indicated that 265 genes were expressed highly in VpSMCs, while another 301 genes were highly expressed in ApSMCs. PCA and signal pathway analyses demonstrated clear differences in gene expression and pathway activation between VpSMCs and ApSMCs. For instance, cell migration, cell junction and cell communication pathways were activated in VpSMCs while collagen-containing extracellular matrix and extracellular matrix pathways were activated in ApSMCs. Importantly, different sets of genes were utilized even within the same activated pathways. Western blot results demonstrated that FAK protein levels were increased in VpSMCs while Paxillin and Vinculin levels were similar in both cell types. ECM proteins, such as fibronectin and vitronectin proteins were produced more by VpSMCs. Importantly, VpSMCs proliferated faster than ApSMCs (1.80±0.21 vs 1.56±0.19 fold after 48hr in growth media). However, their response to PDGF in serum free media was similar (1.53±0.16 vs 1.49±0.17 fold after 48hr). Migration assays revealed that VpSMCs had a faster migration as compared to ApSMCs in growth media and serum-free media. Conclusion: Differential gene expression and pathway activation in VpSMCs and ApSMCs.are likely responsible for their differences in cell proliferation and migration, suggesting a much needed precision medicine approach to AVF maturation failure. Funding: NIDDK Support