Introduction:
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 cell (SMC) 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. We herein attempt to identify the differences and similarities of venous versus arterial SMCs.
Methods:
Pig arterial SMCs (ApSMCs) and pig venous SMCs (VpSMC) were cultured in DMEM containing 10% FBS and 1% P/S as well as normal glucose (5 mM). Total RNA was isolated for bulk RNA sequencing. Cell lysates were collected for analyzing protein expression levels. Western blot results were quantified using ImageJ. 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 as compared to ApSMCs, while another 301 genes were highly expressed in ApSMCs as compared to VpSMCs. 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 for focal adhesion proteins demonstrated that FAK protein levels were increased in VpSMCs compared to ApSMCs while Paxillin and Vinculin levels were similar in both cell types. Interestingly, ECM proteins, such as fibronectin and vitronectin proteins were produced more by VpSMCs as compared to ApSMCs, which contradicted with RNA sequencing results. 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 (115±59 vs 100±60 cells/field) and serum-free media (e.g. 67±9 vs 55±14 cells/field).
Conclusions:
Differential gene expression and pathway activation in VpSMCs and ApSMCs.are likely responsible for their differences in cell proliferation and migration. This information could be the first step towards the development of future therapies for AVF maturation failure that specifically target those genes and pathways activated in venous SMCs as opposed to an extrapolation from the arterial SMCs data. In other words, a much needed precision medicine approach to AVF maturation failure.
Some of this data would have been presented at ASN Kidney Week 2024
I have no potential conflict of interest to disclose.
I did not use generative AI and AI-assisted technologies in the writing process.