Transcriptomic Analysis Identifies Differentially Expressed Genes Associated with Vascular Cuffing and Chronic Inflammation Mediating Early Thrombosis in Arteriovenous Fistula

Abstract

Arteriovenous fistula (AVF) is vascular access created for hemodialysis in end-stage renal disease patients. AVF creation causes increased blood flow in the outflow vein with increased pressure. Increased blood flow, blood volume, and shear stress causes outward remodeling so that the outflow vein can withstand the increased pressure. Outward remodeling of the vein involved in AVF is necessary for AVF maturation, however, inward remodeling due to excessive neointimal hyperplasia (NIH) and chronic inflammation may end up with vessel thrombosis and AVF maturation failure. Early thrombosis of the vessel may be due to the luminal factors including NIH and chronic inflammation or due to chronic inflammation of the adventitial due to perivascular cuffing. Inflammation may either be due to an immune response to the vascular injury during AVF creation or injury to the surrounding muscles and fascia. Several studies have discussed the role of inflammation in vascular thrombosis due to intimal injury during AVF creation, but there is limited information on the role of inflammation due to surrounding factors like a muscle injury. The concept of perivascular cuffing has been reported in the nervous system, but there is no study of perivascular cuffing in AVF early thrombosis. We performed the bulk RNA sequencing of the femoral arterial tissue and contralateral arteries as we found thrombosed arteries after AVF creation. RNA sequencing revealed several significantly differentially expressed genes (DEGs) related to chronic inflammation and perivascular cuffing, including tripartite motif-containing protein 55 (TRIM55). Additionally, DEGs like myoblast determination protein 1 (MYOD1) increased after muscle injury and relates to skeletal muscle differentiation, and network analysis revealed regulation of various genes regulating inflammation via MYOD1. The findings of this study revealed multiple genes with increased expression in the AVF femoral artery and may provide potential therapeutic targets or biomarkers of early thrombosis in AVF maturation failure. Thus, not only the luminal factors but also the surrounding factors mediating vascular cuffing contribute to vessel thrombosis and AVF failure via early thrombosis, and targeting the key regulatory factors may have therapeutic potential.

Transcriptional and Epigenetic Factors Associated with Early Thrombosis of Femoral Artery Involved in Arteriovenous Fistula

“…Excessive intimal hyperplasia is also a result of increased ROS production and inflammation, mediated by macrophages that are deficient in MED1 [ 30 ]. Inhibited MED1 ( Table 6 ) and the infiltration of pro-inflammatory immune cells, including macrophages, in the thrombosed and intimal hyperplasia region of femoral arteries in this study support the notion of the presence of inflammation [ 12 ] and a the possible role of MED1-deficient macrophages in early thrombosis and AVF failure. Inflammation within the evolving plaque is associated with the activation of signal transducers and activators of transcription (STAT)1 and STAT3, and STATs function as intracellular regulators of vascular remodeling [ 31 ].…”

“…Intimal injury during AVF creation induces NIH, which helps in vascular remodeling and makes vessel suitable for increased blood flow through the outflow vein. However, excessive NIH and early thrombosis of the inflow artery or excessive NIH in the outflow vein can lead to vessel stenosis and AVF maturation failure [ 12 ]. Neointimal reendothelialization, with the aim of obtaining an intact endothelium, is necessary for the proper healing and AVF maturation and involves endothelial cell (EC) proliferation and migration and the formation of tight adherence junctions.…”

“…The material and methods involved in creating AVFs, assessing pre-and post-operative femoral vessels involved in AVFs, tissue harvesting, tissue processing, histology, immunostaining, and bulk RNA sequencing have been described previously [ 12 ]. We choose the swine model because the swine model represents the best choice for studies of occlusive arterial and venous diseases and there are similarities in the anatomy, physiology and pathological responses of the human and porcine cardiovascular systems [ 19 ].…”

“…The total RNA, isolated using the TRIzol method from three thrombosed femoral arteries and three contralateral femoral arteries, was sent for bulk RNA sequencing. RNA samples with RIN > 6 were subjected to library preparation and sequencing using the NEBNext Ultra II RNA Library Prep Kit for Illumina and Illumina HiSeq instruments at Genewiz LLC (Plainfield, NJ, USA) as mentioned in [ 12 ]. IPA was used for the analyses of microRNAs and transcription factors, as detailed in the following sections.…”

“…To attenuate vessel stenosis after AVF creation, we targeted chronic inflammation by inhibiting TREM-1 using LR-12 peptides and TLR4 with TAK-242 and performed sequencing in collected tissues to investigate the changes in gene expression associated with stenosis and thrombosis. Recently, we reported on differentially expressed genes (DEGs) associated with stenosis and thrombosis of the femoral artery involved in AVF [ 12 ].…”