Significance Statement: The arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis, but several studies have shown that the primary patency rate at 1 year is approximately 60%. Percutaneous transluminal angioplasty (PTA) is the first line of treatment for AVF stenosis. However, the rate of restenosis after angioplasty is high and the primary patency rate after PTA treatment in AVFs at 6, 12, and 24 months is 61%, 42%, and 35%, respectively. Currently, no durable therapies prolong patency after angioplasty. This paper describes the use of adipose-derived mesenchymal stem cells to reduce stenosis after PTA treatment in a murine AVF model and provides rationale for potential clinical use. Background: Percutaneous transluminal angioplasty (PTA) is the first line of treatment for stenosis in the arteriovenous fistula (AVF) created to provide access for hemodialysis, but resenosis still occurs. Transplants of adipose-derived mesenchymal stem cells (AMSCs) labeled with green fluorescent protein (GFP) to the adventitia could reduce pro-inflammatory gene expression, possibly restoring patency in a murine model of PTA for venous stenosis. Methods: Partial nephrectomy of male C57BL/6J mice induced CKD. Placement of the AVF was 28 days later and, 14 days after that, PTA of the stenotic outflow vein was performed with delivery of either vehicle control or AMSCs (5×10⁵) to the adventitia of the vein. Mice were euthanized 3 days later and gene expression for interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha TNF-α) analyzed, and histopathologic analysis performed on day 14 and 28. GFP (+) AMSCs were tracked after transplantation for up to 28 days and Doppler ultrasound performed weekly after AVF creation. Results: Gene and protein expression of IL-1β and TNF-α, fibrosis, proliferation, apoptosis and smooth muscle actin decreased, and the proportions of macrophage types (M2/M1) shifted in a manner consistent with less inflammation in AMSC-transplanted vessels compared to controls. After PTA, AMSC-treated vessels had significantly higher wall shear stress, average peak, and mean velocity, with increased lumen vessel area and decreased neointima/media area ratio compared to the control group. At 28 days after delivery, GFP (+) AMSC were present in the adventitia of the outflow vein. Conclusions: AMSC-treated vessels had improved vascular remodeling with decreased proinflammatory gene expression, inflammation, and fibrotic staining compared to untreated vessels.

Significance Statement: Expression of immediate early response gene X-1 (Iex-1), also known as Ier-3, is increased in venous neointimal hyperplasia (VNH) and stenosis in hemodialysis arteriovenous fistulas (AVF). In a porcine model, 1α,25(OH)₂D₃, an inhibitor of Ier3, reduced VNH/stenosis formation. The 1α,25(OH)₂D₃ released in the perivascular AVF space from poly(lactic-co-glycolic acid) nanoparticles embedded in a Pluronic F127 hydrogel (1,25 NP) reduced Ier3 gene and protein expression, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining. Concomitantly, blood flow, lumen area, pulse wave velocity, and Young’s modulus increased, and neointima area, peak systolic velocity, and wall shear stress decreased in 1,25 NP–treated vessels. RNA sequencing analysis identified 242 genes involved in inflammatory and apoptotic pathways that had decreased expression in 1,25 NP vessels. Background: Few therapies prevent venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation in arteriovenous fistulas (AVF). Expression of the immediate early response gene X-1 (Iex-1), also known as Ier3, is associated with VNH and stenosis in murine AVFs. The study aimed to determine if local release of Ier3 long-acting inhibitor 1α,25(OH)₂D₃ from poly(lactic-co-glycolic acid) (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (1,25 NP) could affect VNH/VS formation in a large animal model. Methods: Immediately after AVF creation in a porcine model of renal failure, 1,25 NP or vehicle control was injected into the adventitia space of AVF outflow veins. Scanning electron microscopy and dynamic light scattering characterized drug and control nanoparticles. Animals were sacrificed 3 and 28 days later for gene expression, immunohistologic, magnetic resonance imaging and angiography, and ultrasound analyses. Whole transcriptome RNA sequencing with differential gene expression analysis was performed on outflow veins of AVF. Results: Encapsulation of 1α,25(OH)₂D₃ in PLGA nanoparticles formed nanoparticles of uniform size that were similar to nanoparticles without 1α,25(OH)₂D₃. The 1,25 NP–treated AVFs exhibited lower VNH/VS, Ier3 gene expression, and IER-3, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining, fibrosis, and proliferation. Blood flow and lumen area increased significantly, whereas peak systolic velocity and wall shear stress decreased. Treatment increased Young’s modulus and correlated with histologic assessment of fibrosis and with no evidence of vascular calcification. RNA sequencing analysis showed changes in the expression of genes associated with inflammatory, TGFβ1, and apoptotic pathways. Conclusions: Local release of 1,25 NP improves AVF flow and hemodynamics, and reduces stenosis in association with reduction in inflammation, apoptosis, and fibrosis in a porcine model of arteriovenous fistula.

The molecular mechanism of hemodialysis access arteriovenous fistula (AVF) failure due to venous neointimal hyperplasia (VNH) is not known. The role of microRNA-21 (miR-21) in VNH associated with AVF failure was investigated by performing in vivo and in vitro experiments. In situ hybridization results revealed that miR-21 expression increased and was associated with fibroblasts in failed AVFs from patients. In a murine AVF model, qRT-PCR gene expression results showed a significant increase in miR-21 and a decrease in miR-21 target genes in graft veins (GVs) compared to contralateral veins in mouse AVF. miR-21 knockdown in GVs was performed using a lentivirus-mediated small hairpin RNA (shRNA), and this improved AVF patency with a decrease in neointima compared to control GVs. Moreover, loss of miR-21 in GVs significantly decreased the Tgfβ1, Col-Ia, and Col-Iva genes. Immunohistochemistry demonstrated a significant decrease in myofibroblasts and proliferation with an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in miR-21-knockdown vessels, along with a decrease in hypoxia-inducible factor-1 alpha (HIF-1α) and phospho-SMAD2 (pSMAD-2) and phospho-SMAD3 (pSMAD-3) and an increase in phosphatase and tensin homolog (PTEN) staining. Hypoxic fibroblast knockdown for miR-21 showed a significant decrease in Tgfβ-1 expression and pSMAD-2 and -3 levels and a decrease in myofibroblasts. These results indicate that miR-21 upregulation causes VNH formation by fibroblast-to-myofibroblast differentiation.

Significance Statement: Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration into the vasculature. In this study, we used humanized mice knocked in with the human CX3CR1 gene and inhibited CX3CR1 signaling using a variable domains of camelid heavy-chain-only molecule (BI 655088) to test the hypothesis that blockade of CX3CR1 results in less of the venous neointimal hyperplasia formation that is associated with arteriovenous fistula (AVF) failure. We also used human samples removed from failed AVFs combined with cell culture experiments. Our results demonstrate a novel role for CX3CR1 in reducing venous stenosis formation in AVFs. Background: Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration and accumulation, causing venous neointimal hyperplasia (VNH)/venous stenosis (VS) in arteriovenous fistula (AVF). The effect of blocking CX3CR1 using an anti–human variable VHH molecule (hCX3CR1 VHH, BI 655088) on VNH/VS was determined using a humanized mouse in which the human CX3CR1 (hCX3CR1) gene was knocked in (KI). Methods: Whole-transcriptomic RNA sequencing with bioinformatics analysis was used on human stenotic AVF samples, C57BL/6J, hCX3CR1 KI mice with AVF and CKD, and in in vitro experiments to identify the pathways involved in preventing VNH/VS formation after hCX3CR1 VHH administration. Results: Accumulation of CX3CR1 and CD68 was significantly increased in stenotic human AVFs. In C57BL/6J mice with AVF, there was increased Cx3cr1, Cx3cl1, Cd68, and Tnf-α gene expression, and increased immunostaining of CX3CR1 and CD68. In hCX3CR1-KI mice treated with hCX3CR1 VHH molecule (KI-A), compared with vehicle controls (KI-V), there was increased lumen vessel area and patency, and decreased neointima in the AVF outflow veins. RNA-seq analysis identified TNF-α and NF-κB as potential targets of CX3CR1 inhibition. In KI-A–treated vessels compared with KI-V, there was decreased gene expression of Tnf- α, Mcp-1, and Il-1 β; with reduction of Cx3cl1, NF-κB, and Cd68; decreased M1, Ly6C, smooth muscle cells, fibroblast-activated protein, fibronectin, and proliferation; and increased TUNEL and M2 staining. In cell culture, monocytes stimulated with PMA and treated with hCX3CR1 VHH had decreased TNF- α, CD68, proliferation, and migration. Conclusions: CX3CR1 blockade reduces VNH/VS formation by decreasing proinflammatory cues.