Arteriovenous fistulas (AVFs) used for hemodialysis fail because of venous neointimal hyperplasia (VNH). There are 1,500,000 patients that have end stage renal disease worldwide and the majority requires hemodialysis. In the present study, the role of the intermediate early response gene X-1 (IEX-1), also known as IER-3 in the pathogenesis of VNH was evaluated. In human samples removed from failed AVF, there was a significant increase in IEX-1 expression localized to the adventitia. In Iex-1 −/− mice and wild type (WT) controls, chronic kidney disease was induced and an AVF placed 28 days later by connecting the carotid artery to jugular vein. The outflow vein was removed three days following the creation of the AVF and gene expression analysis demonstrated a significant decrease in vascular endothelial growth factor-A (Vegf-A) and monocyte chemoattractant protein-1 (Mcp-1) gene expression in Iex-1 −/− mice when compared to WT mice (P<0.05). At 28 days after AVF placement, histomorphometric and immune-histochemical analyses of the outflow vein demonstrated a significant decrease in neointimal hyperplasia with an increase in average lumen vessel area associated with a decrease in fibroblast, myofibroblast, and Ly6C staining. There was a decrease in proliferation (Ki-67) and an increase in the TUNEL staining in Iex-1 KO mice compared to WT. In addition, there was a decrease in Vegf-A, Mcp-1, and matrix metalloproteiniase-9 (Mmp-9) staining. Iex-1 expression was reduced in vivo and in vitro using nanoparticles coated with calcitriol, an inhibitor of Iex-1 that demonstrated that Iex-1 reduction results in decrease in Vegf-A. In aggregate, these results indicate that the absence of IEX-1 gene results in reduced VNH accompanied with a decrease in proliferation, reduced fibroblast, myofibroblast, and Ly6C staining accompanied with increased apoptosis mediated through a reduction in Vegf-A/Mcp-1 axis and Mmp-9. Adventitial delivery of nanoparticles coated with calcitriol reduced Iex-1 and VNH.
Introduction: Arteriovenous fistulas (AVFs) used for hemodialysis vascular access will fail because of venous neointimal hyperplasia (VNH) that causes stenosis formation at the venous outflow tract. As a result, only 60% of all AVFs are patent at 1-year. There are several factors which have been hypothesized to contribute to VNH including shear stress, hypoxia, and both local and systemic inflammation. Mesenchymal stem cells (MSCs) have been demonstrated to reduce inflammation while having antiproliferitive properties that aid abrogating vascular injury. Hypothesis: We hypothesized that adventitial transplantation of human adipose derived MSCs to the outflow vein of AVF created in CD1-FoxN1 would attenuate VNH failure by reducing pro-inflammatory mediators including tissue necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-1 beta (IL-1β). Methods: Adventitial transplantation of human MSC was performed in CD1-FoxN1 mice. A carotid artery to ipsilateral jugular AVF was placed and animals were divided into three groups: MSCs (M), CorMatrix ECM scaffold (S, CorMatrix Scaffold, Roswell, GE), or scaffold embedded with 250,000 MSCs (S+M) which were delivered to the adventitia. The mice were then sacrificed at 7 and 28 days post AVF placement and the outflow veins were harvested for histomorphometric and qPCR expression analysis. Results: The average lumen vessel area was significantly increased in the outflow veins removed from the S + M group compared to S or M alone at days 7 and 21 (both, P<0.05). The average area of neointima was significantly decreased in the S+M group when compared to S or M groups (P<0.05) while cell density and Ki-67 index were also significantly decreased at day 7 between S and S+M groups (P<0.05). RNA expression analysis revealed that MCP-1, IL-1β, and TNF-α were all significantly decreased in the outflow vein removed from the S+M group when compared to the S or M group (P<0.05). Conclusion: Adventitial transplantation of MSCs to the outflow vein of the AVF at the time of placement attenuates VNH mediated through a reduction of MCP-1, IL-1β, and TNF-α.
Purpose: The majority of patients with end stage renal disease who require hemodialysis use a arteriovenous fistula (AVF) for their vascular access. However, only 40% of AVFs are clinically usable after 1 year. Arteriovenous fistulas fail because of venous neointimal hyperplasia (VNH) that develops in the proximal outflow vein. The immediate early gene, IEX-1 (IER-3), is increased in response to shear stress and hypoxia and both factors contribute to VNH. IEX-1 expression was increased in stenotic venous stenoses removed from patients with hemodialysis vascular access compared to controls. We hypothesized that reducing the expression of IEX-1 using a knockout mouse model with chronic kidney disease (CKD) and AVFs would have decreased VNH compared to wild type (WT) mice. Methods: CKD was induced by performing a nephrectomy. Four weeks later, an AVF was placed to connect the carotid artery to ipsilateral jugular vein. The mice were sacrificed 3 and 28 days after fistula creation and outflow veins were harvested for gene expression and histomorphometric analyses. Results: By day 3, the average gene expression of vascular endothelial growth factor-A (VEGF-A) was significantly lower in the IEX-1 KO vs. WT mice (average reduction: 40%, P<0.05). At day 28, the average lumen vessel area was increased significantly (average increase: 133%, P<0.05, IEX-1 KO vs. WT, respectively) while the average area of the neointima was significantly decreased (average reduction: 20%, IEX-1 KO vs. WT, respectively). Accompanied with this, there was a significant decrease in α-SMA staining and Ki-67 proliferation while the average TUNEL staining was significantly increased (all P<0.05, IEX-1 KO vs. WT). Finally, there was a significant decrease in the average staining intensity for VEGF-A and matrix metalloproteinase-9 (MMP-9) in the KO vs. WT mice (both, P<0.05). Conclusion: A reduction in IEX-1 gene expression results in positive vascular remodeling accompanied with a reduction in proliferation and α-SMA density mediated through a VEGF-A/MMP-9 pathway. Taken collectively, these findings indicate that reducing IEX-1 expression is protective for VNH formation and provide a rationale for use of anti-IEX-1 therapies aimed at prolonging AVF function.
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