McCall, Deborah

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.

The exact molecular mechanisms involved in hemodialysis arteriovenous fistula (AVF) failure caused by venous neointimal hyperplasia (VNH) are not clear. It has been observed that there is an accumulation of extracellular matrix and up regulation of pro-fibrotic genes accompanied with presence of fibroblasts, smooth muscle cells, and inflammatory cells in the stenotic veins. Previous studies have demonstrated that adventitial and medial fibroblasts have a pivotal role(s) in VNH formation. MicroRNA-21 (miR-21) contributes to fibroblast to myofibroblast differentiation and dysregulation of miR-21 plays a pathological role in failure of coronary artery bypass grafts. The aim of the present study was to determine the role of miR-21 in VNH associated with AVF. We assessed miR-21 expression using qRT-PCR in the outflow veins of AVFs compared to control (contralateral jugular veins) veins in the C57BL/6J mice with chronic kidney disease (CKD). MiR-21 expression was upregulated accompanied with down regulation of miR-21 target genes; PPAR-α, PTEN and TIMP-3. In addition, gene expression of fibroblast specific protein (FSP) -1, TGF (transforming growth factor) -β1, matrix metalloproteinases (MMP)-2, -9, collagen-I, and IV were significantly increased at day 7 after AVF creation. Immunohistochemistry revealed that there was a significant increase in proliferating cell index (Ki-67) and fibroblast index (FSP-1) in the outflow veins of AVFs. Hypoxia has been shown to increase fibroblast to myofibroblast differentiation and this is predicted to be an early step in VNH formation. Therefore we assessed miR-21 expression in hypoxic (1%O 2 ) mouse pulmonary vein fibroblasts compared to normoxic cells in vitro and it was found that miR-21and TGF-β1 significantly elevated with down regulation of miR-21 target genes PTEN and TIMP-3. Furthermore, miR-21 knockdown in hypoxic fibroblasts attenuated TGF-β1 expression with a significant upregulation of genes targeted by miR-21 compared to controls. Together these results indicate that upregulation of miR-21 expression may result in fibroblast to myofibroblast differentiation resulting in VNH formation.

Introduction: Venous neointimal hyperplasia (VNH) is a major cause of hemodialysis arteriovenous fistula (AVF) vascular access failure. CX3CR1 mediates macrophage infiltration into the vasculature. Mice were used which had been genetically engineered to knock in the human CX3CR1 gene. Hypothesis: The hypothesis to be tested is that increased CX3CR1 gene expression results in VNH formation associated with AVF. Methods: We used 50 CX3CR1 knock in mice which were divided into 2 groups: 1. CX3CR1 antibody (30 mg/kg administered intraperitoneally two times per week) or vehicle (equal amount of volume used for CX3CR1 antibody administered intraperitoneally). AVFs were created by connecting the carotid artery to ipsilateral jugular vein 28 days after nephrectomy was performed to induce chronic kidney disease. Histology and Immunohistochemistry analysis at the outflow vein of AVF were performed to assess the pathological changes. Results: There was a significant decrease in neointima area in the outflow veins of AVF in CX3CR1 antibody group compared to the vehicle group at day 28 (43085 ± 12678 vs. 58963.4 ± 9273 μm 2 , P<0.05). There was a significant decrease in the ratio of neointima/media+adventitia area in the outflow veins of AVF in CX3CR1 antibody group compared to the vehicle group at day 28 (0.34 ± 0.1 vs. 0.61 ± 0.1, P<0.05). Cell density in neointima area in CX3CR1 group outflow veins was significantly lower than the vehicle group at day 14 (9600 ± 1000 vs. 13000 ± 3000 /mm 2 , P<0.01). There was a significant decrease in the average CD68-positive cell density in the CX3CR1 group outflow veins compared to the vehicle group (0.96 ± 0.34 vs.15.9 ±10, P<0.001) at day 14. Conclusion: Decreasing CX3CR1 significantly reduces macrophages infiltration and results in a significant reduction in VNH. This study provides a rationale for using CX3CR1 antibody in reducing VNH formation.

Mechanical ventilation may cause harm by straining lungs at a time they are particularly prone to injury from deforming stress. The objective of this study was to define the relative contributions of alveolar overdistension and cyclic recruitment and “collapse” of unstable lung units to membrane wounding of alveolar epithelial cells. We measured the interactive effects of tidal volume (V T ), transpulmonary pressure (P TP ), and of airspace liquid on the number of alveolar epithelial cells with plasma membrane wounds in ex vivo mechanically ventilated rat lungs. Plasma membrane integrity was assessed by propidium iodide (PI) exclusion in confocal images of subpleural alveoli. Cyclic inflations of normal lungs from zero end-expiratory pressure to 40 cmH 2 O produced V T values of 56.9 ± 3.1 ml/kg and were associated with 0.12 ± 0.12 PI-positive cells/alveolus. A preceding tracheal instillation of normal saline (3 ml) reduced V T to 49.1 ± 6 ml/kg but was associated with a significantly greater number of wounded alveolar epithelial cells (0.52 ± 0.16 cells/alveolus; P < 0.01). Mechanical ventilation of completely saline-filled lungs with saline (V T = 52 ml/kg) to pressures between 10 and 15 cmH 2 O was associated with the least number of wounded epithelial cells (0.02 ± 0.02 cells/alveolus; P < 0.01). In mechanically ventilated, partially saline-filled lungs, the number of wounded cells increased substantially with V T , but, once V T was accounted for, wounding was independent of maximal P TP . We found that interfacial stress associated with the generation and destruction of liquid bridges in airspaces is the primary biophysical cell injury mechanism in mechanically ventilated lungs.