Endothelial TGF-ß Signaling Regulates Endothelial-Mesenchymal Transition During Arteriovenous Fistula Remodeling in Mice With Chronic Kidney Disease.

BACKGROUND: Arteriovenous fistulae (AVFs) are the preferred vascular access for hemodialysis in patients with end-stage kidney disease. Chronic kidney disease (CKD) is associated with endothelial injury, impaired AVF maturation, and reduced patency, as well as utilization. Because CKD is characterized by multiple pathophysiological processes that induce endothelial-to-mesenchymal transition (EndMT), we hypothesized that CKD promotes EndMT during venous remodeling and that disruption of endothelial TGF (transforming growth factor)-ß signaling inhibits EndMT to prevent AVF failure even in the end-stage kidney disease environment. METHODS: The mouse 5/6 nephrectomy and aortocaval fistula models were used. CKD was created via 5/6 nephrectomy, with controls of no (0/6) or partial (3/6) nephrectomy in C57BL/6J mice. AVFs were created in mice with knockdown of TGF-ßR1/R2 (TGF-ß receptors type 1/2) in either smooth muscle cells or endothelial cells. AVF diameters and patency were measured and confirmed by serial ultrasound examination. AVF, both murine and human, were examined using Western blot, histology, and immunofluorescence. Human and mouse endothelial cells were used for in vitro experiments. RESULTS: CKD accelerates TGF-ß activation and promotes EndMT that is associated with increased AVF wall thickness and reduced patency in mice. Inhibition of TGF-ß signaling in both endothelial cells and smooth muscle cells decreased smooth muscle cell proliferation in the AVF wall, attenuated EndMT, and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Human AVF also showed increased TGF-ß signaling and EndMT. CONCLUSIONS: CKD promotes EndMT and reduces AVF patency. Inhibition of TGF-ß signaling, especially disruption of endothelial cell-specific TGF-ß signaling, attenuates EndMT and improves AVF patency in mouse AVF. Inhibition of EndMT may be a therapeutic approach of translational significance to improve AVF patency in human patients with CKD.

Disturbed flow in the juxta-anastomotic area of an arteriovenous fistula correlates with endothelial loss, acute thrombus formation, and neointimal hyperplasia.

Clinical failure of arteriovenous neointimal hyperplasia (NIH) fistulae (AVF) is frequently due to juxta-anastomotic NIH (JANIH). Although the mouse AVF model recapitulates human AVF maturation, previous studies focused on the outflow vein distal to the anastomosis. We hypothesized that the juxta-anastomotic area (JAA) has increased NIH compared with the outflow vein. AVF was created in C57BL/6 mice without or with chronic kidney disease (CKD). Temporal and spatial changes of the JAA were examined using histology and immunofluorescence. Computational techniques were used to model the AVF. RNA-seq and bioinformatic analyses were performed to compare the JAA with the outflow vein. The jugular vein to carotid artery AVF model was created in Wistar rats. The neointima in the JAA shows increased volume compared with the outflow vein. Computational modeling shows an increased volume of disturbed flow at the JAA compared with the outflow vein. Endothelial cells are immediately lost from the wall contralateral to the fistula exit, followed by thrombus formation and JANIH. Gene Ontology (GO) enrichment analysis of the 1,862 differentially expressed genes (DEG) between the JANIH and the outflow vein identified 525 overexpressed genes. The rat jugular vein to carotid artery AVF showed changes similar to the mouse AVF. Disturbed flow through the JAA correlates with rapid endothelial cell loss, thrombus formation, and JANIH; late endothelialization of the JAA channel correlates with late AVF patency. Early thrombus formation in the JAA may influence the later development of JANIH.NEW & NOTEWORTHY Disturbed flow and focal endothelial cell loss in the juxta-anastomotic area of the mouse AVF colocalizes with acute thrombus formation followed by late neointimal hyperplasia. Differential flow patterns between the juxta-anastomotic area and the outflow vein correlate with differential expression of genes regulating coagulation, proliferation, collagen metabolism, and the immune response. The rat jugular vein to carotid artery AVF model shows changes similar to the mouse AVF model.

Sex hormones impact early maturation and immune response in the arteriovenous fistula mouse model.

Arteriovenous fistulae (AVF) fail to mature more frequently in female patients compared with male patients, leading to inferior outcomes and decreased utilization. Since our mouse AVF model recapitulates sex differences in human AVF maturation, we hypothesized that sex hormones mediate these differences during AVF maturation. C57BL/6 mice (9-11 wk) were treated with aortocaval AVF surgery and/or gonadectomy. AVF hemodynamics were measured via ultrasound (days 0-21). Blood was collected for FACS and tissue for immunofluorescence and ELISA (days 3 and 7); wall thickness was assessed by histology (day 21). Inferior vena cava shear stress was higher in male mice (P = 0.0028) after gonadectomy, and they had increased wall thickness (22.0 ± 1.8 vs. 12.7 ± 1.2 µm; P < 0.0001). Conversely, female mice had decreased wall thickness (6.8 ± 0.6 vs. 15.3 ± 0.9 µm; P = 0.0002). Intact female mice had higher proportions of circulating CD3+ T cells on day 3 (P = 0.0043), CD4+ (P = 0.0003) and CD8+ T cells (P = 0.005) on day 7, and CD11b+ monocytes on day 3 (P = 0.0046). After gonadectomy, these differences disappeared. In intact female mice, CD3+ T cells (P = 0.025), CD4+ T cells (P = 0.0178), CD8+ T cells (P = 0.0571), and CD68+ macrophages (P = 0.0078) increased in the fistula wall on days 3 and 7. This disappeared after gonadectomy. Furthermore, female mice had higher IL-10 (P = 0.0217) and TNF-α (P = 0.0417) levels in their AVF walls than male mice. Sex hormones mediate AVF maturation, suggesting that hormone receptor signaling may be a target to improve AVF maturation.NEW & NOTEWORTHY After arteriovenous fistula creation, females have lower rates of maturation and higher rates of failure than males. In a mouse model of venous adaptation that recapitulates human fistula maturation, sex hormones may be mechanisms of the sexual dimorphism: testosterone is associated with reduced shear stress, whereas estrogen is associated with increased immune cell recruitment. Modulating sex hormones or downstream effectors suggests sex-specific therapies and could address disparities in sex differences in clinical outcomes.

Endothelial Cell TGF-ß (Transforming Growth Factor-Beta) Signaling Regulates Venous Adaptive Remodeling to Improve Arteriovenous Fistula Patency.

BACKGROUND: Arteriovenous fistulae (AVF) are the gold standard for vascular access for hemodialysis. Although the vein must thicken and dilate for successful hemodialysis, excessive wall thickness leads to stenosis causing AVF failure. Since TGF-ß (transforming growth factor-beta) regulates ECM (extracellular matrix) deposition and smooth muscle cell (SMC) proliferation-critical components of wall thickness-we hypothesized that disruption of TGF-ß signaling prevents excessive wall thickening during venous remodeling. METHODS: A mouse aortocaval fistula model was used. SB431542-an inhibitor of TGF-ß receptor I-was encapsulated in nanoparticles and applied to the AVF adventitia in C57BL/6J mice. Alternatively, AVFs were created in mice with conditional disruption of TGF-ß receptors in either SMCs or endothelial cells. Doppler ultrasound was performed serially to confirm patency and to measure vessel diameters. AVFs were harvested at predetermined time points for histological and immunofluorescence analyses. RESULTS: Inhibition of TGF-ß signaling with SB431542-containing nanoparticles significantly reduced p-Smad2-positive cells in the AVF wall during the early maturation phase (days 7-21) and was associated with decreased AVF wall thickness that showed both decreased collagen density and decreased SMC proliferation. SMC-specific TGF-ß signaling disruption decreased collagen density but not SMC proliferation or wall thickness. Endothelial cell-specific TGF-ß signaling disruption decreased both collagen density and SMC proliferation in the AVF wall and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. CONCLUSIONS: Endothelial cell-targeted TGF-ß inhibition may be a translational strategy to improve AVF patency.

PD-L1 (Programmed Death Ligand 1) Regulates T-Cell Differentiation to Control Adaptive Venous Remodeling.

OBJECTIVE: Patients with end-stage renal disease depend on hemodialysis for survival. Although arteriovenous fistulae (AVF) are the preferred vascular access for hemodialysis, the primary success rate of AVF is only 30% to 50% within 6 months, showing an urgent need for improvement. PD-L1 (programmed death ligand 1) is a ligand that regulates T-cell activity. Since T cells have an important role during AVF maturation, we hypothesized that PD-L1 regulates T cells to control venous remodeling that occurs during AVF maturation. Approach and results: In the mouse aortocaval fistula model, anti-PD-L1 antibody (200 mg, 3×/wk intraperitoneal) was given to inhibit PD-L1 activity during AVF maturation. Inhibition of PD-L1 increased T-helper type 1 cells and T-helper type 2 cells but reduced regulatory T cells to increase M1-type macrophages and reduce M2-type macrophages; these changes were associated with reduced vascular wall thickening and reduced AVF patency. Inhibition of PD-L1 also inhibited smooth muscle cell proliferation and increased endothelial dysfunction. The effects of anti-PD-L1 antibody on adaptive venous remodeling were diminished in nude mice; however, they were restored after T-cell transfer into nude mice, indicating the effects of anti-PD-L1 antibody on venous remodeling were dependent on T cells. CONCLUSIONS: Regulation of PD-L1 activity may be a potential therapeutic target for clinical translation to improve AVF maturation.

Inhibition of T-Cells by Cyclosporine A Reduces Macrophage Accumulation to Regulate Venous Adaptive Remodeling and Increase Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) are the preferred vascular access for hemodialysis, but the primary success rate of AVF remains poor. Successful AVF maturation requires vascular wall thickening and outward remodeling. A key factor determining successful AVF maturation is inflammation that is characterized by accumulation of both T-cells and macrophages. We have previously shown that anti-inflammatory (M2) macrophages are critically important for vascular wall thickening during venous remodeling; therefore, regulation of macrophage accumulation may be an important mechanism promoting AVF maturation. Since CD4+ T-cells such as T-helper type 1 cells, T-helper type 2 cells, and regulatory T-cells can induce macrophage migration, proliferation, and polarization, we hypothesized that CD4+ T-cells regulate macrophage accumulation to promote AVF maturation. Approach and Results: In a mouse aortocaval fistula model, T-cells temporally precede macrophages in the remodeling AVF wall. CsA (cyclosporine A; 5 mg/kg, sq, daily) or vehicle (5% dimethyl sulfoxide) was administered to inhibit T-cell function during venous remodeling. CsA reduced the numbers of T-helper type 1 cells, T-helper type 2, and regulatory T-cells, as well as M1- and M2-macrophage accumulation in the wall of the remodeling fistula; these effects were associated with reduced vascular wall thickening and increased outward remodeling in wild-type mice. However, these effects were eliminated in nude mice, showing that the effects of CsA on macrophage accumulation and adaptive venous remodeling are T-cell-dependent. CONCLUSIONS: T-cells regulate macrophage accumulation in the maturing venous wall to control adaptive remodeling. Regulation of T-cells during AVF maturation may be a strategy that can improve AVF maturation. Graphic Abstract: A graphic abstract is available for this article.

TGFß (Transforming Growth Factor-Beta)-Activated Kinase 1 Regulates Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) are the optimal conduit for hemodialysis access but have high rates of primary maturation failure. Successful AVF maturation requires wall thickening with deposition of ECM (extracellular matrix) including collagen and fibronectin, as well as lumen dilation. TAK1 (TGFß [transforming growth factor-beta]-activated kinase 1) is a mediator of noncanonical TGFß signaling and plays crucial roles in regulation of ECM production and deposition; therefore, we hypothesized that TAK1 regulates wall thickening and lumen dilation during AVF maturation. Approach and Results: In both human and mouse AVF, immunoreactivity of TAK1, JNK (c-Jun N-terminal kinase), p38, collagen 1, and fibronectin was significantly increased compared with control veins. Manipulation of TAK1 in vivo altered AVF wall thickening and luminal diameter; reduced TAK1 function was associated with reduced thickness and smaller diameter, whereas activation of TAK1 function was associated with increased thickness and larger diameter. Arterial magnitudes of laminar shear stress (20 dyne/cm2) activated noncanonical TGFß signaling including TAK1 phosphorylation in mouse endothelial cells. CONCLUSIONS: TAK1 is increased in AVF, and TAK1 manipulation in a mouse AVF model regulates AVF thickness and diameter. Targeting noncanonical TGFß signaling such as TAK1 might be a novel therapeutic approach to improve AVF maturation.

Stimulation of Caveolin-1 Signaling Improves Arteriovenous Fistula Patency.

Objective- Arteriovenous fistulae (AVF) are the most common access created for hemodialysis; however, many AVF fail to mature and require repeated intervention, suggesting a need to improve AVF maturation. Eph-B4 (ephrin type-B receptor 4) is the embryonic venous determinant that is functional in adult veins and can regulate AVF maturation. Cav-1 (caveolin-1) is the major scaffolding protein of caveolae-a distinct microdomain that serves as a mechanosensor at the endothelial cell membrane. We hypothesized that Cav-1 function is critical for Eph-B4-mediated AVF maturation. Approach and Results- In a mouse aortocaval fistula model, both Cav-1 mRNA and protein were increased in the AVF compared with control veins. Cav-1 KO (knockout) mice showed increased fistula wall thickening ( P=0.0005) and outward remodeling ( P<0.0001), with increased eNOS (endothelial NO synthase) activity compared with WT (wild type) mice. Ephrin-B2/Fc inhibited AVF outward remodeling in WT mice but not in Cav-1 KO mice and was maintained in Cav-1 RC (Cav-1 endothelial reconstituted) mice (WT, P=0.0001; Cav-1 KO, P=0.7552; Cav-1 RC, P=0.0002). Cavtratin-a Cav-1 scaffolding domain peptide-decreased AVF wall thickness in WT mice and in Eph-B4 het mice compared with vehicle alone (WT, P=0.0235; Eph-B4 het, P=0.0431); cavtratin also increased AVF patency (day 42) in WT mice ( P=0.0275). Conclusions- Endothelial Cav-1 mediates Eph-B4-mediated AVF maturation. The Eph-B4-Cav-1 axis regulates adaptive remodeling during venous adaptation to the fistula environment. Manipulation of Cav-1 function may be a translational strategy to enhance AVF patency.

Transforming Growth Factor-ß1 Inhibits Pseudoaneurysm Formation After Aortic Patch Angioplasty.

OBJECTIVE: Pseudoaneurysms remain a significant complication after vascular procedures. We hypothesized that TGF-ß (transforming growth factor-ß) signaling plays a mechanistic role in the development of pseudoaneurysms. APPROACH AND RESULTS: Rat aortic pericardial patch angioplasty was associated with a high incidence (88%) of pseudoaneurysms at 30 days, with increased smad2 phosphorylation in small pseudoaneurysms but not in large pseudoaneurysms; TGF-ß1 receptors were increased in small pseudoaneurysms and preserved in large pseudoaneurysms. Delivery of TGF-ß1 via nanoparticles covalently bonded to the patch stimulated smad2 phosphorylation both in vitro and in vivo and significantly decreased pseudoaneurysm formation (6.7%). Inhibition of TGF-ß1 signaling with SB431542 decreased smad2 phosphorylation both in vitro and in vivo and significantly induced pseudoaneurysm formation by day 7 (66.7%). CONCLUSIONS: Normal healing after aortic patch angioplasty is associated with increased TGF-ß1 signaling, and recruitment of smad2 signaling may limit pseudoaneurysm formation; loss of TGF-ß1 signaling is associated with the formation of large pseudoaneurysms. Enhancement of TGF-ß1 signaling may be a potential mechanism to limit pseudoaneurysm formation after vascular intervention.

Early thrombus formation is required for eccentric and heterogeneous neointimal hyperplasia under disturbed flow.

BACKGROUND: Anticoagulation and antiplatelet therapy effectively inhibit neointimal hyperplasia (NIH) in both arterial and venous systems but not in arteriovenous fistulae (AVF). The main site of AVF failure is the juxta-anastomotic area that is characterized by disturbed flow compared with laminar flow in the arterial inflow and the venous outflow. OBJECTIVES: We hypothesized that early thrombus formation is required for eccentric and heterogeneous NIH in the presence of disturbed flow. METHODS: Needle puncture and sutured AVF were created in C57BL/6 mice, in PF4-Cre × mT/mG reporter mice, and in Wistar rats. Human AVF samples were second-stage basilic vein transpositions. The tissues were examined by histology, immunofluorescence, immunohistochemistry, and en face staining. RESULTS: In the presence of disturbed flow, both mouse and human AVF showed eccentric and heterogeneous NIH. Maladapted vein wall was characterized by eccentric and heterogeneous neointima that was composed of a different abundance of thrombus and smooth muscle cells. PF4-cre × mT/mG reporter mice AVF showed that green fluorescent protein-labeled platelets deposit on the wall directly facing the fistula exit with endothelial cell loss and continue to accumulate in the presence of disturbed flow. Neither disturbed flow with limited endothelial cell loss nor nondisturbed flow induced heterogeneous neointima in different animal models. CONCLUSION: Early thrombus contributes to late heterogeneous NIH in the presence of disturbed flow. Disturbed flow, large area of endothelial cell loss, and thrombus formation are critical to form eccentric and heterogeneous NIH. Categorization of adapted or maladapted walls may be helpful for therapy targeting heterogeneous NIH.

Heterogeneous gene expression during early arteriovenous fistula remodeling suggests that downregulation of metabolism predicts adaptive venous remodeling.

Clinical outcomes of arteriovenous fistulae (AVF) for hemodialysis remain inadequate since biological mechanisms of AVF maturation and failure are still poorly understood. Aortocaval fistula creation (AVF group) or a sham operation (sham group) was performed in C57BL/6 mice. Venous limbs were collected on postoperative day 7 and total RNA was extracted for high throughput RNA sequencing and bioinformatic analysis. Genes in metabolic pathways were significantly downregulated in the AVF, whereas significant sex differences were not detected. Since gene expression patterns among the AVF group were heterogenous, the AVF group was divided into a 'normal' AVF (nAVF) group and an 'outliers' (OUT) group. The gene expression patterns of the nAVF and OUT groups were consistent with previously published data showing venous adaptive remodeling, whereas enrichment analyses showed significant upregulation of metabolism, inflammation and coagulation in the OUT group compared to the nAVF group, suggesting the heterogeneity during venous remodeling reflects early gene expression changes that may correlate with AVF maturation or failure. Early detection of these processes may be a translational strategy to predict fistula failure and reduce patient morbidity.

A central arteriovenous fistula reduces systemic hypertension in a mouse model.

Objective: A central arteriovenous fistula (AVF) has been proposed as a potential novel solution to treat patients with refractory hypertension. We hypothesized that venous remodeling after AVF creation in the hypertensive environment reduces systemic blood pressure but results in increased AVF wall thickness compared with remodeling in the normotensive environment. Methods: A central AVF was performed in C57BL6/J mice previously made hypertensive with angiotensin II (Ang II); mice were sacrificed on postoperative day 7 or 21. Results: In mice treated with Ang II alone, the mean systolic blood pressure increased from 90 ± 5 mmHg to 160 ± 5 mmHg at day 21; however, in mice treated with both Ang II and an AVF, the blood pressure decreased with creation of an AVF. There were significantly more PCNA-positive cells, SM22α/PCNA-positive cells, collagen I deposition, and increased Krüppel-like Factor 2 immunoreactivity in hypertensive mice with an AVF compared with normotensive mice with an AVF. Conclusions: These data show that a central AVF decreases systemic hypertension as well as induces local alterations in venous remodeling.

Interferon-gamma induces prolyl hydroxylase (PHD)3 through a STAT1-dependent mechanism in human endothelial cells.

OBJECTIVE: We previously reported that interferons (IFNs) regulate transcription of HIF-1alpha in human endothelial cells (ECs), linking immunity and hypoxia. Prolyl hydroxylases (PHDs) regulate expression of HIF-1alpha in response to hypoxia. We examined whether IFNs affect PHD expression and whether PHDs regulate the EC response to IFNs. METHODS AND RESULTS: Human cell cultures were treated with various cytokines, and PHD expression was examined using qRT-PCR and immunoblotting. IFNgamma and, to a lesser extent, IFNalpha significantly induced PHD3, but not PHD1 or 2, mRNA, and protein expression selectively in ECs directly via a JAK/STAT1 pathway as demonstrated by pharmacological inhibition, siRNA knockdown, and chromatin immunoprecipitation. Inhibition of PHD activity with dimethyloxallyl glycine or desferroxamine reduced IFNg-dependent responses in these same cells. CONCLUSIONS: IFNgamma induces PHD3 through a JAK/STAT1-dependent mechanism in human ECs. Induction is independent of HIF-1alpha and may contribute to expression of IFNgamma-dependent genes.

Altered hemodynamics during arteriovenous fistula remodeling leads to reduced fistula patency in female mice.

OBJECTIVE: The arteriovenous fistula (AVF) is the preferred method of dialysis access because of its proven superior long-term outcomes.However, women havelower rates of AVF patency andutilizationthan men.We used a novel mouseAVF model that recapitulates human AVF maturation to determine whether there are differences in AVF patency in female and male mice. METHODS: Aortocaval fistulas were created in female and male C57BL/6 mice (9-10 weeks). At days 0, 3, 7, and 21, infrarenal inferior vena cava (IVC) and aortic diameters and flow velocity were monitored by Doppler ultrasound and used to calculate the vessel diameter, blood flow, and shear stress. AVF were harvested, and expression of proteins was examined by proteomic analysis and immunofluorescence and of messenger RNA by quantitative polymerase chain reaction analysis. RESULTS: At baseline, female mice weighed less and had lower IVC velocity and smaller magnitudes of shear stress, but there was no significant difference in IVC diameter and thickness. After AVF creation, both female and male mice had similar IVC dilation and thickening with no significant differences in IVC wall thickness at day 21. However, female mice had diminished AVF patency by day 42 (25.7% vs 64.3%; P = .039). During fistula remodeling, female mice had lower IVC mean velocity and shear stress magnitude and increased spectral broadening (days 0-21). Messenger RNA and protein expression of Krüppel-like factor 2, endothelial nitric oxide synthase, and vascular cell adhesion molecule 1 was similar at baseline in female and male mice but increased in the AVF only in male mice but not in female mice (day 21). Proteomic analysis of female and male mice detected 56 proteins expressed at significantly higher levels in the IVC of female mice and 67 proteins expressed at significantly higher levels in the IVC of male mice (day 7); function-specific analysis showed that the IVC of male mice overexpressed proteins that belong to pathways implicated in the regulation of vascular function, thrombosis, response to flow, and vascular remodeling. CONCLUSIONS: AVF in female mice have diminished patency, preceded by lower velocity, reduced magnitudes of shear stress, and less laminar flow during remodeling. There is also sex-specific differential expression of proteins involved in thrombosis, response to laminar flow, inflammation, and proliferation. These findings suggest that hemodynamic changes during fistula maturation may play an important role underlying the diminished rates of AVF utilization in women. CLINICAL RELEVANCE: Women have lower rates of arteriovenous fistula (AVF) utilization than men. Using a mouse AVF model that recapitulates human AVF maturation, we show that female mice have similar AVF remodeling but diminished patency. AVF remodeling in female mice is associated with reduced shear stress and laminar flow; lack of increased transcription and translation of several anti-inflammatory, antiproliferative, and laminar flow response proteins (endothelial nitric oxide synthase, Krüppel-like factor 2, and vascular cell adhesion molecule 1); and different patterns of expression of pathways that regulate thrombosis and venous remodeling. Identifying downstream targets involved in these mechanisms may improve AVF outcomes in female patients.

A mouse model of stenosis distal to an arteriovenous fistula recapitulates human central venous stenosis.

OBJECTIVE: Central venous stenosis (CVS) is a major cause of arteriovenous fistula (AVF) failure. However, central veins are relatively inaccessible to study with conventional Doppler ultrasound methods. To understand mechanisms underlying AVF failure owing to CVS, an animal model was established that creates a stenosis distal to an AVF. We hypothesized that this mouse model will show comparable morphology and physiology to human CVS. METHODS: An aortocaval fistula was created between the distal aorta and inferior vena cava (IVC); a stenosis was then created distal to the fistula by partial IVC ligation. Sham-operated animals, AVF without venous stenosis, and venous stenosis without AVF were used as controls. Physiologic properties of the IVC, both upstream and downstream of the stenosis, or the corresponding sites in models without stenosis, were assessed with ultrasound examination on days 0 to 21. The spectral broadening index was measured to assess the degree of disturbed shear stress. The IVC was harvested at day 21 and specimens were analyzed with immunofluorescence. RESULTS: The IVC diameter of mice with an AVF and stenosis showed increased upstream (P = .013), but decreased downstream diameter (P = .001) compared with mice with an AVF but without a stenosis, at all postoperative times (days 3-21). IVC wall thickness increased in mice with an AVF, compared with IVC without an AVF (upstream of stenosis: 13.9 µm vs 11.0 µm vs 4.5 µm vs 3.9 µm; P = .020; downstream of stenosis: 6.0 µm vs 6.6 µm vs µm 4.5 µm vs 3.8 µm; P = .002; AVF with stenosis, AVF, stenosis, sham, respectively). AVF patency significantly decreased in mice with an AVF and stenosis by day 21 (50% vs 90%; P = .048). The IVC of mice with AVF and stenosis showed a venous waveform with pulsatility as well as enhanced velocity at and downstream of the stenosis; similar waveforms were observed in a human case of CVS. Downstream to the stenosis, the spectral broadening index was significantly higher compared with mice with AVF alone (1.06 vs 0.78; P = .011; day 21), and there was a trend towards less immunoreactivity of both Krüppel-like factor 2 and phosphorylated-endothelial nitric oxide synthase compared with mice with an AVF alone. CONCLUSIONS: Partial IVC ligation distal to a mouse aortocaval fistula alters the fistula diameter and wall thickness, decreases patency, and increases distal disturbed flow compared with fistulae without a distal stenosis. Our mouse model of stenosis distal to an AVF may be a faithful representation of human CVS that shows similar morphology and physiology, including disturbed shear stress.

Author Correction: Inhibition of the Akt1-mTORC1 Axis Alters Venous Remodeling to Improve Arteriovenous Fistula Patency.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibition of the Akt1-mTORC1 Axis Alters Venous Remodeling to Improve Arteriovenous Fistula Patency.

Arteriovenous fistulae (AVF) are the most common access created for hemodialysis, but up to 60% do not sustain dialysis within a year, suggesting a need to improve AVF maturation and patency. In a mouse AVF model, Akt1 regulates fistula wall thickness and diameter. We hypothesized that inhibition of the Akt1-mTORC1 axis alters venous remodeling to improve AVF patency. Daily intraperitoneal injections of rapamycin reduced AVF wall thickness with no change in diameter. Rapamycin decreased smooth muscle cell (SMC) and macrophage proliferation; rapamycin also reduced both M1 and M2 type macrophages. AVF in mice treated with rapamycin had reduced Akt1 and mTORC1 but not mTORC2 phosphorylation. Depletion of macrophages with clodronate-containing liposomes was also associated with reduced AVF wall thickness and both M1- and M2-type macrophages; however, AVF patency was reduced. Rapamycin was associated with improved long-term patency, enhanced early AVF remodeling and sustained reduction of SMC proliferation. These results suggest that rapamycin improves AVF patency by reducing early inflammation and wall thickening while attenuating the Akt1-mTORC1 signaling pathway in SMC and macrophages. Macrophages are associated with AVF wall thickening and M2-type macrophages may play a mechanistic role in AVF maturation. Rapamycin is a potential translational strategy to improve AVF patency.

Molecular Targets for Improving Arteriovenous Fistula Maturation and Patency.

The increasing prevalence of chronic and end-stage renal disease creates an increased need for reliable vascular access, and although arteriovenous fistulae (AVF) are the preferred mode of hemodialysis access, 60% fail to mature and only 50% remain patent at one year. Fistulae mature by diameter expansion and wall thickening; this outward remodeling of the venous wall in the fistula environment relies on a delicate balance of extracellular matrix (ECM) remodeling, inflammation, growth factor secretion, and cell adhesion molecule upregulation in the venous wall. AVF failure occurs via two distinct mechanisms with early failure secondary to lack of outward remodeling, that is insufficient diameter expansion or wall thickening, whereas late failure occurs with excessive wall thickening due to neointimal hyperplasia (NIH) and insufficient diameter expansion in a previously functional fistula. In recent years, the molecular basis of AVF maturation and failure are becoming understood in order to develop potential therapeutic targets to aide maturation and prevent access loss. Erythropoietin-producing hepatocellular carcinoma (Eph) receptors, along with their ligands, ephrins, determine vascular identity and are critical for vascular remodeling in the embryo. Manipulation of Eph receptor signaling in adults, as well as downstream pathways, is a potential treatment strategy to improve the rates of AVF maturation and patency. This review examines our current understanding of molecular changes occurring following fistula creation, factors predictive of fistula success, and potential areas of intervention to decrease AVF failure.

Mutations in Chromatin Modifier and Ephrin Signaling Genes in Vein of Galen Malformation.

Normal vascular development includes the formation and specification of arteries, veins, and intervening capillaries. Vein of Galen malformations (VOGMs) are among the most common and severe neonatal brain arterio-venous malformations, shunting arterial blood into the brain's deep venous system through aberrant direct connections. Exome sequencing of 55 VOGM probands, including 52 parent-offspring trios, revealed enrichment of rare damaging de novo mutations in chromatin modifier genes that play essential roles in brain and vascular development. Other VOGM probands harbored rare inherited damaging mutations in Ephrin signaling genes, including a genome-wide significant mutation burden in EPHB4. Inherited mutations showed incomplete penetrance and variable expressivity, with mutation carriers often exhibiting cutaneous vascular abnormalities, suggesting a two-hit mechanism. The identified mutations collectively account for ∼30% of studied VOGM cases. These findings provide insight into disease biology and may have clinical implications for risk assessment.

Autologous tissue patches acquire vascular identity depending on the environment.

Vascular identity is genetically determined, but can be altered during surgical procedures. We hypothesized that the environment of the procedure critically alters the identity of autologous tissue patches implanted into the arterial or venous environment. Autologous jugular vein or carotid artery was used as a patch to repair a rat aorta or inferior vena cava. In the aortic environment patches contained neointimal cells that were CD34/Ephrin-B2-dual positive but not CD34/Eph-B4-dual positive; patches expressed Ephrin-B2, notch-4 and dll-4 but not Eph-B4 and COUP-TFII. In the venous environment patches contained neointimal cells that were CD34/Eph-B4-dual positive but not CD34/Ephrin-B2-dual positive; patches expressed Eph-B4 and COUP-TFII but not Ephrin-B2, notch-4 and dll-4. These data show that autologous tissue patches heal by acquisition of the vascular identity determined by the environment into which they are implanted, suggesting some plasticity of adult vascular identity.