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.

[Silver Ion Decreases Foreign Body Reaction and Venous Neointimal Hyperplasia through the Inhibition of Interleukin-33 Expression].

INTRODUCTION: Vascular prosthetic grafts are widely used in vascular surgery; however, graft infection remains a major concern. Silver-coated vascular grafts have demonstrated anti-infection properties in clinical settings; however, whether the silver irons influence foreign body reaction or neointimal hyperplasia remains unclear. METHODS: Sodium alginate and hyaluronic acid (SA/HA) hydrogel patches loaded with rhodamine, with or without silver, were fabricated. Patches were implanted in the subcutaneous or abdominal cavity and inferior vena cava of rats. Samples were harvested on day 14 and examined via immunohistochemical and immunofluorescence analyses. RESULTS: Silver hydrogel was found to decrease the foreign body reaction; after subcutaneous and abdominal cavity implantation in rats, the capsule was found to be thinner in the silver hydrogel group than in the control hydrogel group. The silver hydrogel group had fewer CD68-positive cells and proliferating cell nuclear antigen and interleukin-33 (IL-33) dual-positive cells than the control hydrogel group. Additionally, the silver hydrogel patch reduced the neointimal thickness after patch venoplasty in rats, and the number of IL-33- and IL-1ß-positive cells was lower than that in the control patch. CONCLUSION: Silver-loaded SA/HA hydrogel patches decreased the foreign body reaction and venous neointimal hyperplasia in rats by the inhibition of IL-33 expression.

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.

Intramural injection of pluronic gel loaded with drugs to alleviate arterial injury.

BACKGROUND: Balloon angioplasty, stent implantation, and application of an arterial clamp during surgery can induce artery injury such as elastin breaks and endothelium injury, but there is little research focused on the injury induced by these therapeutic manipulations. We established a simple and reproducible small animal aortic injury model and examined intramural injection as a potential therapeutic method to alleviate injury. MATERIALS AND METHODS: The abdominal aorta of male Sprague Dawley (SD) rats or C57BL/6 J mice was clamped sequentially throughout its length. Transforming growth factor ß1 (TGFß1), SB431542, lipopolysaccharide (LPS), Necrostatin-1 (Nec-1), rapamycin, or MHY1485 contained in Pluronic gel was injected intramurally at day 0 or day 7. Animals were fed with chow containing 0.25% beta-aminopropionitrile (BAPN) to evaluate the influence of BAPN. All samples were harvested and examined by immunohistochemistry and immunofluorescence. RESULTS: The clamped rat aorta showed luminal dilation, elastin fiber breaks, neointimal hyperplasia, and dissection (days 0-90). Intramural injection of TGFß1, rapamycin and Nec-1 showed a protective effect on the injured aorta, whereas SB431542, MHY1485 and LPS showed more severe wall damage. The aortic lumen in rats fed with BAPN was significantly larger than in control rats (day 7). Mouse aorta showed similar injury with neointimal hyperplasia and elastin fiber breaks. CONCLUSIONS: The rodent arterial injury model is reproducible and may mimic early changes of arterial injury. The model accommodates intramural injection of different drugs that may show mechanisms of arterial injury. Although this is a preliminary animal model, the intramural injection method may have potential clinical application in the future.

Biodegraded PCl and gelatin fabricated vascular patch in rat aortic and inferior vena cava angioplasty.

Novel synthetic prosthesis materials for patch angioplasty are continuously under development and optimization. When a nonwoven-based gelatin membrane is coupled with an electrospun layer of polycaprolactone (PCL), these biohybrid polymer membranes (BHMs) possess higher mechanical properties in aqueous environments. We hypothesized that BHMs can also be used as vascular patches, and we tested our hypothesis in a rat IVC venoplasty and aortic arterioplasty model. Patch venoplasty and arterioplasty were performed in SD rats (200 g), the patches were harvested at day 14, and samples were analyzed by immunohistochemistry and immunofluorescence. The BHM patches were almost degraded, with few parts remaining after 14 days. There was a line of CD34- and nestin-positive cells on the endothelium, with some cells were CD34 and nestin dual-positive, macrophages and leukocytes also participated in the patch healing process. There were PCNA-positive cells in the neointima and peri-patch area, with some cells were also PCNA and α-actin dual-positive. Arterial neointimal endothelial cells were Ephrin-B2- and dll-4-positive, and venous neointimal endothelial cells were Eph-B4- and COUP-TFII-positive. BHM shares a similar healing process like other patch materials, and BHM may have potential applications in vascular surgery.

Nonatheromatous Popliteal Artery Disease.

OBJECTIVE: Peripheral artery disease (PAD) is often caused by atherosclerosis. However, causes other than atherosclerosis is often overlooked. Popliteal artery entrapment syndrome (PAES) and popliteal artery adventitial cystic disease (PACD) are two common nonatheromatous causes of claudication and critical limb ischemia. The purpose of this study is to present early results of treatment of PAES and PACD involving the lower limbs. METHODS: From December 2019 to February 2021, 10 patients with PAES underwent surgeries, and 1 patient with PAES received conservative treatment. 2 patients with PACD underwent surgery. Patient data including age, gender, etiology of vascular pathology, diseased vessel, surgical method, and hemodynamic status were collected retrospectively. RESULTS: The mean follow-up duration was 5.64 ± 3.72 months (range, 1-12 months). All patients had their symptoms improved or resolved. The success rate of surgery was 100%, the rate of freedom from reintervention for any reason was 100%. There were no death, bleeding, embolism, or skin ulcers during late follow-up. CONCLUSIONS: PAES and PACD require early diagnosis and intervention, and early surgery may lead to good early- and mid-term results.

A Systematic Review and Meta-Analysis of Seasonal and Monthly Variability in the Incidence of Acute Aortic Dissection.

BACKGROUND: This study explored seasonal and monthly variations of the incidence of acute aortic dissection (AAD). METHODS: MEDLINE, EMBASE, and the Cochrane Library databases were searched up to July 2021. Temporal variation in the incidence of AAD was analyzed including all studies analyzing seasonal and monthly aggregations. Then, we performed subgroup analyses according to the type of AAD. Two authors independently reviewed and extracted data. RESULTS: Twenty-seven studies for a total of 128,101 patients were included. Our results showed that the incidence of AAD was highest in winter and lowest in summer. Regardless of type A or type B, the incidence of AAD was significantly higher in winter than in summer and autumn. Nonetheless, there was no significant difference between spring and winter, and between summer and autumn. Results may be limited by the quality of the included articles. However, in the sensitivity analysis that excluded low-quality studies, results did not change significantly. In addition, the pooled incidence was highest in January and lowest in August. CONCLUSIONS: Our data strongly support the presence of distribution patterns in the incidence of AAD, characterized by significantly higher risk in winter and in January. These distribution patterns of AAD incidence may help to develop better prevention strategies.

Early Outcomes of Complex Vascular Reconstructions in Lower Extremities Using Spiral and Panel Vein Grafts.

BACKGROUND: Spiral saphenous vein grafts (SSVG) or paneled vein grafts (PVG) can be used when the diameter of the autologous great saphenous vein does not match the vessel that needs to be repaired. This study aimed to present early results of complex vascular reconstruction with SSVGs and PVGs in the lower extremities. METHODS: From May 2019 through January 2021, 6 SSVGs and 3 PVGs were used for vascular reconstruction in 9 patients. Patient data were collected retrospectively, including age, gender, cause of vascular pathology, target vessels, concomitant injury, surgical method, additional surgical methods, and hemodynamic status. The Kaplan-Meier method was used to calculate the rate of freedom from reintervention. RESULTS: Among these patients, 7 had trauma, 1 had graft infection, and 1 had vascular reconstruction after tumor excision. The mean duration of follow-up was 6 ± 6.6 months (range 1-19 months). The rate of freedom from reintervention for any reason was 77.8% at 1 year. Two patients underwent amputation after vascular reconstruction with patent vascular reconstructions. One of the 2 amputations was performed because of infection, and the other was due to ischemia >24 hr. The success rate of reconstruction was 100%, and the primary patency rate was 100%. The rate of limb salvage was 77.8%. There was no death, bleeding, embolism, skin ulcers, graft-related complication, or aneurysmal dilation during follow-up. CONCLUSIONS: SSVG and PVG were associated with low infection rates and satisfactory short-term patency rates. Both 2 grafts may be good choices when there is a diameter mismatch in vascular reconstructions.

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.

Adult Human Vein Grafts Retain Plasticity of Vessel Identity.

BACKGROUND: The spiral saphenous vein graft is an excellent choice for venous reconstruction after periphery vein injury, but only few cases have been reported. We implanted a segment of a single saphenous vein into both the popliteal vein as a venous vein graft and into the popliteal artery as an arterial vein graft at the same time in a trauma patient; we then had an extraordinary opportunity to harvest and examine both patent venous and arterial vein grafts at 2 weeks after implantation. METHODS: A spiral saphenous vein graft was made as previously described and implanted into the popliteal vein and artery as interposition grafts; because of the patient's serious injuries, an amputation was performed at day 18 after vascular reconstruction. The grafts were harvested, fixed, and examined using histology and immunohistochemistry. RESULTS: Both grafts were patent, and there was a larger neointimal area in the venous graft compared to the arterial graft. There were CD31- and vWF-positive cells on both neointimal endothelia, with subendothelial deposition of α-actin-, CD3-, CD45-, and CD68-positive cells. There were fewer cells in the venous graft neointima compared to the arterial graft neointima; however, there were more inflammatory cells in the neointima of the venous graft. Some of the neointimal cells were PCNA-positive, whereas very few cells were cleaved caspase-3 positive. The venous graft neointimal endothelial cells were Eph-B4 and COUP-TFII positive, while the arterial graft neointimal endothelial cells were dll-4 and Ephrin-B2 positive. CONCLUSIONS: The spiral saphenous vein graft remains a reasonable choice for vessel reconstruction, especially in the presence of diameter mismatch. Both the venous and arterial grafts showed similar re-endothelialization and cellular deposition; the venous graft had more neointimal hyperplasia and inflammation. At an early time, endothelial cells showed venous identity in the venous graft, whereas endothelial cells showed arterial identity in the arterial graft. CLINICAL RELEVANCE: Veins can be used as venous or arterial vein grafts but venous grafts have more neointimal hyperplasia and inflammation; vein grafts acquire different vessel identity depending on the environment into which they are implanted.

A rat arteriovenous graft model using decellularized vein.

BACKGROUND: The high rate of clinical failure of prosthetic arteriovenous grafts continues to suggest the need for novel tissue-engineered vascular grafts. We tested the hypothesis that the decellularized rat jugular vein could be successfully used as a conduit and that it would support reendothelialization as well as adaptation to the arterial environment. MATERIALS AND METHODS: Autologous (control) or heterologous decellularized jugular vein (1 cm length, 1 mm diameter) was sewn between the inferior vena cava and aorta as an arteriovenous graft in Wistar rats. Rats were sacrificed on postoperative day 21 for examination. RESULTS: All rats survived, and grafts had 100% patency in both the control and decellularized groups. Both control and decellularized jugular vein grafts showed similar rates of reendothelialization, smooth muscle cell deposition, macrophage infiltration, and cell turnover. The outflow veins distal to the grafts showed similar adaptation to the arteriovenous flow. Both CD34, CD90 and nestin positive cells, as well as M1-type and M2-type macrophages accumulated around the graft. CONCLUSIONS: This model shows that decellularized vein can be successfully used as an arteriovenous graft between the rat aorta and the inferior vena cava. Several types of cells, including progenitor cells and macrophages, are present in the host response to these grafts in this model. This model can be used to test the application of arteriovenous grafts before conducting large animal experiments.

Decellularized Carotid Artery Functions as an Arteriovenous Graft.

BACKGROUND: Prosthetic arteriovenous grafts (AVG) continue to have a high rate of failure in clinical use, yet there is continued clinical demand for them. However, there is no small animal model of AVG to test novel tissue-engineered vascular grafts. We established a new rat arteriovenous graft model to compare the healing of decellularized carotid artery (CA) to autologous CA. MATERIALS AND METHODS: The infrarenal vena cava and aorta of Wistar rats were exposed and dissected free below renal artery. A longitudinal 1 mm venotomy and arteriotomy were made on the anterior walls. The conduit was either autologous CA or heterologous decellularized CA; a conduit was sewn to the inferior vena cava and aorta in end-to-side fashion. Rats were sacrificed on postoperative day 21 for examination. RESULTS: All rats survived without heart failure. Conduits had 100% patency rate (day 21) in both the control and decellularized CA groups (n = 6). Both control and decellularized CA showed similar rates of reendothelialization, inflammatory cell infiltration, and cell turnover. The outflow vein beyond the autologous or decellularized conduits showed similar neointimal thickness and cell turnover. CONCLUSIONS: Decellularized CA may be a viable tissue engineering graft for use as an arteriovenous graft for dialysis access. The rat aorta-vena cava graft is a useful model to test new materials including tissue-engineered grafts for use as AVG.

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.

Adipose-derived mesenchymal stem cells accelerate diabetic wound healing in a similar fashion as bone marrow-derived cells.

We have previously shown that bone marrow-derived mesenchymal stem cells (BMSC) accelerate wound healing in a diabetic mouse model. In this study, we hypothesized that adipose tissue-derived stem cells (ADSC), cells of greater translational potential to human therapy, improve diabetic wound healing to a similar extent as BMSC. In vitro, the characterization and function of murine ADSC and BMSC as well as human diabetic and nondiabetic ADSC were evaluated by flow cytometry, cell viability, and VEGF expression. In vivo, biomimetic collagen scaffolds containing murine ADSC or BMSC were used to treat splinted full-thickness excisional back wounds on diabetic C57BL/6 mice, and human healthy and diabetic ADSC were used to treat back wounds on nude mice. Wound healing was evaluated by wound area, local VEGF-A expression, and count of CD31-positive cells. Delivery of murine ADSC or BMSC accelerated wound healing in diabetic mice to a similar extent, compared with acellular controls ( P < 0.0001). Histological analysis showed similarly increased cellular proliferation ( P < 0.0001), VEGF-A expression ( P = 0.0002), endothelial cell density ( P < 0.0001), numbers of macrophages ( P < 0.0001), and smooth muscle cells ( P < 0.0001) with ADSC and BMSC treatment, compared with controls. Cell survival and migration of ADSC and BMSC within the scaffolds were similar ( P = 0.781). Notch signaling was upregulated to a similar degree by both ADSC and BMSC. Diabetic and nondiabetic human ADSC expressed similar levels of VEGF-A ( P = 0.836) in vitro, as well as in scaffolds ( P = 1.000). Delivery of human diabetic and nondiabetic ADSC enhanced wound healing to a similar extent in a nude mouse wound model. Murine ADSC and BMSC delivered in a biomimetic-collagen scaffold are equivalent at enhancing diabetic wound healing. Human diabetic ADSC are not inferior to nondiabetic ADSC at accelerating wound healing in a nude mouse model. This data suggests that ADSC are a reasonable choice to evaluate for translational therapy in the treatment of human diabetic wounds.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. APPROACH AND RESULTS: AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 µm; P=0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P=0.0015; interleukin-10: 7.6-fold, P=0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 µm; P=0.0306), increased collagen density (2.4-fold; P=0.0432), and increased number of M2 macrophages (2.1-fold; P=0.0335). CONCLUSIONS: CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation.

Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase.

OBJECTIVE: Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor Eph-B4, the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be through regulation of downstream eNOS activity. METHODS: Mouse lung endothelial cells were stimulated with ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using an NO-specific chemiluminescence analyzer. Cell migration was assessed using a Transwell assay. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type or eNOS knockout mice. RESULTS: Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release (n = 3; P < .01) as well as increased endothelial cell migration (n = 6; P < .05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS knockout mice showed less dilation and less wall thickening compared with wild-type vein grafts (n = 7; P < .05). CONCLUSIONS: eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation in vitro and may mediate vein graft adaptation by regulation of eNOS activity in vivo.

Increased Oxidative Stress and Hypoxia Inducible Factor-1 Expression during Arteriovenous Fistula Maturation.

BACKGROUND: The poor clinical results that are frequently reported for arteriovenous fistulae (AVF) for hemodialysis are typically due to failure of AVF maturation. We hypothesized that early AVF maturation is associated with generation of reactive oxygen species and activation of the hypoxia-inducible factor-1 (HIF-1) pathway, potentially promoting neointimal hyperplasia. We tested this hypothesis using a previously reported mouse AVF model that recapitulates human AVF maturation. METHODS: Aortocaval fistulae were created in C57Bl/6 mice and compared with sham-operated mice. AVFs or inferior vena cavas were analyzed using a microarray, Amplex Red for extracellular H2O2, quantitative polymerase chain reaction, immunohistochemistry, and immunoblotting for HIF-1α and immunofluorescence for NOX-2, nitrotyrosine, heme oxygenase-1 (HO-1), and vascular endothelial growth factor (VEGF)-A. RESULTS: Oxidative stress was higher in AVF than that in control veins, with more H2O2 (P = 0.007) and enhanced nitrotyrosine immunostaining (P = 0.005). Immunohistochemistry and immunoblot showed increased HIF-1α immunoreactivity in the AVF endothelium; HIF-1 targets NOX-2, HO-1 and VEGF-A were overexpressed in the AVF (P < 0.01). AVF expressed increased numbers of HIF-1α (P < 0.0001) and HO-1 (P < 0.0001) messenger RNA transcripts. CONCLUSIONS: Oxidative stress increases in mouse AVF during early maturation, with increased expression of HIF-1α and its target genes NOX-2, HO-1, and VEGF-A. These results suggest that clinical strategies to improve AVF maturation could target the HIF-1 pathway.

Membrane-mediated regulation of vascular identity.

Vascular diseases span diverse pathology, but frequently arise from aberrant signaling attributed to specific membrane-associated molecules, particularly the Eph-ephrin family. Originally recognized as markers of embryonic vessel identity, Eph receptors and their membrane-associated ligands, ephrins, are now known to have a range of vital functions in vascular physiology. Interactions of Ephs with ephrins at cell-to-cell interfaces promote a variety of cellular responses such as repulsion, adhesion, attraction, and migration, and frequently occur during organ development, including vessel formation. Elaborate coordination of Eph- and ephrin-related signaling among different cell populations is required for proper formation of the embryonic vessel network. There is growing evidence supporting the idea that Eph and ephrin proteins also have postnatal interactions with a number of other membrane-associated signal transduction pathways, coordinating translation of environmental signals into cells. This article provides an overview of membrane-bound signaling mechanisms that define vascular identity in both the embryo and the adult, focusing on Eph- and ephrin-related signaling. We also discuss the role and clinical significance of this signaling system in normal organ development, neoplasms, and vascular pathologies.

Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro.

BACKGROUND: Vein bypass is an essential therapy for patients with advanced peripheral and coronary artery disease despite development of neointimal hyperplasia. We have shown that stimulation of the receptor tyrosine kinase ephrin type-B receptor 4 (Eph-B4) with its ligand ephrin-B2 prevents neointimal hyperplasia in murine vein grafts. This study determines whether Eph-B4 in adult human veins is capable of phosphorylation and activation of downstream signaling pathways, as well as functional to release nitric oxide (NO) and prevent neointimal hyperplasia in vitro. METHODS: Discarded human saphenous veins were taken from the operating room and placed in organ culture without or with ephrin-B2/Fc (2 µg/mL) for 14 days, and the neointima/media ratio was measured in matched veins. Primary human umbilical vein endothelial cells were treated with ephrin-B2/Fc (2 µg/mL) and examined with quantitative polymerase chain reaction, Western blot, immunoassays, and for release of NO. Ephrin-B2/Fc (2 µg/mL) was placed on the adventitia of saphenous veins treated with arterial shear stress for 24 hours in a bioreactor and activated Eph-B4 examined with immunofluorescence. RESULTS: The baseline intima/media ratio in saphenous vein rings was 0.456 ± 0.097, which increased to 0.726 ± 0.142 in untreated veins after 14 days in organ culture but only to 0.630 ± 0.132 in veins treated with ephrin-B2/Fc (n = 19, P = .017). Ephrin-B2/Fc stimulated Akt, endothelial NO synthase and caveolin-1 phosphorylation, and NO release (P = .007) from human umbilical vein endothelial cells (n = 6). Ephrin-B2/Fc delivered to the adventitia stimulated endothelial Eph-B4 phosphorylation after 24 hours of arterial stress in a bioreactor (n = 3). CONCLUSIONS: Eph-B4 is present and functional in adult human saphenous veins, with intact downstream signaling pathways capable of NO release and prevention of neointimal hyperplasia in vitro. Adventitial delivery of ephrin-B2/Fc activates endothelial Eph-B4 in saphenous veins treated with arterial shear stress in vitro. These results suggest that stimulation of Eph-B4 function may be a candidate strategy for translation to human clinical trials designed to inhibit venous neointimal hyperplasia.