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.

Omentin reduces venous neointimal hyperplasia in arteriovenous fistula through hypoxia-inducible factor-1 alpha inhibition.

Arteriovenous fistula (AVF) failure often involves venous neointimal hyperplasia (VNH) driven by elevated hypoxia-inducible factor-1 alpha (HIF-1α) in the venous wall. Omentin, known for its anti-inflammatory and anti-hyperplasia properties, has an uncertain role in early AVF failure. This study investigates omentin's impact on VNH using a chronic renal failure (CRF) rabbit model. The CRF rabbit model of AVF received omentin-expressing adenoviral vector or control ß-gal vector to assess omentin's effects on VNH. Human vascular smooth muscle cells (HVSMCs), stimulated with tumor necrosis factor-α (TNF-α), were exposed to recombinant human omentin (Rh-OMT) to study its influence on cell proliferation and migration. The AMP-activated protein kinase (AMPK) inhibitor compound C and the mammalian target of rapamycin (mTOR) activator MHY1485 were employed to explore omentin's mechanisms in VNH reduction through HIF-1α inhibition. Omentin treatment reduced VNH in CRF rabbits, concomitant with HIF-1α down-regulation and the suppression of downstream factors, including vascular endothelial growth factor and matrix metalloproteinases. Rh-OMT inhibited TNF-α-induced HVSMC proliferation and migration by modulating both cell cycle and cell adhesion proteins. Additionally, omentin reduced HIF-1α expression through the AMPK/mTOR pathway activation. Notably, the blockade of AMPK/mTOR signaling reversed omentin-mediated inhibition of VNH, cell proliferation, and migration, both in vivo and in vitro. In conclusion, omentin mitigates VNH post-AVF creation by restraining HIF-1α via AMPK/mTOR signaling. Strategies boosting circulating omentin levels may offer promise in averting AVF failure.

Atorvastatin Reduces In Vivo Fibrin Deposition and Macrophage Accumulation, and Improves Primary Patency Duration and Maturation of Murine Arteriovenous Fistula.

BACKGROUND: Arteriovenous fistulas placed surgically for dialysis vascular access have a high primary failure rate resulting from excessive inward remodeling, medial fibrosis, and thrombosis. No clinically established pharmacologic or perisurgical therapies currently address this unmet need. Statins' induction of multiple anti-inflammatory and antithrombotic effects suggests that these drugs might reduce arteriovenous fistula failure. Yet, the in vivo physiologic and molecular effects of statins on fistula patency and maturation remain poorly understood. METHODS: We randomized 108 C57Bl/6J mice to receive daily atorvastatin 1.14 mg/kg or PBS (control) starting 7 days before end-to-side carotid artery-jugular vein fistula creation and for up to 42 days after fistula creation. We then assessed longitudinally the effects of statin therapy on primary murine fistula patency and maturation. We concomitantly analyzed the in vivo arteriovenous fistula thrombogenic and inflammatory macrophage response to statin therapy, using the fibrin-targeted, near-infrared fluorescence molecular imaging agent FTP11-CyAm7 and dextranated, macrophage-avid nanoparticles CLIO-VT680. RESULTS: In vivo molecular-structural imaging demonstrated that atorvastatin significantly reduced fibrin deposition at day 7 and macrophage accumulation at days 7 and 14, findings supported by histopathologic and gene-expression analyses. Structurally, atorvastatin promoted favorable venous limb outward remodeling, preserved arteriovenous fistula blood flow, and prolonged primary arteriovenous fistula patency through day 42 (P<0.05 versus control for all measures). CONCLUSIONS: These findings provide new in vivo evidence that statins improve experimental arteriovenous fistula patency and maturation, indicating that additional clinical evaluation of statin therapy in patients on dialysis undergoing arteriovenous fistula placement is warranted.

Vasoreactivity of the Murine External Jugular Vein and Carotid Artery.

INTRODUCTION: Impaired venous reactivity has potential to contribute to clinically significant pathologies such as arteriovenous fistula (AVF) maturation failure. Vascular segments commonly used in murine preclinical models of AVF include the carotid artery and external jugular vein. Detailed descriptions of isometric procedures to evaluate function of murine external jugular vein ex vivo have not been previously published. OBJECTIVE: To establish isometric procedures to measure naive murine external jugular vein reactivity ex vivo. METHODS: Vasomotor responses of external jugular veins and ipsilateral common carotid arteries from C57BL/6 mice were evaluated using isometric tension procedures. RESULTS: External jugular veins developed tension (p < 0.05) to potassium chloride and U-46619, but not to phenylephrine, whereas common carotid arteries responded to all 3 agents (p < 0.05). While maximal responses to acetylcholine (ACh) were similar between the venous and arterial segments, the dose required to achieve this value was lower (p < 0.05) in the artery versus vein. Nitric oxide synthase inhibition attenuated (p < 0.05) but did not abolish ACh-evoked vasorelaxation in both vascular segments, whereas cyclooxygenase blockade had no effect. Endothelium-independent vasorelaxation to sodium nitroprusside was similar in the artery and vein. CONCLUSION: Vasorelaxation and vasocontraction can be reliably assessed in the external jugular vein in C57BL/6 mice using isometric procedures.

Lymphatic Distribution of Etanercept Following Intravenous and Subcutaneous Delivery to Rats.

PURPOSE: The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein. METHODS: Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days. RESULTS: Model-based SC bioavailability of etanercept was 65.2% in the control group. In lymph-cannulated rats, etanercept concentration in the lymph was consistently lower than in serum following IV dosing; and the concentration in the lymph was significantly higher than in serum after SC injection. The absorption occurred predominantly through the lymphatic pathway (82.7%), and only 17.3% by direct uptake into the central compartment (blood pathway). Lymphatic cannulation reduced the area under the serum concentration-time curve by 28% in IV group and by 91% in SC group. A mechanistic pharmacokinetic model that combined dual absorption pathways with redistribution of the systemically available protein drug into lymph was developed. The model successfully captured serum and lymph data in all groups simultaneously, and all parameters were estimated with sufficient precision. CONCLUSIONS: Lymphatic system was shown to play an essential role in systemic disposition and SC absorption of etanercept.

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.

MicroRNA-365 promotes the contractile phenotype of venous smooth muscle cells and inhibits neointimal formation in rat vein grafts.

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA-365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus-mediated miR-365 was able to attenuate neointimal formation in rat vein grafts. We found that miR-365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced grafting-induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR-365 in vein grafts. Specially, to increase the efficiency of miR-365 gene transfection, a 30% poloxamer F-127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus-mediated miR-365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR-365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019.

Evaluation of Venous Stenosis Angioplasty in a Murine Arteriovenous Fistula Model.

PURPOSE: To develop a clinically relevant model of percutaneous transluminal angioplasty (PTA) of venous stenosis in mice with arteriovenous fistula (AVF); to test the hypothesis that there is increased wall shear stress (WSS) after PTA; and to histologically characterize the vessels. MATERIALS AND METHODS: Thirteen C57BL/6J male mice, 6-8 weeks old, underwent partial nephrectomy to create chronic kidney disease. Twenty-eight days later, an AVF was created from the right external jugular vein to the left carotid artery. Fourteen days later, an angioplasty or sham procedure was performed, and the mice were sacrificed 14 days later for histologic evaluation to identify the cells contributing to the vascular remodeling (α-SMA, FSP-1, CD31, and CD68), proliferation (Ki-67), cell death (TUNEL), and hypoxia staining (HIF-1α). Histomorphometric analysis was performed to assess lumen area, neointima+media area, and cellular density. Ultrasound was performed weekly after creation of the AVF. RESULTS: Venous stenosis occurred 14 days after the creation of an AVF. PTA-treated vessels had significantly higher WSS; average peak systolic velocity, with increased lumen vessel area; and decreased neointima + media area compared to sham controls. There was a significant decrease in the staining of smooth muscle cells, fibroblasts, macrophages, HIF-1α, proliferation, and apoptosis and an increase in CD31-(+) cells. CONCLUSIONS: A clinically relevant model of PTA of venous stenosis in mice was created. PTA-treated vessels had increased lumen vessel area and WSS. The alterations in tissue markers of vascular remodeling, tissue hypoxia, proliferation, and cell death may be implications for future design of drug and device development.

Improved Patency of ePTFE Grafts as a Hemodialysis Access Site by Seeding Autologous Endothelial Cells Expressing Fibulin-5 and VEGF.

Small caliber synthetic vascular grafts used for dialysis access sites have high failure rates due to neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. We tested the use of ePTFE grafts seeded with autologous ECs expressing fibulin-5 and vascular endothelial growth factor (VEGF), as a dialysis access site in a porcine model. We connected the carotid arteries and jugular veins of 12 miniature pigs using 7-mm ePTFE grafts; five grafts were seeded with autologous venous ECs modified to express fibulin-5 and VEGF, and seven unseeded grafts were implanted at the same location and served as controls. At 6 months, after completion of angiography, the carotid arteries and jugular veins with the connecting grafts were excised and fixed. Autologous EC isolation and transduction and graft seeding were successful in all animals. At 3 months, 4 of 5 seeded grafts and 3 of 7 control grafts were patent. At 6 months, 4 of 5 (80%) seeded grafts and only 2 of 7 (29%) control grafts were patent. Seeding ePTFE vascular grafts with genetically modified ECs improved long term small caliber graft patency. The biosynthetic grafts offer a novel therapeutic modality for vascular access in hemodialysis.

Changes in expression profiles of internal jugular vein wall and plasma protein levels in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. METHODS: We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. RESULTS: Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P < 0.05), the immune-related CD86 (8.5 fold-change, P = 0.002) emerged among the up regulated genes (N = 409). Several genes encoding HOX transcription factors and histones potentially regulated by blood flow, were overexpressed. Smooth muscle contraction and cell adhesion processes emerged among down regulated genes (N = 515), including the neuronal cell adhesion L1CAM as top scorer (5 fold-change, P = 5 × 10- 4). Repeated measurements in jugular/peripheral plasma and overtime in peripheral plasma showed conserved individual plasma patterns for immune-inflammatory (CCL13, CCL18) and adhesion (NCAM1, VAP1, SELL) proteins, despite significant variations overtime (SELL P < 0.0001). Both age and MS disease phenotypes were determinants of VAP1 plasma levels. Data supported cerebral related-mechanisms regulating ANGPT1 levels, which were remarkably lower in jugular plasma and correlated in repeated assays but not between jugular/peripheral compartments. CONCLUSIONS: This study provides for the first time expression patterns of the IJV wall, suggesting signatures of altered vascular mRNA profiles in MS disease also independently from CCSVI. The combined transcriptome-protein analysis provides intriguing links between IJV wall transcript alteration and plasma protein expression, thus highlighting proteins of interest for MS pathophysiology.

Characteristics of jugular bulb oxygen saturation in patients after cardiac arrest: A prospective study.

BACKGROUND: Using cerebral oxygen venous saturation post-cardiac arrest (CA) is limited because of a small sample size and prior to establishment of target temperature management (TTM). We aimed to describe variations in jugular bulb oxygen saturation during intensive care in relation to neurological outcome at 6 months post- CA in cases where TTM 33°C was applied. METHOD: Prospective observational study in patients over 18 years, comatose immediately after resuscitation from CA. Patients were treated with TTM 33°C M and received a jugular bulb catheter within the first 26 hours post-CA. Neurological outcome was assessed at 6 months using the Cerebral Performance Categories (CPC) and dichotomized into good (CPC 1-2) and poor outcome (CPC 3-5). RESULTS: Seventy-five patients were included and 37 (49%) patients survived with a good outcome at 6 months post-CA. No differences were found between patients with good outcome and poor outcome in jugular bulb oxygen saturation. Higher values were seen in differences in oxygen content between central venous oxygen saturation and jugular bulb oxygen saturation in patients with good outcome compared to patients with poor outcome at 6 hours (12 [8-21] vs 5 [-0.3 to 11]% P = .001) post-CA. Oxygen extraction fraction from the brain illustrated lower values in patients with poor outcome compared to patients with good outcome at 96 hours (14 [9-23] vs 31 [25-34]% P = .008). CONCLUSIONS: Oxygen delivery and extraction differed in patients with a good outcome compared to those with a poor outcome at single time points. Based on the present findings, the usefulness of jugular bulb oxygen saturation for prognostic purposes is uncertain in patients treated with TTM 33°C post-CA.

Distinct Vascular Remodeling Pattern of Adult Rats with Carotid-Jugular Shunt.

BACKGROUND: Previous research has revealed that patent vein grafts lose their venous identity Eph-B4 but do not gain arterial identity ephrin-B2 during adaptation to the arterial circulation, and vascular identity marker, for example, the Eph-B4 signaling is a critical determinant of venous wall thickness of vein grafts. But what is the remodeling pattern, especially the remodeling pattern of vascular identity in the venous segment of arteriovenous shunt at a late stage postoperation has not been fully explored. This study was conducted to characterize the remodeling pattern of shear stress, vascular identity, structural composition and morphology, and transcriptional profiles in jugular segment of carotid-jugular (CJ) shunt and/or pulmonary artery (PA), which delivers an increased amount of mixed blood at a late stage postoperation in adult rats. METHODS: CJ shunt was created in adult Wistar rats via end-to-end anastomosis of carotid artery (CA) and jugular vein (JV). At the time of 15 weeks, after hemodynamics test, remodeled jugular segment of CJ shunt, PA, and sham-operated corresponding vessels were isolated. Reverse transcription polymerase chain reaction, microarray, western blot, immunohistochemistry experiments, and morphology analyses were performed. RESULTS: CJ shunt shear stresses have been patterned to some sort of balance with no significant difference in shear stress between carotid segment and jugular segment (P > 0.05). Immunohistochemical analysis reveals that venous identity marker Eph-B4 is lost, but arterial identity markers ephrin-B2 and regulator of G-protein signaling 5 are gained in jugular segment of CJ shunt (P < 0.01), and these 2 arterial identity markers further strengthened in PA (P < 0.01) in shunted rats compared with controls. Jugular segment of CJ shunt undergoes significant intimal hyperplasia with strong expression of smooth muscle cell markers (P < 0.05) and demonstrates a distinct transcriptional profiles which reveals that transcripts of 5 arterial markers are significantly upregulated (P < 0.05 or < 0.01) compared with sham-operated JV; among them, G-protein signaling 5 is exactly the gene with the largest fold change (10.14-fold) in all genes tested by microarray experiment. CONCLUSIONS: Venous identity is lost, but arterial identity is gained in jugular segment of CJ shunt and arterial identity further strengthened in PA in adult shunted rats during late adaptation.

Local application of paeonol prevents early restenosis: a study with a rabbit vein graft model.

BACKGROUND: Neointimal hyperplasia, which is caused by dysfunction of vascular smooth muscle cells and vascular endothelial cells (VECs), is a foundation for later development of vein grafted occlusion. This study investigates whether neointimal hyperplasia could be prevented by the application of paeonol, a phenolic compound having functions of anti-inflammatory, anti-oxidant, and anti-proliferative. METHODS: Autologous jugular veins, which engrafted to carotid arteries in rabbits, were enveloped with paeonol or left untreated. After 0, 2, and 3 wk, vein grafts were respectively harvested. Proliferating cell nuclear antigen, vascular cell adhesion molecule l (VCAM-1), and intercellular cell adhesion molecule 1 were assessed with immunohistochemistry and Western blot. VECs apoptosis was also detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling assay. RESULTS: Paeonol treatment reduced early neointimal hyperplasia by 42%-46% (P < 0.001) and early medial hyperplasia by 18%-22% (P < 0.001) compared with the controls. Immunohistochemical and Western blot results show a significant downregulation of proliferating cell nuclear antigen (P < 0.001) and VCAM-1 (P < 0.001) in paeonol treatment group in the second and third weeks. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling analysis discovered that VECs apoptosis was also reduced by the paeonol treatment in the second and third weeks (P < 0.001). CONCLUSIONS: Paeonol could prevent vein graft early restenosis by suppressing intimal and medial hyperplasia via inhibition of vascular smooth muscle cells proliferation, VCAM-1 expression, and anti-apoptosis of VECs in grafted veins.

Bedside Monitoring of Cerebral Energy State During Cardiac Surgery-A Novel Approach Utilizing Intravenous Microdialysis.

OBJECTIVES: This study investigated whether the lactate-to-pyruvate (LP) ratio obtained by microdialysis (MD) of the cerebral venous outflow reflected a derangement of global cerebral energy state during cardiopulmonary bypass (CPB). DESIGN: Interventional, prospective, randomized study. SETTING: Single-center, university teaching hospital. PARTICIPANTS: The study included 10 patients undergoing primary, elective coronary artery bypass grafting. INTERVENTIONS: Patients were randomized blindly to low mean arterial pressure (MAP) (40-60 mmHg; n = 5) or high MAP (60-80 mmHg; n = 5) during CPB. The MD catheters were positioned in a retrograde direction into the jugular bulb, and a reference catheter was inserted into the brachial artery. The correlations among LP ratio, MAP, data obtained from bifrontal near-infrared spectroscopy (NIRS), and postoperative neurologic outcome measures were assessed. MEASUREMENTS AND MAIN RESULTS: The correlated difference between pooled LP ratio (low and high MAP) of the jugular venous and the arterial blood was significant (LParterial 17 [15-20] v LPvenous 26 [23-27]; p = 0.0001). No cerebral desaturations (decrease in rSO2>20% from baseline) were observed in either group during CPB. In each group, 50% of the patients showed significant cognitive decline (mini-mental state examination, 3 points) 2 days after surgery. CONCLUSION: The LP ratio of cerebral venous blood increased significantly during CPB, indicating compromised cerebral oxidative metabolism. Conventional monitoring of rSO2 by NIRS did not show a corresponding decrease in cerebral oxygenation. As the patients exhibited decreased cognitive functions after CPB, increases in jugular venous LP ratio may be a sensitive indicator of impending cerebral damage.

Role of ammonia, inflammation, and cerebral oxygenation in brain dysfunction of acute-on-chronic liver failure patients.

Hepatic encephalopathy (HE) is a common feature of acute-on-chronic liver failure (ACLF). Although ammonia, inflammation, and cerebral oxygenation are associated with HE in acute liver failure, their roles in ACLF are unknown. The aim of this prospective, longitudinal study was to determine the role of these pathophysiological variables in ACLF patients with and without HE. We studied 101 patients with ACLF admitted to the intensive care unit. Severity of ACLF and HE, arterial ammonia, jugular venous oxygen saturation (JVO2 ), white blood cell count (WCC), and C-reactive protein were measured at days 0, 1, 3, and 7. Patients were followed until death or hospital discharge. Mortality was high (51 patients, 50.5%), especially in patients with HE of whom 35 of 53 (66.0%) died regardless of ACLF severity. At baseline, increased WCC and abnormal JVO2 (high or low) were independent predictors of death. Further deterioration in inflammation, JVO2 , and ammonia were also predictive of mortality. JVO2 deviation and hyperammonemia were associated with the presence and severity of HE; improvement in these parameters was associated with a reduction in HE grade. No direct interaction was observed between these variables in regards to mortality or HE. In conclusion, this study describes potential mechanisms of HE in ACLF indicating that ammonia and abnormal cerebral oxygenation are important. The results suggest that ammonia, JVO2 , and WCC are important prognostic biomarkers and therapeutic targets. The relative roles of these pathophysiological factors in the pathogenesis of HE in ACLF or guiding therapy to improve survival requires future study. Liver Transplantation 22 732-742 2016 AASLD.

Use of Brilliant Blue FCF during vein graft preparation inhibits intimal hyperplasia.

BACKGROUND: Intimal hyperplasia remains the primary cause of vein graft failure for the 1 million yearly bypass procedures performed using human saphenous vein (HSV) grafts. This response to injury is caused in part by the harvest and preparation of the conduit. The use of Brilliant Blue FCF (FCF) restores injury-induced loss of function in vascular tissues possibly via inhibition of purinergic receptor signaling. This study investigated whether pretreatment of the vein graft with FCF prevents intimal hyperplasia. METHODS: Cultured rat aortic smooth muscle cells (A7r5) were used to determine the effect of FCF on platelet-derived growth factor-mediated migration and proliferation, cellular processes that contribute to intimal hyperplasia. The effectiveness of FCF treatment during the time of explantation on preventing intimal hyperplasia was evaluated in a rabbit jugular-carotid interposition model and in an organ culture model using HSV. RESULTS: FCF inhibited platelet-derived growth factor-induced migration and proliferation of A7r5 cells. Treatment with FCF at the time of vein graft explantation inhibited the subsequent development of intimal thickening in the rabbit model. Pretreatment with FCF also prevented intimal thickening of HSV in organ culture. CONCLUSIONS: Incorporation of FCF as a component of vein graft preparation at the time of explantation represents a potential therapeutic approach to mitigate intimal hyperplasia, reduce vein graft failure, and improve outcome of the autologous transplantation of HSV.

The Role of Repeat Administration of Adventitial Delivery of Lentivirus-shRNA-Vegf-A in Arteriovenous Fistula to Prevent Venous Stenosis Formation.

PURPOSE: To determine if a second dose of a lentivirus mediated small hairpin RNA that inhibits Vegf-A gene expression (LV-shRNA-Vegf-A) can improve lumen vessel area (LVA) of the outflow vein of an arteriovenous fistula (AVF) and decrease venous neointimal hyperplasia. MATERIALS AND METHODS: Chronic kidney disease was created in C57BL/6 mice; 28 days later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein. Immediately after AVF creation, 5 × 10(6) plaque-forming units of LV-shRNA-Vegf-A or control shRNA was administered to the adventitia of the outflow vein, and a second dose of the same treatment was administered 14 days later. Animals were sacrificed at 21 days, 28 days, and 42 days after AVF creation for reverse transcription polymerase chain reaction and histomorphometric analyses. RESULTS: By day 21, there was a 125% increase in the average LVA (day 21, P = .11), with a decrease in cell proliferation (day 21, P = .0079; day 28, P = .28; day 42, P = .5), decrease in α-smooth muscle cell actin staining (day 21, P < .0001; day 28, P < .05; day 42, P = .59), and decrease in hypoxic stress (day 21, P < .001; day 28, P = .28; day 42, P = .46) in LV versus control shRNA vessels. CONCLUSIONS: A second dose of LV-shRNA-Vegf-A administration results in a moderate improvement in LVA at day 21.

In vivo nanoparticle assessment of pathological endothelium predicts the development of inflow stenosis in murine arteriovenous fistula.

OBJECTIVE: In vivo assessment of pathological endothelium within arteriovenous fistula (AVF) could provide new insights into inflow stenosis, a common cause of AVF primary failure in end-stage renal disease patients. Here we developed nanoparticle-based imaging strategies to assess pathological endothelium in vivo and elucidate its relationship to neointimal hyperplasia formation in AVF. APPROACH AND RESULTS: Jugular-carotid AVFs were created in C57BL/6 mice (n=38). Pathological endothelium in the AVF was visualized and quantified in vivo using dextranated magnetofluorescent nanoparticles (CLIO-VT680 [cross-linked iron oxide-VivoTag680]). At day 14, CLIO-VT680 was deposited in AVF, but only minimally in sham-operated arteries. Transmission electron microscopy revealed that CLIO-VT680 resided within endothelial cells and in the intimal extracellular space. Endothelial cells of AVF, but not control arteries, expressed vascular cell adhesion molecule-1 and showed augmented endothelial permeability near the anastomosis. Intravital microscopy demonstrated that CLIO-VT680 deposited most intensely near the AVF anastomosis (P<0.0001). The day 14 intravital microscopy CLIO-VT680 signal predicted the subsequent site and magnitude of AVF neointimal hyperplasia at day 42 (r=0.58, P<0.05). CLIO-VT680 deposition in AVF was further visualized by ex vivo MRI. CONCLUSIONS: AVF develop a pathological endothelial response that can be assessed in vivo via nanoparticle-enhanced imaging. AVF endothelium is activated and exhibits augmented permeability, offering a targeting mechanism for nanoparticle deposition and retention in pathological endothelium. The in vivo AVF nanoparticle signal identified and predicted subsequent inflow neointimal hyperplasia. This approach could be used to test therapeutic interventions aiming to restore endothelial health and to decrease early AVF failure caused by inflow stenosis.

Therapeutic Drug Monitoring of Vancomycin in Dermal Interstitial Fluid Using Dissolving Microneedles.

OBJECTIVE: To design an alternative painless method for vancomycin (VCM) monitoring by withdrawing interstitial fluid (ISF) the skin using dissolving microneedles (DMNs) and possibly replace the conventional clinical blood sampling method. METHODS: Male Wistar rats were anesthetized with 50 mg/kg sodium pentobarbital. Vancomycin at 5 mg/mL in saline was intravenously administered via the jugular vein. ISF was collected from a formed pore at 15, 30, 45, 60, 75, 90, and 120 min after the DMNs was removed from the skin. In addition, 0.3 mL blood samples were collected from the left femoral vein. RESULTS: The correlation between the plasma and ISF VCM concentrations was significantly strong (r = 0.676, p < 0.05). Microscopic observation of the skin after application of the DMNs demonstrated their safety as a device for sampling ISF. CONCLUSION: A novel monitoring method for VCM was developed to painlessly determine concentrations in the ISF as opposed to blood sampling.

Tissue Factor Activity in Dialysis Access Grafts.

BACKGROUND: Intimal hyperplasia at the venous anastomosis of dialysis grafts causes early failure. We developed a sheep model of arteriovenous prosthetic grafts that fail rapidly due to intimal hyperplasia with histologic features nearly identical to human access grafts. A prominent feature of lesion development in this model is formation of luminal thrombus that becomes organized into stenosing lesions by macrophage and myofibroblast infiltration. To better understand this process, we examined the presence and activity of tissue factor (TF) in this system. This protein is the physiological initiator of coagulation in vivo and is known to contribute to development of intimal hyperplasia after vascular injury. METHODS: Expanded polytetrafluorethylene (ePTFE) grafts were placed between the carotid artery and external jugular vein in sheep. Grafts were examined for luminal TF activity using a novel ex vivo assay. In a separate series of grafts, immunohistochemistry was used to localize smooth muscle cells, monocytes, and TF protein. RESULTS: At 2 days, luminal TF activity already was higher in the venous and arterial end of the graft than in the adventitia. This high level of activity persisted at 8 weeks. TF activity was higher in the venous end of the grafts than in the arterial end at 2 and 8 weeks (40% and 47% increase, n = 5, n = 3, respectively, P < 0.05). Immunohistochemistry revealed TF protein localized in regions with or adjacent to fibrin accumulation and often in regions close to the lumen. CONCLUSIONS: This study further examines the development of intimal hyperplasia in ePTFE dialysis access grafts. In this model, TF levels on the luminal surface were increased throughout the arteriovenous grafts and the adjacent vessels as early as 2 days after engraftment and for as long as 8 weeks thereafter. The highest levels of activity were found in the venous end of the graft, where hyperplasia is most robust. Increased activity of TF is associated with luminal thrombus, which provides a scaffolding for development of intimal hyperplasia. These findings present an opportunity to develop strategies to limit TF activity within the graft. Further studies using agents delivered locally or incorporated into the graft matrix to block the luminal activity of TF are warranted.