Mineral Metabolism Disturbances and Arteriovenous Fistula Maturation.

BACKGROUND: The arteriovenous fistula (AVF) is central to haemodialysis treatment, but up to half of surgically created AVF fail to mature. Chronic kidney disease often leads to mineral metabolism disturbances that may interfere with AVF maturation through adverse vascular effects. This study tested associations between mineral metabolism markers and vein histology at AVF creation and unassisted and overall clinical AVF maturation. METHODS: Concentrations of fibroblast growth factor 23, parathyroid hormone, calcium, phosphate, and vitamin D metabolites: 1,25(OH)2D, 24,25(OH)2D, 25(OH)D, and bioavailable 25(OH)D were measured in pre-operative serum samples from 562 of 602 participants in the Haemodialysis Fistula Maturation Study, a multicentre, prospective cohort study of patients undergoing surgical creation of an autologous upper extremity AVF. Unassisted and overall AVF maturation were ascertained for 540 and 527 participants, respectively, within nine months of surgery or four weeks of dialysis initiation. Study personnel obtained vein segments adjacent to the portion of the vein used for anastomosis, which were processed, embedded, and stained for measurement of neointimal hyperplasia, calcification, and collagen deposition in the medial wall. RESULTS: Participants in this substudy were 71% male, 43% black, and had a mean age of 55 years. Failure to achieve AVF maturation without assistance occurred in 288 (53%) participants for whom this outcome was determined. In demographic and further adjusted models, mineral metabolism markers were not significantly associated with vein histology characteristics, unassisted AVF maturation failure, or overall maturation failure, other than a biologically unexplained association of higher 24,25(OH)2D with overall failure. This exception aside, associations were non-significant for continuous and categorical analyses and relevant subgroups. CONCLUSIONS: Serum concentrations of measured mineral metabolites were not substantially associated with major histological characteristics of veins in patients undergoing AVF creation surgery, or with AVF maturation failure, suggesting that efforts to improve AVF maturation rates should increase attention to other processes such as vein mechanics, anatomy, and cellular metabolism among end stage renal disease patients.

Differential gene expression patterns in vein regions susceptible versus resistant to neointimal hyperplasia.

Arteriovenous hemodialysis graft (AVG) stenosis results in thrombosis and AVG failure, but prevention of stenosis has been unsuccessful due in large part to our limited understanding of the molecular processes involved in neointimal hyperplasia (NH) formation. AVG stenosis develops chiefly as a consequence of highly localized NH formation in the vein-graft anastomosis region. Surprisingly, the vein region just downstream of the vein-graft anastomosis (herein termed proximal vein region) is relatively resistant to NH. We hypothesized that the gene expression profiles of the NH-prone and NH-resistant regions will be different from each other after graft placement, and analysis of their genomic profiles may yield potential therapeutic targets to prevent AVG stenosis. To test this, we evaluated the vein-graft anastomosis (NH-prone) and proximal vein (NH-resistant) regions in a porcine model of AVG stenosis with a porcine microarray. Gene expression changes in these two distinct vein regions, relative to the gene expression in unoperated control veins, were examined at early (5 days) and later (14 days) time points following graft placement. Global genomic changes were much greater in the NH-prone region than in the NH-resistant region at both time points. In the NH-prone region, genes related to regulation of cell proliferation and osteo-/chondrogenic vascular remodeling were most enriched among the significantly upregulated genes, and genes related to smooth muscle phenotype were significantly downregulated. These results provide insights into the spatial and temporal genomic modulation underlying NH formation in AVG and suggest potential therapeutic strategies to prevent and/or limit AVG stenosis.

Autologous fat transplants to deliver glitazone and adiponectin for vasculoprotection.

The insulin sensitizing glitazone drugs, rosiglitazone (ROS) and pioglitazone (PGZ) both have anti-proliferative and anti-inflammatory effects and induce adipose tissue (fat) to produce the vaso-protective protein adiponectin. Stenosis due to intimal hyperplasia development often occurs after placement of arteriovenous synthetic grafts used for hemodialysis. This work was performed to characterize the in vitro and in vivo effects of ROS or PGZ incorporation in fat and to determine if fat/PGZ depots could decrease vascular hyperplasia development in a porcine model of hemodialysis arteriovenous graft stenosis. Powdered ROS or PGZ (6-6000µM) was mixed with fat explants and cultured. Drug release from fat was quantified by HPLC/MS/MS, and adiponectin and monocyte chemotactic protein-1 (MCP-1) levels in culture media were measured by ELISA. The effect of conditioned media from the culture of fat with ROS or PGZ on i) platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferation of human venous smooth muscle cells (SMC) was measured by a DNA-binding assay, and ii) lipopolysaccharide (LPS)-induced human monocyte release of tumor necrosis factor-alpha (TNFα) was assessed by ELISA. In a porcine model, pharmacokinetics of PGZ from fat depots transplanted perivascular to jugular vein were assessed by HPLC/MS/MS, and retention of the fat depot was monitored by MRI. A porcine model of synthetic graft placed between carotid artery and ipsilateral jugular vein was used to assess effects of PGZ/fat depots on vascular hyperplasia development. Both ROS and PGZ significantly induced the release of adiponectin and inhibited release of MCP-1 from the fat. TNF production from monocytes stimulated with LPS was inhibited 50-70% in the presence of media conditioned by fat alone or fat and either drug. The proliferation of SMC was inhibited in the presence of media conditioned by fat/ROS cultures. Fat explants placed perivascular to the external jugular vein were retained, as confirmed by MRI at one week after placement. PGZ was detected in the fat depot, in the external jugular vein wall and in adjacent tissue at clinically relevant levels, whereas levels in plasma were below detection. External jugular vein exposed to fat incorporated with PGZ had increased adiponectin expression compared to vein exposed to fat alone. However, the development of hyperplasia within the arteriovenous synthetic grafts was unchanged by treatment with fat/PGZ depots compared to no treatment.

Intimal Hyperplasia, Stenosis, and Arteriovenous Fistula Maturation Failure in the Hemodialysis Fistula Maturation Study.

Intimal hyperplasia and stenosis are often cited as causes of arteriovenous fistula maturation failure, but definitive evidence is lacking. We examined the associations among preexisting venous intimal hyperplasia, fistula venous stenosis after creation, and clinical maturation failure. The Hemodialysis Fistula Maturation Study prospectively observed 602 men and women through arteriovenous fistula creation surgery and their postoperative course. A segment of the vein used to create the fistula was collected intraoperatively for histomorphometric examination. On ultrasounds performed 1 day and 2 and 6 weeks after fistula creation, we assessed fistula venous stenosis using pre-specified criteria on the basis of ratios of luminal diameters and peak blood flow velocities at certain locations along the vessel. We determined fistula clinical maturation using criteria for usability during dialysis. Preexisting venous intimal hyperplasia, expressed per 10% increase in a hyperplasia index (range of 0%-100%), modestly associated with lower fistula blood flow rate (relative change, -2.5%; 95% confidence interval [95% CI], -4.6% to -0.4%; P=0.02) at 6 weeks but did not significantly associate with stenosis (odds ratio [OR], 1.07; 95% CI, 1.00 to 1.16; P=0.07) at 6 weeks or failure to mature clinically without procedural assistance (OR, 1.07; 95% CI, 0.99 to 1.15; P=0.07). Fistula venous stenosis at 6 weeks associated with maturation failure (OR, 1.98; 95% CI, 1.25 to 3.12; P=0.004) after controlling for case mix factors, dialysis status, and fistula location. These findings suggest that postoperative fistula venous stenosis associates with fistula maturation failure. Preoperative venous hyperplasia may associate with maturation failure but if so, only modestly.

Histopathology of Veins Obtained at Hemodialysis Arteriovenous Fistula Creation Surgery.

Stenosis from venous neointimal hyperplasia is common in native arteriovenous fistulas (AVFs). However, the preexisting histologic characteristics of veins at fistula creation, and associations thereof with baseline patient factors, have not been well characterized. In this study, we conducted histologic analysis of a segment of the vein used for anastomosis creation, obtained during AVF creation from 554 of the 602 participants in the multicenter Hemodialysis Fistula Maturation Cohort Study. We quantified intimal and medial areas and lengths of the internal and external elastic lamina by morphometry and assessed venous wall cells by immunohistochemistry, extracellular matrix with Movat stain, and calcium deposition by alizarin red stain. We also studied a representative subset of veins for markers of monocyte/macrophage content, cell proliferation, apoptosis, and neoangiogenesis. Neointima occupied >20% of the lumen in 57% of fully circumferential vein samples, and neointimal hyperplasia associated positively with age and inversely with black race. The neointima was usually irregularly thickened, sometimes concentric, and contained α-smooth muscle actin-expressing cells of smooth muscle or myofibroblast origin. Proteoglycans admixed with lesser amounts of collagen constituted the predominant matrix in the neointima. In 82% of vein samples, the media of vessel walls contained large aggregates of collagen. A minority of veins expressed markers of inflammation, cell proliferation, cell death, calcification, or neoangiogenesis. In conclusion, we observed preexisting abnormalities, including neointimal hyperplasia and prominent accumulation of extracellular matrix, in veins used for AVF creation from a substantial proportion of this cohort.

Vascular Coupling System for End-to-End Anastomosis: An In Vivo Pilot Case Report.

This paper presents the latest in vivo findings of a novel vascular coupling system. Vascular anastomosis is a common procedure in reconstructive surgeries and traditional hand suturing is very time consuming. The vascular coupling system described herein was designed to be used on arteries for a rapid and error-free anastomosis. The system consists of an engaging ring made from high density polyethylene using computer numerical control machining and a back ring made from polymethylmethacrylate using laser cutting. The vascular coupling system and its corresponding installation tools were tested in a pilot animal study to evaluate their efficacy in completing arterial anastomosis. A segment of expanded polytetrafluoroethylene (ePTFE) tubing was interposed into a transected carotid artery by anastomosis using two couplers in a pig. Two end-to-end anastomoses were accomplished. Ultrasound images were obtained to evaluate the blood flow at the anastomotic site immediately after the surgery. MRI was also performed 2 weeks after the surgery to evaluate vessel and ePTFE graft patency. This anastomotic system demonstrated high efficacy and easy usability, which should facilitate vascular anastomosis procedures in trauma and reconstructive surgeries.

Vascular Function at Baseline in the Hemodialysis Fistula Maturation Study.

BACKGROUND: End-stage renal disease is accompanied by functional and structural vascular abnormalities. The objective of this study was to characterize vascular function in a large cohort of patients with end-stage renal disease, using noninvasive physiological measurements, and to correlate function with demographic and clinical factors. METHODS AND RESULTS: We analyzed cross-sectional baseline data from the Hemodialysis Fistula Maturation Study, a multicenter prospective observational cohort study of 602 patients with end-stage renal disease from 7 centers. Brachial artery flow- and nitroglycerin-mediated dilation, carotid-femoral and -radial pulse wave velocity, and venous occlusion plethysmography were performed prior to arteriovenous fistula creation. Relationships of these vascular function measures with demographic, clinical, and laboratory factors were evaluated using linear mixed-effects models. Arterial function, as assessed by flow- and nitroglycerin-mediated dilation and carotid-femoral pulse wave velocity, worsened with increasing age and diabetes mellitus. Venous capacitance decreased with diabetes mellitus but not with age. Flow-mediated dilation was higher among patients undergoing maintenance dialysis than for those at predialysis, and a U-shaped relationship between serum phosphorus concentration and flow-mediated dilation was evident. Partial correlations among different measures of vascular function, adjusting for demographic factors, diabetes mellitus, and clinical center, were modest or essentially nonexistent. CONCLUSIONS: Multiple demographic and clinical factors were associated with the functions of vessels of different sizes and types in this large cohort of patients with end-stage renal disease. Low correlations between the different measures, controlling for demographic factors, diabetes mellitus, and center, indicated that these different types of vascular function otherwise vary heterogeneously across patients.

Rivaroxaban improves patency and decreases inflammation in a mouse model of catheter thrombosis.

INTRODUCTION: Dysfunction of indwelling central venous catheters (CVC) due to tissue ingrowth or clotting is common. The study objective was to determine if the oral anticoagulant rivaroxaban (RIVA) improved CVC patency and inflammation in mice. MATERIALS AND METHODS: Polyurethane catheters (0.5cm length) were placed unilaterally into the external jugular vein (EJV) of mice, which subsequently underwent daily gavage with either vehicle or RIVA (5mg/kg). CVC patency, as assessed by B-mode and Doppler ultrasound, and hematocrit were measured at 3, 7, 14 or 21days (n=8-11 mice/group/time-point). Plasma monocyte chemotactic protein-1 (MCP-1) levels were assessed by ELISA, EJV matrix metalloproteinase-9 (MMP-9) levels by western immunoblotting, and cell proliferation (anti-Ki67), macrophage infiltration (anti-MAC387) by immunostaining of EJV tissues. RESULTS AND CONCLUSIONS: CVC patency was significantly improved in RIVA-treated mice compared to vehicle-treated (93.8% vs. 62.9%) with the probability of patency in RIVA-treated mice being 1.5 times that in vehicle-treated (relative risk [RR], 1.50, 95% confidence interval [CI], 1.14-1.95, p=0.002). Plasma MCP-1 levels were lower in RIVA-treated mice vs. vehicle-treated at 21days (389±260 vs. 804±292ng/mL, p=0.005). Cell proliferation was less at day 7 in EJV from the RIVA-treated mice than vehicle-treated (5.0%±3.0 vs. 11.5%±3.6, p=0.0006), as were MMP-9 protein levels. There were no differences in hematocrit between vehicle and RIVA-treated groups at any time point. In conclusion, these data indicate RIVA lowers inflammation and improves CVC patency in a mouse model, supporting future studies to assess RIVA for improving CVC patency in patients.

Association between Preoperative Vascular Function and Postoperative Arteriovenous Fistula Development.

Arteriovenous fistula (AVF) maturation failure is the primary cause of dialysis vascular access dysfunction. To evaluate whether preoperative vascular functional properties predict postoperative AVF measurements, patients enrolled in the Hemodialysis Fistula Maturation Study underwent up to five preoperative vascular function tests (VFTs): flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NMD), carotid-femoral pulse wave velocity, carotid-radial pulse wave velocity, and venous occlusion plethysmography. We used mixed effects multiple regression analyses to relate each preoperative VFT to ultrasound measurements of AVF blood flow rate and venous diameter at 1 day, 2 weeks, and 6 weeks after AVF placement. After controlling for AVF location, preoperative ultrasound measurements, and demographic factors (age, sex, race, and dialysis status), greater NMD associated with greater 6-week AVF blood flow rate and AVF diameter (per absolute 10% difference in NMD: change in blood flow rate =14.0%; 95% confidence interval [95% CI], 3.7% to 25.3%; P<0.01; change in diameter =0.45 mm; 95% CI, 0.25 to 0.65 mm; P<0.001). Greater FMD also associated with greater increases in 6-week AVF blood flow rate and AVF diameter (per absolute 10% difference in FMD: change in blood flow rate =11.6%; 95% CI, 0.6% to 23.9%; P=0.04; change in diameter =0.31 mm; 95% CI, 0.05 to 0.57 mm; P=0.02). None of the remaining VFT parameters exhibited consistent statistically significant relationships with both postoperative AVF blood flow rate and diameter. In conclusion, preoperative NMD and FMD positively associated with changes in 6-week AVF blood flow rate and diameter, suggesting that native functional arterial properties affect AVF development.

Assessment of Novel Anti-thrombotic Fusion Proteins for Inhibition of Stenosis in a Porcine Model of Arteriovenous Graft.

BACKGROUND: Hemodialysis arteriovenous synthetic grafts (AVG) provide high volumetric blood flow rates shortly after surgical placement. However, stenosis often develops at the vein-graft anastomosis contributing to thrombosis and early graft failure. Two novel fusion proteins, ANV-6L15 and TAP-ANV, inhibit the tissue factor/factor VIIa coagulation complex and the factor Xa/factor Va complex, respectively. Each inhibitor domain is fused to an annexin V domain that targets the inhibitor activity to sites of vascular injury to locally inhibit thrombosis. This study's objective was to determine if these antithrombotic proteins are safe and effective in inhibiting AVG stenosis. METHODS: A bolus of either TAP-ANV or ANV-6L15 fusion protein was administered intravenously immediately prior to surgical placement of a synthetic graft between the external jugular vein and common carotid artery in a porcine model. At surgery, the vein and artery were irrigated with the anti-thrombotic fusion protein. Control animals received intravenous heparin. At 4 weeks, MRI was performed to evaluate graft patency, the pigs were then euthanized and grafts and attached vessels were explanted for histomorphometric assessment of neointimal hyperplasia at the vein-graft anastomosis. Blood was collected at surgery, immediately after surgery and at euthanasia for serum metabolic panels and coagulation chemistries. RESULTS: No acute thrombosis occurred in the control group or in either experimental group. No abnormal serum chemistries, activated clotting times or PT, PTT values were observed after treatment in experimental or control animals. However, at the vein-graft anastomosis, there was no difference between the control and experimental groups in cross-sectional lumen areas, as measured on MRI, and no difference in hyperplasia areas as determined by histomorphometry. These results suggest that local irrigation of TAP-ANV or ANV-6L15 intra-operatively was as effective in inhibiting acute graft thrombosis as intravenous administration of heparin, but failed to inhibit hyperplasia development and stenosis in AVG.

Prevention of Venous Neointimal Hyperplasia by a Multitarget Receptor Tyrosine Kinase Inhibitor.

BACKGROUND/AIMS: Venous neointimal hyperplasia (NH) is the predominant cause of stenosis in hemodialysis arteriovenous grafts (AVG), but there is currently no clinically used therapy to prevent NH. METHODS: A porcine AVG model was used to identify potential pharmacological targets to prevent NH. Sunitinib, a broad-spectrum tyrosine kinase inhibitor, was examined as a potential anti-NH drug utilizing in vitro and ex vivo models. RESULTS: In an in vivo porcine model, PDGF, VEGF and their receptors PDGFR-α and VEGFR-2 were upregulated at the venous anastomosis within 2 weeks after AVG placement, with NH development by 4 weeks. Sunitinib inhibited PDGF-stimulated proliferation, migration, phosphorylation of MAPK and PI3K/Akt proteins and changes in the expression of cell-cycle regulatory proteins in vascular smooth-muscle cells as well as VEGF-stimulated endothelial cell proliferation in vitro. In an ex vivo model, significant NH was observed in porcine vein segments perfused for 12 days under pathological shear stress. Sunitinib (100 nM) inhibited NH formation, with the intima-to-lumen area ratio decreasing from 0.45 ± 0.25 to 0.04 ± 0.02 (p < 0.05) with treatment. CONCLUSION: These findings demonstrate sunitinib to be a potential NH-preventive drug as well as the utility of an ex vivo model to investigate pharmacotherapies under pathophysiological flow conditions.

Aberrant soluble epoxide hydrolase and oxylipin levels in a porcine arteriovenous graft stenosis model.

Synthetic arteriovenous grafts (AVGs) used for hemodialysis frequently fail due to the development of neointimal hyperplasia (NH) at the vein-graft anastomosis. Inflammation and smooth-muscle cell (SMC) and myofibroblast proliferation and migration likely play an important role in the pathogenesis of NH. Epoxyeicosatrienoic acids (EETs), the products of the catabolism of arachidonic acid by cytochrome P450 enzymes, possess anti-inflammatory, antiproliferative, antimigratory and vasodilatory properties that should reduce NH. The degradation of vasculoprotective EETs is catalyzed by the enzyme, soluble epoxide hydrolase (sEH). sEH upregulation may thus contribute to NH development by the enhanced removal of vasculoprotective EETs. In this study, sEH, cytochrome P450 and EETs were examined after AVG placement in a porcine model to explore their potential roles in AVG stenosis. Increased sEH protein expression, decreased P450 epoxygenase activity and dysregulation of 5 oxylipin mediators were observed in the graft-venous anastomotic tissues when compared to control veins. Pharmacological inhibitors of sEH decreased the growth factor-induced migration of SMCs and fibroblasts, although they had no significant effect on the proliferation of these cells. These results provide insights on epoxide biology in vascular disorders and a rationale for the development of novel pharmacotherapeutic strategies to prevent AVG failure due to NH and stenosis.

Novel therapies for hemodialysis vascular access dysfunction: myth or reality?

Hemodialysis vascular access dysfunction is a major source of morbidity for patients with ESRD. Development of effective approaches to prevent and treat vascular access failure requires an understanding of the underlying mechanisms, suitable models for preclinical testing, systems for targeted delivery of interventions to maximize efficacy and minimize toxicity, and rigorous clinical trials that use appropriate outcome measures. This article reviews the substantial progress and ongoing challenges in developing novel treatments for arteriovenous vascular access failure and focuses on localized rather than systemic interventions.

Serial analysis of lumen geometry and hemodynamics in human arteriovenous fistula for hemodialysis using magnetic resonance imaging and computational fluid dynamics.

The arteriovenous fistula (AVF) is the preferred form of vascular access for maintenance hemodialysis, but it often fails to mature to become clinically usable, likely due to aberrant hemodynamic forces. A robust pipeline for serial assessment of hemodynamic parameters and subsequent lumen cross-sectional area changes has been developed and applied to a data set from contrast-free MRI of a dialysis patient's AVF collected over a period of months after AVF creation surgery. Black-blood MRI yielded images of AVF lumen geometry, while cine phase-contrast MRI provided volumetric flow rates at the in-flow and out-flow locations. Lumen geometry and flow rates were used as inputs for computational fluid dynamics (CFD) modeling to provide serial wall shear stress (WSS), WSS gradient, and oscillatory shear index (OSI) profiles. The serial AVF lumen geometries were co-registered at 1mm intervals using respective lumen centerlines, with the anastomosis as an anatomical landmark. Lumen enlargement was limited at the vein region near the anastomosis and a downstream vein valve, potentially attributed to the physical inhibition of wall expansion at those sites. This work is the first serial and detail study of lumen and hemodynamic changes in human AVF using MRI and CFD. This novel protocol will be used for a multicenter prospective study to identify critical hemodynamic factors that contribute to AVF maturation failure.

Soluble epoxide hydrolase expression in a porcine model of arteriovenous graft stenosis and anti-inflammatory effects of a soluble epoxide hydrolase inhibitor.

Synthetic arteriovenous (AV) grafts, placed between an artery and vein, are used for hemodialysis but often fail due to stenosis, typically at the vein-graft anastomosis. This study recorded T lymphocyte and macrophage accumulation at the vein-graft anastomosis, suggesting a role for inflammation in stenosis development. Epoxyeicosatrienoic acids (EETs), products of cytochrome P-450 epoxidation of arachidonic acid, have vasculoprotective and anti-inflammatory effects including inhibition of platelet activation, cell migration, and adhesion. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to less active diols. The effects of a specific inhibitor of sEH (sEHI) on cytokine release from human monocytes and mouse bone marrow-derived macrophages (BMMΦ) from wild-type (WT) and sEH knockout (KO) animals were investigated. Expression of sEH protein increased over time at the anastomosis as evaluated by immunohistochemistry. Pre-exposure of adherent human monocytes to sEHI (5 µM) significantly inhibited lipopolysaccharide-induced release of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α and enhanced the EET-to-diol ratio. Release of MCP-1 from WT BMMΦ was significantly inhibited but release from sEH KO BMMΦ was not attenuated indicating the specificity of the sEHI. In contrast, sEHI did not inhibit the release of macrophage inflammatory protein-1 or interleukin-6. Nuclear translocation of NF-κB, as assessed by immunocytochemical staining, was not decreased with sEHI in monocytes, but the phosphorylation of JNK was completely abrogated, suggesting this pathway is the target of sEHI effects in monocytes. These results suggest that sEHI may be useful for inhibition of inflammation and subsequently stenosis in AV grafts.

A biodegradable perivascular wrap for controlled, local and directed drug delivery.

Perivascular delivery of anti-proliferative agents is an attractive approach to inhibit hyperplasia that causes stenosis of synthetic hemodialysis grafts and other vascular grafts. Perivascular drug delivery systems typically release drugs to both the vascular wall and non-target extravascular tissue. The objective of this study was to develop a biodegradable, perivascular delivery system for localized, sustained and unidirectional drug release in the context of synthetic arteriovenous (AV) grafts used for chronic hemodialysis. To this end, a dense non-porous polymer barrier layer was laminated to either i) a drug-loaded non-porous polymer layer or ii) a porous polymer layer. To provide tunability, the porous layer could be loaded with drug during casting or later infused with a drug-loaded hydrogel. The polymer bilayer wraps were prepared by a solvent casting, thermal-phase inversion technique using either polylactide-co-glycolide (PLGA) or polycaprolactone (PCL). Sunitinib, a multi-target receptor tyrosine kinase inhibitor, was used as a model drug. In a modified transwell chamber system, the barrier function of the non-porous PLGA backing was superior to the non-porous PCL backing although both markedly inhibited drug diffusion. As assessed by in vitro release assays, drug release duration from the drug-loaded non-porous PCL construct was almost 4-fold greater than release from the porous PCL construct infused with drug-laden hydrogel (22 days vs. 5 days); release duration from the drug-loaded non-porous PLGA construct was prolonged approximately 3-fold over release from the porous PLGA construct infused with drug-laden hydrogel (9 days vs. 3 days). Complete in vitro degradation of the PLGA porous and non-porous constructs occurred by approximately 35 days whereas the PCL constructs remained intact even after most of the drug was released (49 days). The PLGA non-porous bilayer wrap containing 143±5.5mg sunitinib in the inner layer was chosen for further pharmacokinetic assessment in vivo where the construct was placed around the external jugular vein in a porcine model. At 1 week, no drug was detected by HPLC/MS/MS in any examined extravascular tissue whereas high levels of drug were detected in the wrapped vein segment (1048 ng g⁻¹ tissue). At 4 weeks, drug was detected in adjacent muscle (52 ng g⁻¹ tissue) but 13-fold greater amounts were detected in the wrapped vein segment (1742 ng g⁻¹ tissue). These results indicate that the barrier layer effectively impedes extravascular drug loss. Tensile testing showed that the initially flexible PLGA construct stiffened with hydration, a phenomenon also observed after in vivo placement. This characteristic may be useful to resist undue circumferential venous tensile stress produced in AV grafting. The PLGA wrap bilayer formulation is a promising perivascular drug delivery design for local treatment of hemodialysis AV graft hyperplasia and possibly other hyperplastic vascular disorders.

In vivo evaluation of the delivery and efficacy of a sirolimus-laden polymer gel for inhibition of hyperplasia in a porcine model of arteriovenous hemodialysis graft stenosis.

Synthetic arteriovenous (AV) hemodialysis grafts are plagued by hyperplasia resulting in occlusion and graft failure yet there are no clinically available preventative treatments. Here the delivery and degradation of a sirolimus-laden polymer gel were monitored in vivo by magnetic resonance imaging (MRI) and its efficacy for inhibiting hyperplasia was evaluated in a porcine model of AV graft stenosis. Synthetic grafts were placed between the carotid artery and ipsilateral jugular vein of swine. A biodegradable polymer gel loaded with sirolimus (2.5mg/mL) was immediately applied perivascularly to the venous anastomosis, and reapplied by ultrasound-guided injections at one, two and three weeks. Control grafts received neither sirolimus nor polymer. The lumen cross-sectional area at the graft-vein anastomosis was assessed in vivo by non-invasive MRI. The explanted tissues also underwent histological analysis. A specifically developed MRI pulse sequence provided a high contrast-to-noise ratio (CNR) between the polymer and surrounding tissue that allowed confirmation of gel location after injection. Polymer signal decreased up to 80% at three to four weeks after injection, slightly faster than its degradation kinetics in vitro. The MR image of the polymer was confirmed by visual assessment at necropsy. On histological assessment, the mean hyperplasia surface area of the treated graft was 52% lower than that of the control grafts (0.43mm(2) vs. 0.89mm(2); p<0.003), while the minimum cross-sectional lumen area, as measured on MRI, was doubled (5.3mm(2) vs 2.5mm(2); p<0.05). In conclusion, customized MRI allowed non-invasive monitoring of the location and degradation of drug delivery polymer gels in vivo. Perivascular application of sirolimus-laden polymer yielded a significant decrease in hyperplasia development and an increase in lumen area at the venous anastomosis of AV grafts.

PDGF-induced proliferation in human arterial and venous smooth muscle cells: molecular basis for differential effects of PDGF isoforms.

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum-starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF-AA, venous SMCs showed no proliferative response to PDGF-AA, but instead demonstrated a significantly greater proliferative response to PDGF-BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5-fold higher (P < 0.05) density of PDGF receptor-α (PDGF-Rα) and a 6.6-fold lower (P < 0.05) density of PDGF-Rß expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF-AA in arterial SMCs was a marked PDGF-Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c-Jun NH2-terminal kinase (JNK), and a significant reduction in expression of the cell-cycle inhibitor p27(kip1). This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF-Rα was detected after venous SMC exposure to PDGF-AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF-BB stimulation of venous SMC resulted in PDGF-Rß activation as well as transactivation of epidermal growth factor receptor (EGF-R); transactivation of EGF-R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins.

Correlation of tissue drug concentrations with in vivo magnetic resonance images of polymer drug depot around arteriovenous graft.

Sustained delivery of anti-proliferative drugs to the perivascular area using an injectable polymeric platform is a strategy to inhibit vascular hyperplasia and stenosis. In this study, the concentrations of sirolimus in vascular tissues were evaluated after delivery using an injectable platform made of poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA). In order to optimize the drug release profile, the effect of two solvents or solid loading of the sirolimus into the polymer gel was first examined in vitro. The early release was slower with loading of dry drug into the polymer, compared to drug dissolution in solvents. Dry sirolimus was therefore used to load the polymer and applied to the perivascular surface of the graft-venous anastomosis at the time of surgical placement of a carotid-jugular synthetic hemodialysis graft in a porcine model. This was replenished by ultrasound-guided injection of additional drug-laden polymer at one, two and three weeks post-operatively. Magnetic resonance imaging (MRI) using pulse sequences specifically designed for optimal detection of the polymeric gel showed that the polymer injected post-operatively remained at the juxta-anastomotic perivascular site at two weeks. Sirolimus was extracted from various segments of the juxta-anastomotic tissues and the drug concentrations were determined using HPLC MS/MS. Tissue sirolimus concentrations at one and two weeks were highest near the venous anastomosis, which were approximately 100- to 500-fold greater than the concentrations necessary to inhibit vascular smooth muscle cell proliferation in vitro. Drug concentrations remained above the inhibitory concentrations for at least six weeks post-operatively. Thus, serial injections of sustained-delivery polymer gel loaded with sirolimus can provide high localized concentrations at target vascular tissues and thus may be useful for the prevention and treatment of vascular proliferative disorders such as hemodialysis graft stenosis. In addition, MRI is useful for the monitoring of the location of the drug depot.

Using the stochastic collocation method for the uncertainty quantification of drug concentration due to depot shape variability.

Numerical simulations entail modeling assumptions that impact outcomes. Therefore, characterizing, in a probabilistic sense, the relationship between the variability of model selection and the variability of outcomes is important. Under certain assumptions, the stochastic collocation method offers a computationally feasible alternative to traditional Monte Carlo approaches for assessing the impact of model and parameter variability. We propose a framework that combines component shape parameterization with the stochastic collocation method to study the effect of drug depot shape variability on the outcome of drug diffusion simulations in a porcine model. We use realistic geometries segmented from MR images and employ level-set techniques to create two alternative univariate shape parameterizations. We demonstrate that once the underlying stochastic process is characterized, quantification of the introduced variability is quite straightforward and provides an important step in the validation and verification process.