Cellular and morphological changes during neointimal hyperplasia development in a porcine arteriovenous graft model.

BACKGROUND: Implantation of a haemodialysis arteriovenous graft is often followed by the development of neointimal hyperplasia (NH) at the venous anastomosis. The nature of the proliferating cells in these lesions is not well understood. A better understanding of the cells contributing to NH is important to the development of preventive strategies. METHODS: Carotid-jugular PTFE grafts were placed in 21 pigs and characterized at various time points following implantation. Venous anastomotic tissues were harvested at 1, 7, 14, 21, 28 or 49 post-operative days for histology and immunohistochemistry. RESULTS: Van Gieson staining of the tissues showed that NH was apparent as early as day 7 and progressed over time. Even by day 1, there were cells expressing the proliferation marker Ki-67 in the venous adventitia, but not the media, at the anastomosis. Double immunohistochemical staining showed that these cells were positive for alpha-smooth muscle actin (alpha-SMA), but negative for smooth muscle myosin heavy chain (SM MHC), suggesting that the proliferating cells were myofibroblasts rather than smooth muscle cells. By day 7, proliferating cells were abundant in the adventitia and began to appear in the media, surrounded by extracellular matrix visualized using Trichrome staining. By day 49, alpha-SMA-positive, SM MHC-negative cells were predominant in the NH, and Ki-67 staining had largely vanished. CONCLUSIONS: These results are consistent with the hypothesis that adventitial fibroblasts are transformed into myofibroblasts and begin to proliferate within hours after graft placement. Migration of these cells towards the vessel lumen with subsequent proliferation appears to be a major contributor to NH formation. The pivotal role of the adventitial fibroblasts in the pathogenesis of NH provides a compelling rationale for therapies that target the transformation, proliferation and migration of these cells to prevent arteriovenous graft stenosis.     

Perivascular paclitaxel wraps block arteriovenous graft stenosis in a pig model.

BACKGROUND: Haemodialysis vascular access dysfunction is currently a huge clinical problem. In an attempt to reduce the morbidity associated with haemodialysis vascular access dysfunction, we have previously developed and validated a local perivascular paclitaxel release system that has been shown to release paclitaxel for at least 3 weeks. The aim of the current study was to evaluate the in vivo use of these perivascular wraps (for both safety and efficacy) at different time points in our pig model of arteriovenous graft stenosis. METHODS: Paclitaxel-loaded ethylene vinyl acetate wraps were placed around the graft-vein anastomosis on one side, with control polymers being placed on the contralateral side in our pig model of arteriovenous graft stenosis. Animals were sacrificed at early (10-11 days), middle (23-24 days) and late (32-38 days) time points. The entire graft-vein anastomosis was removed at the time of sacrifice and assessed for the extent of luminal stenosis using histomorphometric techniques. RESULT: Graft-vein anastomoses treated with the paclitaxel-loaded polymers had an almost complete absence of luminal stenosis at the middle (23-24 days) and late (32-38 days) time points (when one would expect the development of neointimal hyperplasia) as compared with the contralateral control graft-vein anastomoses (37.90% luminal stenosis in the controls vs 0.10% in the paclitaxel group). There were minimal local side effects from this procedure. CONCLUSIONS: Our results demonstrate the safety and efficacy of paclitaxel-loaded perivascular wraps in the setting of a pig model of arteriovenous graft stenosis. We believe that such a local approach which could be easily applied at the time of surgery is ideally suited for use in the clinical setting of haemodialysis vascular access dysfunction. It is likely that this novel approach could result in a significant reduction in the huge economic and health morbidity costs currently associated with this recalcitrant clinical problem.     

Paclitaxel-coated expanded polytetrafluoroethylene haemodialysis grafts inhibit neointimal hyperplasia in porcine model of graft stenosis.

BACKGROUND: The main pathology of haemodialysis graft stenosis is venous neointimal hyperplasia at graft-venous anastomoses. Neointimal hyperplasia is also observed in cases of coronary artery in-stent restenosis. Paclitaxel is a chemotherapeutic agent used to treat cancer, and has been proven to inhibit neointimal hyperplasia of coronary artery in-stent restenosis. In this study, we examined whether a paclitaxel-coated haemodialysis graft could inhibit neointimal hyperplasia and prevent stenosis. METHODS: We dip-coated paclitaxel on expanded polytetrafluoroethylene (ePTFE) grafts at a dose density of 0.59 microg/mm(2). In vitro release tests showed an initial paclitaxel burst followed by a long-term slow release. Using ePTFE grafts with (coated group, n = 8) or without a paclitaxel coating (control group, n = 11), we constructed arteriovenous (AV) grafts connecting the common carotid artery and the external jugular vein in Landrace pigs. RESULTS: After excluding seven pigs for technical failure, cross-sections of graft-venous anastomoses obtained 6 weeks after placing the AV grafts were analysed. Percentage luminal stenosis, ratios of intima to media in whole cross-sections, areas of intima in the peri-junctional areas (within 2 mm above and 2 mm below the graft-venous junction), and the mean thickness of intima within venous sides of cross-sections, were 60.5% (range, 41.5-60.7), 13.0 (range, 8.6-20.4), 23.7 mm(2) (range, 10.8-32.1) and 2.1 mm (range, 1.1-3.0), respectively, in the control group, whereas corresponding median values in the coated group were 10.4% (range, 1.0-17.8), 1.0 (range, 0.7-5.1), 1.6 mm(2) (range, 0.2-8.0) and 0.3 mm (range, 0.1-2.2). All parameters were significantly different between the two groups (P<0.05 by Mann-Whitney test). CONCLUSION: Paclitaxel-coated ePTFE grafts could prevent neointimal hyperplasia and the stenosis of AV haemodialysis grafts.     

重组arresten蛋白对自体移植静脉内膜增生的抑制作用

目的 观察原核表达人arresten重组蛋白对自体移植静脉内膜增生的抑制作用.方法 用pRSET原核表达系统表达并纯化人arresten重组蛋白.将Wistar大鼠颈外静脉移植于腹主动脉,建立大鼠自体静脉移植模型,实验分3组:假手术组、移植对照组和移植实验组.自术后第3天起,皮下注射给予arresten重组蛋白(每日4 mg/kg体重)处理.4周后取移植静脉组织标本,进行病理组织学观察与免疫组织化学染色分析.结果 移植组移植静脉均呈现典型的内膜增生、肥厚,导致血管管腔狭窄;新生内膜主要有过度增殖的α-SMA染色阳性平滑肌细胞组成.移植实验组移植静脉内膜增生受到明显抑制,新生内膜面积(0.12±0.07)mm2及新生内膜/中膜面积比(0.373±0.085)均显著低于对照组[(0.38±0.11)mm2,1.621±0.086,P<0.01];并且实验组移植静脉新生内膜细胞PCNA标记指数显著低于对照组[(15.62±3.97)%比(56.36±3.49)%,P<0.01].结论 重组arresten蛋白通过抑制新生内膜平滑肌细胞的增殖能有效抑制自体移植静脉内膜增生的发生发展,在防治血管重建术后再狭窄方面显示出良好的应用前景.     

Pharmacologic inhibition of vein graft neointimal hyperplasia

Although arterial conduits are widely used and have improved the long-term results of coronary artery bypass grafting, vein grafts remain important additional conduits in coronary surgery. Newer studies show a saphenous vein graft patency of 60% or more at 10 years postoperatively. The pathology of vein graft disease consists of thrombosis, neointimal hyperplasia, and vein graft atherosclerosis, which limit graft longevity. Therapeutic strategies to prevent vein graft disease include external stenting, pharmacotherapy, and gene therapy. The potential benefits of a pharmacologic approach are as follows: (1) Drugs with a broad clinical experience can be used; (2) side effects of systemic application can be minimized by local therapy; and (3) no vascular injury, such as pressurizing the vein for a viral transfection approach, is necessary. The different sites for pharmacotherapy in vein graft disease are reviewed in this article.     

Locally applied cilostazol suppresses neointimal hyperplasia and medial thickening in a vein graft model.

BACKGROUND: Pathological changes in vein grafts begin immediately after arterial circulation is applied to the grafts. Chemical mediator stimulation and mechanical strain induce neointimal hyperplasia and medial thickening of the vein grafts, resulting in their failure. We investigated the inhibitory effect of locally applied cilostazol, an inhibitor of cyclic adenosine monophosphate phosphodiesterase III, on neointimal hyperplasia and medial thickening of the grafts. METHODS AND RESULTS: We established a distal anastomotic stricture model of femoral vein-abdominal aorta interposition grafting in rats. In this model, neointimal hyperplasia was observed not only at the distal anastomotic sites, but also in the graft body at postoperative day 14 and was markedly progressed at day 28. A strong expression of tenascin-C was found in the media and neointima of the graft body. In the grafts around which cilostazol was administered locally using Pluronic gel, neointimal hyperplasia was significantly suppressed compared with control grafts treated with the gel alone, with the mean neointimal cross-sectional area reduced by 87.1% for the graft body and by 78.9% for the distal anastomotic sites and mean medial cross-sectional area of the graft body reduced by 54.2% at day 28 versus the control. Cilostazol treatment decreased cell proliferation and the number of tenascin-C-producing cells seen by in situ hybridization, but the expression of tenascin-C protein was not suppressed. CONCLUSION: We concluded that a single perivascular application of cilostazol inhibits neointimal hyperplasia and medial thickening of vein grafts in a rat model.     

Efficacy of local dipyridamole therapy in a porcine model of arteriovenous graft stenosis

Perivascular delivery of antiproliferative drugs has been proposed as an approach to prevent neointimal hyperplasia associated with hemodialysis polytetrafluoroethylene (PTFE) grafts. We examined this approach to deliver dipyridamole in a porcine graft model. PTFE grafts were implanted between the carotid artery and external jugular vein bilaterally in pigs. During the surgery or 1 week post-graft placement, dipyridamole (0.26-52 mg) alone or incorporated in microspheres was mixed with an injectable polymeric gel and applied to the graft-arterial and graft-venous anastomoses on one side, whereas the contralateral control graft received no treatment. Three or four weeks after operation, the grafts and adjacent vessels were explanted en bloc and cross-sections of the anastomoses were examined histologically. The degree of neointimal hyperplasia was quantified by planimetry. In separate experiments, dipyridamole was extracted from the explanted tissues and assayed by spectrofluorometry. The normalized median hyperplasia areas of the treated and control graft-venous anastomoses were 0.45 (25th-75th percentile, 0.30-0.86) and 0.24 (0.21-0.30), respectively (N=7; P=0.08). The median hyperplasia areas of the treated and control graft-arterial anastomoses were 0.12 (0.07-0.39) and 0.11 (0.09-0.13), respectively (N=7; P=0.31). The dipyridamole levels in the vascular walls around the anastomoses were at or above the in vitro inhibitory concentrations for approximately 3 weeks. These results suggest that the local perivascular sustained delivery of dipyridamole, even at high dosages, was ineffective in inhibiting neointimal hyperplasia associated with PTFE grafts in a porcine model.     

Rapid,arteriovenous graft failure due to intimal hyperplasia: a porcine,bilateral, carotid arteriovenous graft model

BACKGROUND: The loss of patency constitutes the major complication of arteriovenous (AV) polytetrafluoroethylene hemodialysis grafts. In most cases, this graft failure is due to intimal hyperplasia at the venous outflow tract, including proliferation of vascular, smooth muscle cells and fibroblasts with deposition of extracellular matrix proteins. Thus far, procedures developed for improving patency have proven unsuccessful, which can be partly explained by the lack of relevant animal models. For this purpose, we developed a porcine model for AV graft failure that will allow the assessment of promising therapeutic strategies in the near future. MATERIALS AND METHODS: In 14 pigs, AV grafts were created bilaterally between the carotid artery and the jugular vein using expanded polytetrafluoroethylene. Two, 4 or 8 weeks after AV shunting, the grafts and adjacent vessels were excised and underwent histologic analysis. RESULTS: From 2 weeks onwards, a thick neo-intima developed at the venous anastomosis, predominantly consisting of alpha-actin-positive vascular smooth muscle cells (VSMC). Intimal area increased over time, coinciding with a decreased graft flow. Grafts remained patent for at least 4 weeks. At 8 weeks, patency rates declined to less than 50% due to thrombus formation superimposed on progressive neo-intima formation. CONCLUSIONS: Implantation of an AV graft between the carotid artery and jugular vein in pigs causes a rapid neo-intimal response, accompanied by a loss of patency of 50% at 8 weeks after surgery. This model offers a suitable tool to study local interventions aimed at the improvement of AV graft patency rates.     

Expression of C-reactive protein in myointimal hyperplasia in a porcine arteriovenous graft model.

BACKGROUND: The migration and proliferation of myofibroblasts are prominent features of myointimal hyperplasia associated with haemodialysis polytetrafluoroethylene (PTFE) grafts. Since C-reactive protein (CRP) possesses functional activities on vascular smooth muscle cells (SMCs), we examined the expression of this protein in PTFE grafts early in the course of myointimal hyperplasia development in a porcine model. METHOD: Bilateral carotid-jugular PTFE loop grafts were placed in pigs. After euthanasia at 2-4 weeks, the graft-venous and graft-arterial anastomoses and the adjacent blood vessels were excised en bloc and subjected to immunohistochemical analyses and in situ hybridization for CRP. The ability of CRP to stimulate proliferation was examined in cultured porcine venous SMCs using the bromodeoxyuridine assay after incubation for 48 h. RESULTS: Severe myointimal hyperplasia was found at 3 weeks after graft placement at both graft-venous and graft-arterial anastomoses. Compared to normal tissues, staining for CRP was far more intense in cells in the hyperplastic lesions at both anastomoses, which also stained positive for smooth muscle alpha-actin. In situ hybridization showed that these cells also expressed mRNA for CRP. At 1 microg/ml, CRP increased the proliferation of cultured porcine venous SMCs by 45.9+/-5.8%. CONCLUSION: CRP was produced in venous and arterial SMCs and its expression was enhanced in the hyperplastic lesions associated with arteriovenous PTFE grafts in a porcine model. Together with the ability of CRP to promote SMC proliferation, these data suggest that CRP might play a pathogenic role in the development of myointimal hyperplasia in PTFE grafts.     

A novel mouse model of autologous venous graft intimal hyperplasia

BACKGROUND: To investigate the molecular mechanism of autologous venous graft intimal hyperplasia, a mouse model is needed. Currently only vein to carotid artery mouse models are available and are hampered by a high thrombosis rate. We hypothesized that operating on the aorta would lead to intimal hyperplasia with decreased risk of thrombosis. MATERIALS AND METHODS: In C57BL/6J mice, the left external jugular vein was grafted into the infrarenal abdominal aorta by end-to-end anastomosis with 11-0 Ethilon. Grafts harvested at 1, 2, 4, 8, and 16 weeks postoperatively were subjected to histological and immunohistochemical analysis. RESULTS: Thirty-one of 35 mice survived; 2 mice were sacrificed secondary to thrombosis. The percentage lumen narrowing (+/-SE) was 7.8 +/- 0.3, 16.4 +/- 0.9, 19.2 +/- 0.9, 22.3 +/- 0.8, and 23.9 +/- 1.6% at 1, 2, 4, 8 and 16 weeks, respectively. Nuclear density decreased with each successive time point. The percentage of alpha-smooth-muscle actin-positive cells within the neointima peaked at 16 weeks (53%), and the percentage of cells positive for proliferating cell nuclear antigen peaked at 2 weeks (39%). CONCLUSIONS: We thus report on a novel mouse model of intimal hyperplasia in autologous venous grafts with a low thrombosis rate. Further studies using this model, coupled with genetic and bone marrow transplantation mouse models, should lead to significant enhancement in understanding of the mechanism of intimal hyperplasia.     

A pig model of venous neoinitimal hyperplasia in polytetrafluoroethylene dialysis grafts

Purpose: Vascular access dysfunction is a major cause of morbidity and hospitalization in the hemodialysis population. In the United States, $1 billion per year is spent managing the vascular complications of hemodialysis. Venous neointimal hyperplasia (VNH), with subsequent venous stenosis and thrombosis at the graft-vein anastomosis, constitutes 75% of failed polytetrafluoroethylene vascular grafts. To understand both the pathogenesis and potential therapeutic interventions of VNH, we have validated a model of arteriovenous graft stenosis in pigs.Methods: Goretex loop grafts, 7 cm long × 4 mm ID, were placed bilaterally from the femoral artery to the femoral vein in 12 pigs, using standard operative vascular techniques. Grafts were harvested at 2-, 4-, 7-, 14-, and 28-day time points, and H&E-stained specimens were evaluated for neointimal encroachment into the graft. Immunohistochemistry was performed to demonstrate the expression of factor VIII, smooth muscle α-actin, and Ki67 (a marker of cell proliferation).Results: Venous neointimal hyperplasia was present postoperatively within 14 days at the graft-vein anastomosis and within the proximal vein. It was characterized by prominent angiogenesis within the thickened neointima, a perigraft macrophage layer, extracellular matrix components, and the proliferation of smooth muscle cells and myofibroblasts.Conclusion: The pathognomonic features of VNH visualized in the pig specimens are similar to those identified in human hemodialysis patients, which demonstrates the clinical relevance of this model. Each of these characterized lesions represents a potential site for novel prophylactic and therapeutic interventions, which will diminish the significant human and economic costs associated with dialysis graft dysfunction.     

L-arginine polymers inhibit the development of vein graft neointimal hyperplasia

OBJECTIVE: We sought to determine whether L -arginine polymer treatment of vein grafts enhances vascular production of nitric oxide and inhibits the development of neointimal hyperplasia. METHODS: External jugular veins of New Zealand White rabbits (n = 42) were harvested; treated intraluminally for 15 minutes with phosphate-buffered saline solution or L -arginine polymer 5, 7, or 9 at either 10 or 100 micromol/L; and then grafted into the contralateral carotid artery. Rabbits were killed after 28 days, and 5-microm sections of vessels were stained with hematoxylin and scored for intima/media ratio by using computerized morphometric analysis. Separate veins were treated in a similar fashion with biotinylated polymers and phosphate-buffered saline solution to assess for translocation efficiencies. Finally, vein segments pretreated with either phosphate-buffered saline solution or L -arginine polymers were cultured in Dulbecco's modified Eagle's medium containing lipopolysaccharide (100 microg/mL) and interferon gamma (200 U/mL) for 48 hours before measuring nitric oxide levels by means of the Griess reaction. RESULTS: Biotinylated L -arginine polymers demonstrated a dose- and length-dependent uptake into intimal and medial cells of treated vessels. Nitric oxide levels were significantly higher in vein segments treated with 100 micromol/L of L -arginine polymer 9 compared with control segments. Finally, the intima/media ratio also reflected both length- and concentration-dependent inhibition of neointimal hyperplasia.intima/media ratio PBS R5 R7 R9 10 micromol/L 0.909 +/- 0.072 0.920 +/- 0.073 0.861 +/- 0.138 0.710 +/- 0.122 100 micromol/L 0.924 +/- 0.061 0.581 +/- 0.089* 0.529 +/- 0.093* PBS, Phosphate-buffered saline solution; R, L -arginine polymer. *P <.001 versus phosphate-buffered saline solution and L -arginine polymer 5 controls (Bonferroni-corrected value). CONCLUSIONS: Arginine polymers of sufficient length and concentration were effective in increasing nitric oxide levels and reducing neointimal hyperplasia in this vein graft model.     

An appropriately sized soft polyester external stent prevents enlargement and neointimal hyperplasia of a saphenous vein graft in a canine model

Although the efficacy of external stents for vein grafts in coronary artery bypass grafting has been recognized, the ideal diameter and material of the stent remain controversial. We created a new external stent made of soft polyester mesh and performed an animal experiment using canines. Bilateral saphenous vein grafts were interposed in the bilateral common carotid artery of 10 beagles. The grafts in the left carotid artery were designated as the control group, and those in the right rolled by a soft polyester mesh external stent were designated as mesh group. Two of the 10 animals were sacrificed due to severe wound infection. The other eight were observed by echography for 6 months, and then grafts were extracted and thickness of the neointima of the grafts was measured. The control group showed 146% ± 26% postoperative enlargement of the internal diameter of the vein grafts after 6 months, whereas the mesh group showed only 115% ± 15% after the same duration (P = 0.0003). The median thickness of the neointima in the mesh group (170 µm [range: 150–190]) was significantly thinner than that in the control group (260 µm [range: 220–310], P < 0.0001). Some degree of correlation between the thickness of neointima and proportion of enlargement was noted (r = 0.518, P = 0.0024). A soft polyester mesh external stent for vein grafts successfully suppressed the enlargement of the vein grafts and thickness of the neointima after 6 months.     

Novel microsurgical model of experimental vascular neointimal hyperplasia

Neointimal hyperplasia is a common finding after trauma to blood vessels and also as a primary change in atherosclerosis. In this study we have developed a simple model, using microsurgical techniques, for the initiation of neointimal hyperplasia in the rat. In 24 Wistar rats, a 2 mm-diameter arteriotomy in the aorta was repaired with a ?patch”? of iliolumbar vein, using eight evenly spaced 10-0 Ethilon sutures. The patch overlapped the edges of the arteriotomy, and the sutures fastened the patch to the subjacent aorta. At 2, 6 and 12 weeks after surgery, the venous patch grafts and segments of the adjacent aortae of eight rats were removed. One-half of the specimens were analyzed by scanning electron microscopy and the other one-half by light microscopy. All patch grafts were surgically successful. Endothelial cells regenerated to cover the patch within 2 weeks of insertion. By 6 weeks after surgery, neointimal hyperplasia, consisting predominantly of smooth muscle, had developed in all patches to a thickness that was not significantly different from that of the adjacent aorta. These findings are consistent with data from other more complex experimental models of neointimal hyperplasia in the rat. We consider that this venous patch technique is a simple but effective model for the initiation of neointimal hyperplasia in the rat and may easily be used to study the experimental effects of various injurious or therapeutic agents on neointimal hyperplasia. © 1993 Wiley-Liss Inc.     

MicroRNA-155 promotes neointimal hyperplasia through smooth muscle-like cell-derived RANTES in arteriovenous fistulas

Objective

Arteriovenous fistula (AVF) suffers from a high number of failures caused by insufficient outward remodeling and venous neointimal hyperplasia formation. The aim was to investigate the exact mechanism by which microRNA-155 (miR-155) in the outflow vein of AVF is regulated.