Low molecular weight fucoidan prevents neointimal hyperplasia after aortic allografting

BACKGROUND: Fucoidan, a new low molecular weight sulfated polysaccharide (LMWF), has previously been shown to mobilize bone marrow-derived progenitors cells via stimulation of stromal derived factor (SDF)-1 release. Mobilized progenitor cells have been suggested to repair intimal lesions after immune-mediated endothelial injury and thus prevent intimal proliferation. The aim of this study was to evaluate the effect of LMWF treatment in a rat aortic allograft model of transplant arteriosclerosis (TA). METHODS: Aortic grafts were performed in Brown Norway (BN, donor) and Lewis (Lew, recipient) rats. The recipient rats were treated with LMWF (5 mg/kg/day) and sacrificed at 30 days. To determine the role of SDF-1 in mediating the effects of LMWF, a specific inhibitor of the SDF-1 receptor CXCR4, AMD 3100 (20 microg/kg/day), was used. The grafted segments were evaluated by morphometric (histochemical) analyses. RESULTS: Untreated aortic allografts exhibited severe intimal proliferation, indicative of TA. In contrast, LMWF treatment significantly prevented allograft intimal proliferation as compared with controls (5.7+/-3 vs. 66.2+/-6 microm, P<0.01) and permitted a normalization of the intima/media ratio (0.1+/-0.1 vs. 1.7+/-0.3, P<0.01). Further, LMWF treatment stimulated allograft reendothelialization, as evidenced by strong intimal endothelial nitric oxide synthase antibody and CD31 signals. Unexpectedly, AMD treatment failed to prevent the protective effect of LMWF on intimal thickening and AMD treatment alone was found to reduced intimal proliferation in allografts. CONCLUSIONS: We found that LMWF treatment reduced intimal thickness and induced the presence of an endothelial cell lining in the vascular graft at 30 days. Our findings may suggest a novel therapeutic strategy in the prevention of TA.     

Perivascular delivery of losartan with surgical fibrin glue prevents neointimal hyperplasia after arterial injury

OBJECTIVE: Long-term success of revascularization procedures is limited by recurrent stenosis, a reduction in vascular lumen area that results from neointimal hyperplasia. Inhibitors of the renin-angiotensin system, such as losartan, have potential to prevent recurrent stenosis; however, to date, efficacy has not been demonstrated in either animal models or human beings. While we have previously reported that treatment with a satisfactory dose may be an important element in obtaining efficacy, oral delivery cannot achieve the required concentration. We therefore tested the ability of losartan to restrict neointimal hyperplasia after local delivery of an elevated dose in a fibrin glue. METHODS: The porcine saphenous artery was subjected to balloon angioplasty. Losartan (25 micromol/mL) was applied directly to the adventitial surface of the injured vessel after mixing with 1.0 mL of Tisseel. Neointimal formation was quantified after 14 days with morphometry, and immunologic staining was used to monitor expression of proteins associated with cell proliferation, migration, and phenotypic modulation. RESULTS: A statistically significant decrease of 82% (n = 5) in neointimal area was obtained with losartan, and cell proliferation, as defined by proliferating cell nuclear antigen (PCNA) expression, was inhibited by 97%. Reduced cyclin A expression in losartan-treated vessels confirmed that cell cycle progression was blocked; however, the presence of cytokeratin 8 and tenascin in the media and neointima of injured vessels, regardless of treatment, suggested that losartan does not restrict phenotypic modulation. Inhibition of MT1-MMP (MMP-14) expression by losartan indicated that this inhibitor likely suppresses cell migration as well. CONCLUSIONS: These data demonstrate that losartan can effectively prevent recurrent stenosis when delivered locally with a fibrin glue such as Tisseel. Our results also indicate that losartan may operate by interfering with the expression of proteins required for cell cycle progression and migration. CLINICAL RELEVANCE: Release of angiotensin II in response to vascular injury may promote neointimal hyperplasia, because this hormone can stimulate smooth muscle cell proliferation and migration. This study demonstrates that local application of an angiotensin receptor antagonist, losartan, to the site of injury can effectively prevent neointimal hyperplasia after balloon angioplasty. Application of losartan to the perivascular surface of the injured vessel in a surgical fibrin glue enabled delivery of a dose that exceeds the maximum attainable, via a systemic delivery route. The glue also served as a depot from which the drug was slowly released over time. Treatment with losartan may be a viable approach for controlling neointimal hyperplasia at locations (eg, grafts) that are accessible during a surgical procedure.     

Haemodynamics of the interposition vein cuff

It has been suggested that the use of an interposition vein cuff for anastomosing PTFE grafts to small calibre arteries confers a haemodynamic advantage. Pulsatile blood flow through a standard anastomosis was compared, in vitro, to that incorporating a vein cuff. 6 mm PTFE was anastomosed end-to-side to 10 pairs of cadaver internal mammary arteries matched for length and internal diameter (median 1.8 mm, range 1.6-2.4). Blood flow was significantly higher through the vein cuff anastomosis than the standard anastomosis (comparison of regression analysis, P less than 0.001) when the arterial diameter was less than 2.0 mm (five pairs). These results suggest that the vein cuff does confer a significant haemodynamic advantage when PTFE is anastomosed to arteries less than 2.0 mm diameter and this may be because the compliant vein cuff allows more distension of the anastomosis thus reducing anastomotic resistance.     

Compliance in anastomoses with and without vein cuff interposition.

OBJECTIVE: to compare anastomotic compliance in end-to-side anastomoses with and without vein cuff interposition. Materials polytetrafluoroethylene graft to bovine carotid artery without (standard) and with vein interposition (Linton-patch and Miller-cuff). METHODS: zonewise compliance measurement of end-to-side anastomoses in an in-vitro circulation system. The zone most distal to the suture-line served as reference compliance. RESULTS: directly distal to the suture-line the compliance of the Linton-patch (5.6+/-1.6%/100 mmHg) and Miller-cuff anastomosis (5.2+/-1.1%/100 mmHg) more closely approached reference compliance (standard: 5.0+/-1.2, Linton-patch: 4.5+/-1.5, Miller-cuff: 4.9+/-1.0%/100 mmHg) than that of the standard anastomosis (7.9+/-3.0%/100 mmHg). The maximal compliance values of the Linton-patch (9.5+/-2.3%/100 mmHg) and Miller-cuff anastomoses (9.8+/-2.7%/100 mmHg) were significantly higher than that of the standard end-to-side anastomosis (7.9+/-3.0%/100 mmHg). However, maximal compliance was shifted from the zone directly distal to the suture line in the standard end-to-side anastomosis, to the vein cuff interposition in the Linton-patch and Miller-cuff anastomoses. CONCLUSION: the shift in maximal compliance to the wider portion of the anastomosis in the Miller-cuff and Linton-patch anastomoses may obviate reocclusion.     

Peptide inhibition of neointimal hyperplasia in vein grafts.

Angiopeptin, a novel synthetic octapeptide, was evaluated as a new approach toward the inhibition of neointimal hyperplasia in vein grafts. Male New Zealand white rabbits (n = 22) underwent carotid artery interposition bypass grafting with autologous reversed jugular vein. Nine rabbits were in the treatment group, and 13 were in the control group. The treatment group received angiopeptin 20 micrograms/kg/day by subcutaneous injection beginning 1 day before operation and continuing for 3 weeks until they were killed. At death the vein grafts were fixed in situ with 10% buffered formalyn at 80 mm Hg perfusion pressure. Histologic sections through each vein graft were analyzed by computerized morphometric analysis for area of neointimal hyperplasia (mm2). Neointimal hyperplasia in the control animals was 0.080 + 0.017 mm2 (mean + SEM), whereas neointimal hyperplasia in the group treated with angiopeptin was 0.022 + 0.006 mm2 (mean + SEM) (p = 0.02). This is the first time that peptide inhibition of neointimal hyperplasia has been demonstrated in vein grafts and may have significant implications for future use in vascular surgery.     

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.     

Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts

BACKGROUND: Vascular access dysfunction is the most important cause of morbidity and hospitalization in the hemodialysis population in the United States at a cost of $1 billion per annum. Venous neointimal hyperplasia (VNH) characterized by stenosis and subsequent thrombosis accounts for the overwhelming majority of pathology resulting in polytetrafluoroethylene (PTFE) dialysis graft failure. Despite the magnitude of the problem and the enormity of the cost ($1 billion), there are currently no effective therapies for the prevention or treatment of venous neointimal hyperplasia in PTFE dialysis grafts. METHODS: Tissue samples were collected from the graft-vein anastomosis of stenotic PTFE grafts during surgical revision. Specimens were graded using standard light microscopy and immunohistochemistry for the magnitude of neointimal hyperplasia and for the expression of specific cell types, cytokines, and matrix proteins. RESULTS: VNH was characterized by the (1) presence of smooth muscle cells/myofibroblasts, (2) accumulation of extracellular matrix components, (3) angiogenesis within the neointima and adventitia, and (4) presence of an active macrophage cell layer lining the PTFE graft material. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were expressed by smooth muscle cells/myofibroblasts within the venous neointima, by macrophages lining both sides of the PTFE graft, and by vessels within the neointima and adventitia. CONCLUSIONS: Our results suggest that macrophages, specific cytokines (bFGF, PDGF, and VEGF), and angiogenesis within the neointima and adventitia are likely to contribute to the pathogenesis of VNH in PTFE dialysis grafts. Interventions aimed at these specific mediators and processes may be successful in reducing the very significant human and economic costs of vascular access dysfunction.     

Interleukin-10 fails to modulate low shear stress-induced neointimal hyperplasia.

INTRODUCTION: Overexpression of the anti-inflammatory cytokine interleukin-10 (IL-10) blocks atherosclerotic events in vivo, and IL-10 has been recently hailed as an "immunologic scalpel" for atherosclerosis. Alternatively, mice lacking IL-10 receiving atherogenic diets have increased occlusive lesions. It remains unclear whether such IL-10 modulation broadly applies to other forms of occlusive arterial remodeling. We hypothesized that lack of IL-10 would exacerbate, and exogenous or overexpression of IL-10 would abrogate low shear stress-induced neointimal hyperplasia (NIH). METHODS: Wild-type (WT) and IL-10-deficient (IL-10-/-) mice underwent unilateral common carotid artery (CCA) ligation. Low shear stress in the patent ligated artery results in remodeling and formation of neointima containing BrdU and SMC alpha-actin-positive cells. Additional groups of WT mice underwent CCA ligation and were treated daily with intraperitoneal saline or 1 microg of human IL-10. Chronic delivery gene therapy approaches were also utilized to define the role of IL-10 signaling. WT mice were treated adventitially with 1 x 10(10) adenovirus/green fluorescent protein (Ad/gfp) and an Ad/empty control to confirm the veracity of adventitial delivery. Then, Ad viral IL-10 (vIL-10), Ad/empty, and virus buffer alone were applied directly to the adventitia of the CAA immediately following ligation. In separate experiments, 1 x 10(10) Ad/empty or Ad/vIL-10 was injected intramuscularly. CCAs were perfusion fixed 28 days postligation, the time at which NIH is near maximum. RESULTS: IL-10-/- mice developed identical NIH areas compared to WT controls. Mice receiving IL-10 demonstrated NIH equivalent to saline controls. Mice receiving intramuscular or adventitial Ad/IL-10 developed high serum levels of IL-10 yet formed NIH areas similar to those of controls. Serum IL-10 levels were significantly higher (P = 0.04) with adventitial delivery. Mice treated adventitially with Ad/gfp showed reliable transfection of cells within the adventitia of CAA. No antibody to human IL-10 was found in the sera of intraperitoneal IL-10-treated mice, which failed to attenuate NIH. CONCLUSION: Under the conditions of this experiment, lack of IL-10 does not exacerbate low shear stress-induced NIH, nor does exogenous administration or overexpression of IL-10 attenuate it. Despite high serum levels of vIL-10 in mice treated with ad/vIL-10 adventitially, there appears to be no therapeutic effect despite the confirmed transfection of adventitial cells. Discrepancies between these findings and previous research may be related to IL-10 dosing, inflammation induced by the adenoviral vector, or disparities between the NIH models.     

Does compliance mismatch alone cause neointimal hyperplasia?

To define the relationship between compliance mismatch and the development of neointimal hyperplasia, one 3 cm segment of common iliac artery was externally banded in seven dogs, thereby fixing the arterial diameter at end diastole. To quantify compliance, end-diastole diameter and its change with pulse pressure were measured by induction angiometry. This technique uses intravascular soft trifilar wire probes introduced through distally placed polytetrafluoroethylene sidearms. Compliance was checked in the banded and contralateral undissected unbanded control iliac arteries at 3 and 6 months, at which times the vessels were fixed by perfusion, excised, and examined histologically. Sustained (6-month) compliance mismatch was successfully induced within the banded segments (p less than 0.0001), and no compliance mismatch was seen in the control segments (p = 0.357). The intima of all banded vessels was virtually indistinguishable from that in controls grossly and histologically. Mild focal intimal thickening, less than 3 cell layers thick involving less than 5% of the vessel circumference, was typically seen in both banded and control vessels (range 6.57 +/- 6.80 micron to 38.86 +/- 57.16 micron). In marked contrast, at the sites of the polytetrafluoroethylene-to-femoral artery anastomosis, near-occlusive neointimal hyperplasia (1714 +/- 415.47 micron) was seen in all animals. Residual lumen area in the banded and control vessels was only minimally abnormal (range 98.65% +/- 2.18% to 99.96% +/- 0.08%). These data indicate that compliance mismatch alone is an insufficient stimulus for the development of neointimal hyperplasia in the canine model.     

Cilostazol suppresses neointimal hyperplasia in canine vein grafts

Purpose  To investigate whether cilostazol, a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor, suppresses intimal hyperplasia in canine vein grafts, and to elucidate its mechanisms in terms of cell proliferation and apoptosis. Methods  Bilateral reversed jugular vein interposition grafts of the common carotid artery were performed in 12 beagle dogs. Starting from 7 days before surgery, either cilostazol (30 mg/day; n = 6) or a placebo (n = 6) was given orally twice daily. Vein grafts were harvested at 1 or 4 weeks, and fixed under pressure for histological examination. Results  By 1 week after implantation, the cilostazol group showed significantly less cell proliferation than the placebo group. By 4 weeks after implantation, intimal and medial thickness was significantly thinner in the cilostazol group than in the placebo group. There was significantly more apoptosis in the placebo group than in the cilostazol group at both time points. Conclusion  Cilostazol suppressed the development of intimal hyperplasia in canine autogenous vein grafts. Thus, it may be associated with the modulation of cell proliferation and apoptosis.     

Intraluminal thrombus and neointimal hyperplasia after microvascular surgery

Despite advances in instrumentation, suture materials, and techniques, thrombosis and intimal hyperplasia due to myointimal cell proliferation are still problems in microvascular anastomosis. Platelet factors stimulate smooth-muscle cell proliferation and migration, but little is known about platelet-vessel wall interaction in microvascular surgery. This study evaluates the effect of the disposition of platelet or luminal thrombus, or both, in intimal evolution in autologous venous micrografts interposed in the common carotid artery of 30 rats. Three hours postoperatively, venous graft and suture line endothelium was completely denuded, and the host artery exhibited focal de-endothelialization. Thrombus deposition was observed on suture line and venous graft luminal surfaces, whereas host artery-denuded areas exhibited platelet adhesion. Two weeks postoperatively, intimal thickenings developed in venous graft and suture line, becoming narrower toward the venous graft. The distribution and size of intimal thickening did not change significantly at later observation periods (3-8 months). Intimal thrombus deposition and intimal hyperplasia, appearing in later stages of evolution, show a similar pattern of distribution.     

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.     

Estradiol inhibition of arterial neointimal hyperplasia after balloon injury

Purpose: Estrogens have been shown to protect against cardiovascular disease in postmenopausal women. The mechanisms are unknown. In this study we investigated the effect of estrogen treatment on arterial neointimal proliferation elicited by balloon injury of blood vessels of the rabbit.Methods: The aorta and the common and external iliac arteries of the rabbit underwent balloon injury. 17-ß-Estradiol cypionate (100 μg/kg/day intramuscularly) was administered beginning 1 day before injury and until sacrifice at 22 to 24 days after injury. Angiopeptin (20 μg/kg/day) was administered for the same length of time as estrogen to a group of rabbits to use this peptide as a positive control for morphometric analysis. Angiopeptin was also combined with estradiol to determine whether it was possible to further enhance the effect of estrogen. Morphometric studies were performed to determine measurement of intimal thickening. Inhibition of cell proliferation by estrogen was evaluated by incorporation of tritiated thymidine in vitro into the balloon injured rabbit aorta 72 hours after balloon injury.Results: Treatment of male rabbits with estradiol significantly (p < 0.01) decreased the neointimal thickening of these vessels by 50% to 70%. The somatostatin analog, angiopeptin, was similarly effective in the same circumstances. Estradiol failed to further inhibit neointimal thickening when combined with angiopeptin. Treatment with estradiol for 3 days inhibited both thymidine incorporation (p < 0.01) and DNA content in injured vessels.Conclusion: Estradiol treatment of rabbits undergoing balloon injury of the aorta and iliac arteries, significantly inhibits the myointimal thickening. This effect of estrogen is mediated by inhibition of vascular smooth muscle cell proliferation. (J VASC SURG 1994;19:722-6.)     

Inhibition of neointimal hyperplasia by a specific thrombin inhibitor

Objective—Restenosis secondary to neointimal hyperplasia remains the major limiting factor after vascular interventions. Thrombin generated in high concentrations at the site of vascular injury plays a central role in thrombosis and hemostasis. Thrombin has also been implicated as a mitogen for smooth muscle cell proliferation that contributes to restenosis. This study was designed to determine the effects of a specific thrombin inhibitor on neointimal hyperplasia after balloon injury in a rat carotid artery model.

Design—A total of 47 male Sprague–Dawley rats were divided into five groups. All groups underwent balloon injury of the left carotid artery. A specific thrombin inhibitor, inogatran, was given in four different regimens: low and high dose injections, short‐term infusion for 3?h, and long‐term infusion for 1 week. After 2 weeks the animals were killed and the carotid neointima/media area ratio and the luminal narrowing were calculated.

Results—All treatments significantly reduced the neointimal hyperplasia. Inogatran given as a long‐term infusion for 1 week had the lowest neointima/media ratio compared with the other groups. The percentage of lumen narrowing was also significantly lower in all treatment groups compared with the control group.

Conclusion—A specific direct thrombin inhibitor, inogatran, reduces neointimal hyperplasia after arterial injury in rats. A more prolonged administration of the thrombin inhibitor gave a further reduction of the neointimal hyperplasia. It seems that inhibition of thrombin activity is not only important early after injury, but also later. This could have clinical implications in the treatment of restenosis and needs to be further evaluated.     

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.