Vein Graft Thickening Research Articles

Endothelin-1, endothelin receptor antagonists, and vein graft occlusion in coronary artery bypass surgery: 20 years on and still no journey from bench to bedside.

The saphenous vein is the most commonly used bypass graft in patients with coronary artery disease. During routine coronary artery bypass, grafting the vascular damage inflicted on the vein is likely to stimulate the release of endothelin-1, a potent endothelium-derived vasoconstrictor that also possesses cell proliferation and inflammatory properties, conditions associated with vein graft failure. In both in vitro and in vivo studies, endothelin receptor antagonists reduce neointimal thickening. The mechanisms underlying these observations are multifactorial and include an effect on cell proliferation and cell/tissue damage. Much of the data supporting the beneficial action of endothelin-1 receptor antagonism at reducing intimal thickening and occlusion in experimental vein grafts were published over 20 years ago. The theme of the recent ET-16 conference in Kobe was "Visiting Old and Learning New". This short review article provides an overview of studies showing the potential of endothelin receptor antagonists to offer an adjuvant therapeutic approach for reducing saphenous vein graft failure and poses the question why this important area of research has not been translated from bench to bedside given the potential benefit for coronary artery bypass patients.

The Nogo-B-PirB Axis Controls Macrophage-Mediated Vascular Remodeling

ObjectiveNogo-B mediates vascular protection and facilitates monocyte- and macrophage-dependent vascular remodeling. PirB is an alternate receptor for Nogo-B, but a role for the Nogo-PirB axis within the vascular system has not been previously reported. We examined whether Nogo-B or PirB play a role in regulating macrophage-mediated vascular remodeling and hypothesized that endothelial Nogo-B regulates vein graft macrophage infiltration via its alternate receptor PirB.MethodsVein grafts were performed using Nogo and PirB wild type and knockout mice. Human vein grafts were similarly analyzed. The hindlimb ischemia model was performed in PirB wild type and knockout mice. Accompanying in vitro work included isolation of macrophages from PirB wild type and knockout mice.ResultsIncreased Nogo-B and PirB mRNA transcripts and protein expression were observed within mouse and human vein grafts. Both Nogo knockout and PirB knockout vein grafts showed increased wall thickness and increased numbers of F4/80-positive macrophages. Macrophages derived from PirB knockout mice had increased adhesion to fibronectin, increased EC-specific binding, and increased numbers of mRNA transcripts of M2 markers as well as MMP3 and MMP9. PirB knockout vein grafts had increased active MMP9 compared to wild type vein grafts. PirB knockout mice had increased recovery from hindlimb ischemia and increased macrophage infiltration compared to wild type mice.ConclusionsVein graft adaptation shows increased expression of both Nogo-B and PirB. Loss of PirB, or its endothelial ligand Nogo-B, results in increased inflammatory cell infiltration and vein graft wall thickening. These findings suggest that PirB regulates macrophage activity in vein grafts and that Nogo-B in the vein graft limits macrophage infiltration and vein graft thickening. PirB may play a more general role in regulating macrophage responses to vascular injury. Macrophage inhibition via Nogo-PirB interactions may be an important mechanism regulating vein graft adaptation to the arterial circulation.

Important protective effects of toll like receptor 3 and downstream interferon regulating factors on vein graft remodelling

Purpose: Toll like receptors (TLRs) are key regulators of innate immunity and are highly involved in vein graft remodelling. Activation of most TLRs results in MyD88 and NFκB signalling and induction of inflammatory cytokines. In vascular disease models this leads to pro-inflammatory profiles resulting in augmentation of lesion formation and remodelling. TLR3 however, has protective effects on vascular diseases, although the molecular mechanism is not yet fully clarified. Interestingly, TLR3 is unique among the TLRs since signalling occurs in a MyD88 independent manner; via activation of TRIF and key interferon regulating factors IRF3 and IRF7 resulting in induction of type I interferons. The aim of this study was to investigate the role of TLR3 pathway constituents on vein graft remodelling. Methods: This study was performed in compliance with Dutch government guidelines and principles of laboratory animal care (NIH Publication no. 85-23 revised 1985). Donor caval veins were engrafted in carotid arteries of recipient Tlr3-/-, Irf3-/-, Irf7-/- and control C57BL/6 mice (n=7-10/group). 28 days after surgery vein grafts were harvested and analyzed for vessel wall morphology, cell content and cytokine RNA levels. Results: Tlr3-/- mice developed substantially larger vein grafts than the control mice (155%, p<0.001). Also Irf3-/- and Irf7-/- mice showed increased vein graft thickening (Irf3-/-; 39%, p=0.185, Irf7-/-; 68% p=0.003). The lumen area did not deviate between these strains and their control mice, resulting in outward remodelling in all three strains (Tlr3-/-; 52%, p<0.001, Irf3-/-; 26%, p=0.081, Irf7-/-; 42%, p=0.049). Since macrophages produce large amounts of TLRs, we looked at the influx of these cells in the vein grafts. Tlr3-/-, Irf3-/- and Irf7-/- mice showed significant more influx of macrophages than the control mice. The relative percentage smooth muscle cells and collagen in the vein graft walls did not deviate between the three strains and controls. RNA analysis of vein grafts revealed that pro-inflammatory cytokines TNFα and CCL2 were significantly increased in the Tlr3-/- mice, whereas Irf3-/- and Irf7-/- mice showed no increases in gene expression compared to controls. Conclusions: The TLR3 pathway seems to be protective for vein graft remodelling since Tlr3-/-, as well as the key downstream factors Irf3-/- and Irf7-/- vein grafts show increased vessel wall thickening and outward remodelling. This increased remodelling in the knock out mice is the result of a pro-inflammatory response as reflected by the increase in macrophages and cytokines in the vein graft wall.

Complement factor C5a as mast cell activator mediates vascular remodelling in vein graft disease

Failure of vein graft conduits due to vein graft thickening, accelerated atherosclerosis, and subsequent plaque rupture is applicable to 50% of all vein grafts within 10 years. New potential therapeutic targets to treat vein graft disease may be found in components of the innate immune system, such as mast cells and complement factors, which are known to be involved in atherosclerosis and plaque destabilization. Interestingly, mast cells can be activated by complement factor C5a and, therefore, a direct role for C5a-mediated mast cell activation in vein graft disease is anticipated. We hypothesize that C5a-mediated mast cell activation is involved in the development and destabilization of vein graft lesions. Mast cells accumulated in time in murine vein graft lesions, and C5a and C5a-receptor (CD88) expression was up-regulated during vein graft disease in apolipoprotein E-deficient mice. Mast cell activation with dinitrophenyl resulted in a profound increase in vein graft thickening and in the number of plaque disruptions. C5a application enhanced vein graft lesion formation, while treatment with a C5a-receptor antagonist resulted in decreased vein graft disease. C5a most likely exerts its function via mast cell activation since the mast cell inhibitor cromolyn totally blocked C5a-enhanced vein graft disease. These data provide evidence that complement factor C5a-induced mast cell activation is highly involved in vein graft disease, which identifies new targets to prevent vein graft disease.

Mineralocorticoid receptor antagonism inhibits vein graft remodeling in mice

Vein graft failure rates resulting from adverse graft remodeling remain high with no effective therapy. The mineralocorticoid receptor (MR) plays a role in pathologic arterial remodeling. We demonstrated recently that the MR is upregulated in venous tissues after grafting and hypothesized that MR inhibition would reduce vein graft remodeling. Reverse transcription polymerase chain reaction and immunoblotting were used to examine the expression of the MR and other components of the renin-angiotensin-aldosterone system in human vein and primary human saphenous vein smooth muscle cells (HSVSMC). Adenoviral reporter gene assays were used to explore MR transcriptional activity in HSVSMC. The effect of MR inhibition on vein graft remodeling invivo was characterized in a mouse vein graft model. Messenger RNAs encoding the MR, 11-β-hydroxysteroid dehydrogenase 2, angiotensin type 1 receptor, and the angiotensin-converting enzyme are expressed in whole HSVSMC. MR and 11-β-hydroxysteroid dehydrogenase 2 protein expression is confirmed, and MR-dependent transcriptional regulation is demonstrated at physiologic aldosterone concentrations in HSVSMC. Treatment of mice with the MR antagonist spironolactone, at doses that do not lower blood pressure (20mg/kg per day), reduces maximal vein graft intima-media thickness by 68%, with an associated reduction in graft inflammatory cell infiltration and fibrosis. MR is expressed in human venous tissue and cells and modulates gene expression in HSVSMC in response to physiologic aldosterone concentrations. Invivo, MR inhibition reduces vein graft thickening and inflammation. These preclinical data support the potential to use MR antagonists as novel treatments to preserve vein graft patency.

Oxidative stress and vein graft failure: a focus on NADH oxidase, nitric oxide and eicosanoids

Recent interest has focused on superoxide and the upregulation of NADPH oxidase expression in the aetiology of vein graft failure. Implantation of saphenous vein grafts promotes upregulation of NADPH oxidase through a number of distinct interrelated mechanisms: (a) endothelial denudation, (b) factors released by adherent platelets, monocytes and neutrophils, (c) hypoxia and (d) altered prostacyclin (PGI(2)) and enhanced isoprostane formation. These, in turn, impact on neointima (NI) formation (vascular smooth muscle cell [VSMC] replication and migration) and metalloproteinase (MMP) expression, key events in vein graft thickening. NADPH oxidase in the aetiology of vein graft failure will be discussed in this review with particular reference to nitric oxide and eicosanoids and related drugs that inhibit its activity and expression.

Cytoplasmic Tyrosines 653 and 774 Are Critical for Eph-B4 Signaling to ERK1/2 and Akt

Introduction: Eph-B4, a receptor tyrosine kinase that determines vascular venous fate during vasculogenesis, is downregulated during vein graft adaptation to the arterial circulation. We have previously shown that stimulation of Eph-B4 with Ephrin-B2/Fc prevents vein graft thickening. However, the mechanisms by which Eph-B4 activation prevents vein graft thickening are unknown. Similarly, we have also shown that Eph-B4 phosphorylation is critical to Eph-B4 action; therefore we examined whether several of the 15 phosphorylation sites in the cytoplasmic portion of Eph-B4 are critical to Eph-B4 stimulation of intracellular signal transduction pathways. Methods: A wild-type Eph-B4 plasmid vector was created, followed by three individual point mutations at tyrosine 581, 653, and 774, with substitution of phenylalanine for tyrosine. Mutations were confirmed by sequencing. Cos-7 cells were transfected with wild-type or mutant Eph-B4 (Y581F-, Y653F-, and Y774F-Eph-B4). Transfected cells were starved for 24 hours and then stimulated with Ephrin-B2/Fc (1 min). Cell lysates were examined via Western blotting for differences in tyrosine-phosphorylation, and phospho-Akt, phospho-ERK1/2, and phospho-FAK activity between wild-type and mutant Eph-B4. Double-labeled immunofluorescence of wild-type and mutant Eph-B4 transfected Cos-7 cells was utilized to examine Eph-B4 tyrosine phosphorylation and colocolization of Eph-B4 with caveolin-1. Results: Stimulation of wild-type Eph-B4 in transfected Cos-7 cells results in phosphorylation of Eph-B4, Akt, and ERK1/2, but not FAK. Tyrosine phosphorylation of Eph-B4 is decreased with the loss of Y774, but not with the loss of Y653 and Y581. Loss of Y653 and Y774 resulted in decreased phospho-AKT activity compared to wild-type Eph-B4 and a loss of Y774 resulted in decreased phospho-ERK1/2. phospho-FAK activity was equivalent between wild-type and all mutant Eph-B4 transfected cells. Loss of Y581 did not influence phosphorylation of Akt, ERK1/2, or FAK. Double-labeled immunofluorescence confirmed the above findings. Wild-type Eph-B4, but not mutant Eph-B4, colocalized with caveolin-1 after Ephrin-B2/Fc stimulation. Conclusions: Eph-B4 downstream cell signaling requires tyrosine residues 653 and 774. Their loss results in decreased phospho-ERK1/2 and phospho-Akt, but not phospho-FAK, activity, and also prevents colocalization of activated Eph-B4 with caveolin-1, showing that tyrosine phosphorylation of these residues is critical to Eph-B4 function. These results suggest novel molecular targets to modulate Eph-B4 activity.

Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA

BackgroundProliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in neointimal formation which leads to restenosis of vein graft in venous bypass. STAT-3 is a transcription factor associated with cell proliferation. We hypothesized that silencing of STAT-3 by siRNA will inhibit proliferation of VSMCs and attenuate intimal thickening.MethodsRat VSMCs were isolated and cultured in vitro by applying tissue piece inoculation methods. VSMCs were transfected with STAT 3 siRNA using lipofectamine 2000. In vitro proliferation of VSMC was quantified by the MTT assay, while in vivo assessment was performed in a venous transplantation model. In vivo delivery of STAT-3 siRNA plasmid or scramble plasmid was performed by admixing with liposomes 2000 and transfected into the vein graft by bioprotein gel applied onto the adventitia. Rat jugular vein-carotid artery bypass was performed. On day 3 and7 after grafting, the vein grafts were extracted, and analyzed morphologically by haematoxylin eosin (H&E), and assessed by immunohistochemistry for expression of Ki-67 and proliferating cell nuclear antigen (PCNA). Western-blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression in vivo and in vitro. Cell apoptosis in vein grafts was detected by TUNEL assay.ResultsMTT assay shows that the proliferation of VSMCs in the STAT-3 siRNA treated group was inhibited. On day 7 after operation, a reduced number of Ki-67 and PCNA positive cells were observed in the neointima of the vein graft in the STAT-3 siRNA treated group as compared to the scramble control. The PCNA index in the control group (31.3 ± 4.7) was higher than that in the STAT-3 siRNA treated group (23.3 ± 2.8) (P < 0.05) on 7d. The neointima in the experimental group(0.45 ± 0.04 μm) was thinner than that in the control group(0.86 ± 0.05 μm) (P < 0.05).Compared with the control group, the protein and mRNA levels in the experimental group in vivo and in vitro decreased significantly. Down regulation of STAT-3 with siRNA resulted in a reduced expression of Bcl-2 and cyclin D1. However, apoptotic cells were not obviously found in all grafts on day 3 and 7 post surgery.ConclusionsThe STAT-3 siRNA can inhibit the proliferation of VSMCs in vivo and in vitro and attenuate neointimal formation.

Hemodynamically “insignificant” stenoses stimulate neointimal thickening in experimental vein grafts

Vein graft stenoses <50% are believed to be clinically “insignificant” because they cause little decrement in blood flow. However, the possible deleterious effects of minor flow disturbances on long-term cellular proliferation are unknown. The purpose of this study was to evaluate the effect of artificially created 50% stenoses on neointimal thickening in experimental vein grafts. Fifteen male New Zealand White rabbits underwent carotid interposition bypass grafting using the external jugular vein. Mid-graft 50% stenoses were created in eight grafts by the application of a two-millimeter internal diameter stainless steel clip. After four weeks, the grafts were perfusion-fixed, excised, and histologic sections were examined for neointimal thickening. Application of the clip caused a 50% reduction in external diameter (3.9±0.4 mm to 2.0 mm), causing only slight perturbations in pressure gradient (ΔP=2.1±1.1 to 2.3±1.5 mmHg) and mean blood flow (flow 16±3.0 to 14±4.4 ml/min). After four weeks, four grafts had occluded (two from each group) and one clip had become dislodged, leaving ten grafts for analysis. Neointimal thickness was minimal in control grafts (88±12 µm), but was significantly increased in stenotic grafts both immediately proximal (200±39 µm; p=0.03) and immediately distal to the stenosis (230±50 µm; p=0.02). Hemodynamically “insignificant” stenoses stimulate vein graft neointimal thickening. These results support the continued use of graft surveillance and an aggressive approach to the treatment of “minimal” vein graft lesions.

Eph-B4 prevents venous adaptive remodeling in the adult arterial environment.

Eph-B4 determines mammalian venous differentiation in the embryo but is thought to be a quiescent marker of adult veins. We have previously shown that surgical transposition of a vein into the arterial environment is characterized by loss of venous identity, as indicated by the loss of Eph-B4, and intimal thickening. We used a mouse model of vein graft implantation to test the hypothesis that Eph-B4 is a critical determinant of venous wall thickness during postsurgical adaptation to the arterial environment. We show that stimulation of Eph-B4 signaling, either via ligand stimulation or expression of a constitutively active Eph-B4, inhibits venous wall thickening and preserves venous identity; conversely, reduction of Eph-B4 signaling is associated with increased venous wall thickness. Stimulated Eph-B4 associates with caveolin-1 (Cav-1); loss of Cav-1 or Eph-B4 kinase function abolishes inhibition of vein graft thickening. These results show that Eph-B4 is active in adult veins and regulates venous remodeling. Eph-B4-Cav-1-mediated vessel remodeling may be a venous-specific adaptive mechanism. Controlled stimulation of embryonic signaling pathways such as Eph-B4 may be a novel strategy to manipulate venous wall remodeling in adults.

Short-term effects of double-layer autologous vein graft on restraint of excessive distension and alleviation of neointimal hyperplasia in a porcine saphenous vein graft model

Although the use of external vein graft support seems a promising approach to prevent neointimal hyperplasia and wall thickening in vein grafts, its extensive clinical application still has a long way to go. The aim of this study was to evaluate short-term effects of self-designed double-layer autologous saphenous vein graft on restraining excessive distension of vein graft and alleviating neointimal hyperplasia in a porcine model. Left and right hind femoral arteries of 24 white pigs were randomly divided into an experimental group (double-layer vein graft) and a control group (single-layer vein graft). After 1 h of implantation, then 1, 2, and 4 weeks later, the mean inner diameter of the vein grafts in the experimental group measured by Doppler-ultrasound was 2.7 ± 0.1, 2.8 ± 0.1, 2.9 ± 0.1, and 3.1 ± 0.1 mm, respectively; mean peak blood flow velocity measured by Doppler-ultrasound was 96.7 ± 12.8, 93.7 ± 11.5, 89.4 ± 9.6 and 84.6 ± 10.1 cm/s, respectively, while the mean neointimal thicknesses were 47.1 ± 7.7, 93.7 ± 15.1, and 177.4 ± 25.5 μm at 1, 2 and 4 weeks, respectively. As compared to the control group, inner diameter and neointimal thickness of vein grafts in the experimental group were significantly lower, while mean peak blood flow velocity was significantly higher at 1, 2, and 4 weeks after implantation. The proliferation index in the experimental group was also significantly lower within 4 weeks after implantation. The self-designed double-layer autologous saphenous vein graft restrains early excessive distension of vein graft and alleviates early neointimal hyperplasia.

Annexin A5 Therapy Attenuates Vascular Inflammation and Remodeling and Improves Endothelial Function in Mice

Annexin A5 (AnxA5) has antithrombotic, antiapoptotic, and antiinflammatory properties; we investigated its effectiveness against vascular inflammation, remodeling, and dysfunction in accelerated atherosclerosis. AnxA5 (1 mg/kg per day or vehicle) was investigated in vascular injury models in hypercholesterolemic apolipoprotein E (ApoE)3*Leiden mice. AnxA5 treatment reduced adhesion and infiltration of leukocytes by 71% to 69% (P=0.015, P=0.031) and macrophages by 51% to 87% (P=0.014, P=0.018), as well as monocyte chemotactic protein-1 and tumor necrosis factor-α expression in a femoral artery inflammation model (perivascular cuff for 3 days), indicating reduced vascular inflammation. In a vein graft model, 28 days of AnxA5 treatment reduced vein graft thickening (48%; P=0.006) and leukocyte infiltration (46%; P=0.003). In these mice, reduced plasma concentrations of IFN-γ (-72%; P=0.040), granulocyte colony-stimulating factor (-41%; P=0.010), and macrophage inflammatory protein-1β (MIP-1β) (-66%; P=0.020) were measured, indicating reduced systemic inflammation. An in vitro endothelial cell model shows the importance of AnxA5's anticoagulant properties in reducing vascular inflammation. Endothelium-mediated dilatation in hypercholesterolemic ApoE((-/-)) mice was improved by 3 days of AnxA5 treatment, shown by improved systolic and diastolic blood pressure reductions in response to metacholine, which could be abolished by l-Nitro-Arginine-Methyl Ester (l-NAME), indicating nitric oxide involvement. AnxA5 reduced local vascular and systemic inflammation and vascular remodeling and improved vascular function, indicating that it has a therapeutic potential against atherosclerotic cardiovascular diseases.

Characterization of the Inhibition of Vein Graft Intimal Hyperplasia by a Biodegradable Vascular Stent

Characterization of biodegradable stent vein graft thickening. Polydioxanone vascular sutures (PDSs) were used in a biodegradable arteriovenous bypass model. Twenty-four rabbits underwent carotid interposition bypass via ipsilateral jugular vein. One half received the stent (PDS group) and the remaining half a simple vein graft (controls). Group subsets received external stent removal or sham-control exploration at 4 and 12 weeks. At 4 and 12 weeks, the PDS group had significantly less medial and intimal thickening than the control group (P < 0.05), and there were fewer proliferating smooth muscle cells and extra cellular matrix formation than the control group at every interval. At 12 weeks, partial stent degradation occurred without deleterious effects. Furthermore proliferating cell nuclear antigen (PCNA), angiotensin type 1 receptor (AT1R), and transforming growth factor beta 1 (TGF-β1) levels were significantly lower than the control group. The external stent inhibited medial and intimal hyperplasia, an effect that remains after the material has completely degraded. This PDS stent is feasible option for vein grafts.

A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease

Matrix metalloproteinases (MMP) and plasminogen activator (PA)/plasmin-mediated proteolysis, especially at the cell surface, play important roles in matrix degeneration and smooth muscle cell migration, which largely contributes to vein graft failure. In this study, a novel hybrid protein was designed to inhibit both protease systems simultaneously. MMP and plasmin activity were inhibited at the cell surface by this hybrid protein, consisting of the receptor-binding amino-terminal fragment (ATF) of urokinase-type PA, linked to both the tissue inhibitor of metalloproteinases (TIMP-1) and bovine pancreas trypsin inhibitor (BPTI), a potent protease inhibitor. The effect of overexpression of this protein on vein graft disease was studied. A non-viral expression vector encoding the hybrid protein TIMP-1.ATF.BPTI was constructed and validated. Next, cultured segments of human veins were transfected with this vector. Expressing TIMP-1.ATF.BPTI in vein segments resulted in a mean 36 ± 14% reduction in neointima formation after 4 weeks. In vivo inhibition of vein graft disease by TIMP-1.ATF.BPTI is demonstrated in venous interpositions placed into carotid arteries of hypercholesterolaemic APOE*3Leiden mice. After 4 weeks, vein graft thickening was significantly inhibited in mice treated with the domains TIMP-1, ATF, or BPTI (36-49% reduction). In the TIMP-1.ATF.BPTI-treated mice, vein graft thickening was reduced by 67±4%, which was also significantly stronger when compared with the individual components. These data provide evidence that cell surface-bound inhibition of the PA and MMP system by the hybrid protein TIMP-1.ATF.BPTI, overexpressed in distant tissues after electroporation-mediated non-viral gene transfer, is a powerful approach to prevent vein graft disease.

Periadventitial Rapamycin-Eluting Microbeads Promote Vein Graft Disease in Long-Term Pig Vein-Into-Artery Interposition Grafts

Neointima formation and atherosclerosis compromise long-term graft patency in aortocoronary and peripheral vein bypass grafts. We investigated the short- and long-term effects of periadventitial application of a sustained-release formulation of rapamycin on experimental pig vein grafts with similar dimensions and kinetics to human saphenous vein bypass grafts. Periadventitial application of rapamycin-eluting polyvinyl alcohol microspheres (60 microg . cm(-2)) to porcine saphenous vein-to-carotid artery interposition grafts inhibited vein graft positive and vascular smooth muscle cell proliferation in 1-week grafts. It also decreased neointima formation and wall thickening in 4-week vein grafts compared with controls. The inhibition of vein graft thickening was not sustained; however, a catch-up phenomenon was observed, and there was no therapeutic benefit evident in 12-week grafts. Increasing the dose of rapamycin to 120 microg . cm(-2) was associated with significant local toxicity manifest by high rates of graft rupture (25%), inhibition of adventitial neoangiogenesis, and a paradoxical acceleration of vein graft disease as evidenced by increased vascular smooth muscle cell proliferation. Local toxicity and poor long-term efficacy limits the clinical applicability of locally applied, sustained rapamycin release in vein graft disease.

Folic acid administration reduces neointimal thickening, augments neo-vasa vasorum formation and reduces oxidative stress in saphenous vein grafts from pigs used as a model of diabetes.

There is evidence that plasma homocysteine augments vein graft failure and that it augments both micro- and macro-angiopathy in patients with diabetes mellitus. It is therefore suggested that homocysteine may augment vein graft thickening, a major cause of vein graft failure, in diabetic patients, as well as impairing adaptive growth of a new vasa vasorum, possibly through overproduction of superoxide. In order to test these proposals, the effect of folic acid administration, which lowers plasma homocysteine, on vein graft thickening and microvessel density was studied in pigs used as a model of diabetes. Non-ketotic hyperglycaemia was induced in Landrace pigs by intravenous injection of streptozotocin, and folic acid was fed daily for 1 month. Vein grafts were excised and the thickness of the neointima and media and microvessel density were assessed by planimetry and superoxide formation. Plasma total homocysteine was significantly reduced by folic acid in both control and diabetic pigs, whereas glucose was unchanged. Compared with controls, diabetic pigs showed increased neointimal thickness and superoxide formation and decreased adventitial microvessel density. Folic acid reduced neointimal thickness and superoxide formation and augmented microvessel density in diabetic but not in control pigs. Folic acid administration reduces neointimal thickening, augments vasa vasorum neoformation and reduces oxidative stress in saphenous vein grafts from diabetic pigs. Folic acid may therefore be particularly effective in reducing vein graft failure in diabetic patients.

In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor

Smooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression. Plasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed. Electroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P < .025). Although all constructs reduced vein graft thickening compared with the controls, the luminal area was best preserved in the TIMP-1.ATF-treated mice. Intramuscular electroporation of TIMP-1.ATF inhibits vein graft thickening in vein grafts in carotid arteries of hypercholesterolemic mice. Binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface enhances the inhibitory effect of TIMP-1 on vein graft remodeling in vitro as well as in vivo and may be an effective strategy to prevent vein graft disease.

Local lentiviral short hairpin RNA silencing of CCR2 inhibits vein graft thickening in hypercholesterolemic apolipoprotein E3-Leiden mice

Inflammatory responses to vascular injury are key events in vein graft disease and accelerated atherosclerosis, which may result in bypass failure. The monocyte chemoattractant protein-1 (MCP-1)/CC-chemokine receptor (CCR)-2 pathway is hypothesized to play a central role. A murine model for vein graft disease was used to study the effect of local application of lentiviral short hairpin RNA (shRNA) targeted against CCR2. A venous interposition was placed into the carotid artery of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3-Leiden) mice to induce vein graft thickening with features of accelerated atherosclerosis. To demonstrate the efficacy of the lentiviral shRNA targeting murine CCR2 (shCCR2) in blocking vein graft disease in vivo, lentiviral shCCR2 or a control lentivirus was used to infect the vein graft locally (n = 8). Vascular CCR2 and MCP-1 messenger RNA expression levels were significantly upregulated during lesion progression in the vein graft. Infection of smooth muscle cells (SMCs) with a lentiviral shRNA targeting shCCR2 completely abolished MCP-1-induced SMC migration and inhibited SMC proliferation in vitro (n = 3 per group). Morphometric analysis of sections of grafts showed a significant 38% reduction in vein graft thickening in the shCCR2-treated mice 4 weeks after surgery (control, 0.42 +/- 0.05 mm(2); shCCR2, 0.26 +/- 0.03 mm(2); P = .007). Vascular CCR2 contributes to vein graft disease, and local application of shRNA against CCR2 to the vessel wall prevents vein graft thickening in hypercholesterolemic mice, suggesting that local overexpressing of shRNA using organ-targeted lentiviral gene delivery may be a promising therapeutic tool to improve vein graft disease in bypassed patients. Vein graft disease is an important clinical issue that results from an inflammatory response. The monocyte chemoattractant protein (MCP)-1/CC-chemokine receptor (CCR)-2 pathway plays a key role in the initiation and development of vein graft disease. This study demonstrates that perivascular overexpression of short hairpin RNA, targeted against CCR2, inhibits vein graft thickening. These data show that organ-targeted gene therapy against CCR2 in the vessel wall could be a promising therapeutic tool to improve vein graft patency in bypassed patients.

Slower Onset of Low Shear Stress Leads to Less Neointimal Thickening in Experimental Vein Grafts

Slower Onset of Low Shear Stress Leads to Less Neointimal Thickening in Experimental Vein Grafts

A randomized trial of an external Dacron sheath for the prevention of vein graft disease: The Extent study

A randomized trial of an external Dacron sheath for the prevention of vein graft disease: The Extent study