Experimental Vein Grafts Research Articles

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1β (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

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.

Abstract 329: Systems-based Target Discovery Reveals PPARa as a Key Metabolic Regulator for Macrophage Activation in Vein Graft Disease

Objectives: Vein graft (VG) failure rates after surgical bypass for PAD remain high, which can cause limb loss. Yet, the underlying mechanisms for VG lesion development remain obscure. We took a systems approach to explore key pathways in a mouse model. Approach and Results: We implanted the inferior vena cava into the left carotid artery of fat-fed Ldlr-/- mice and monitored lesion development up to 4 weeks post-surgery. Unbiased proteomic profiling followed by hierarchical clustering showed distinct clusters. One protein cluster is decreased in vein grafts with overt lesions (poor outcome) relative to the group of non-diseased veins devoid of lesions and vein grafts with minimal lesion (favorable outcome). Conversely, this can be viewed as increased in the “favorable” outcome group. Using the “favorable outcome” predominant protein cluster, network analysis and pathway enrichment identified PPARα pathway as a top-ranked key hub node with high betweeness centrality measure. This implies that PPARα may play a key regulatory role in limiting lesion severity. We examined whether network-based prediction of PPARα can also serve as a therapeutic target to attenuate the development of experimental VG lesions. A blinded in vivo loss-of-function study using PPARα siRNA encapsulated in macrophage-targeted lipid nanoparticles (LNP) demonstrated higher plaque volume as seen by 3D ultrasound and histologic morphometry in the test group (n=7) versus the control group (n=8). In contrast, a randomized drug study on another set of VG mice using the new PPARα-selective agonist pemafibrate demonstrated less plaque lesions and macrophage accumulation in the drug group (n=11) compared to the control group (n=11). In vitro validation studies (alongside loss-of-function) including high-throughput qPCR, single cell qPCR, CyTOF, ELISA, FACS, and metabolic assays all suggested that pemafibrate-induced PPARα activation promotes a phenotype shift of activated macrophages into a less inflammatory state through metabolic reprogramming. Conclusion: Systems-based target discovery on VG demonstrates that PPARα agonism reduces lesion development and inflammatory burden. Targeting this pathway might furnish a novel therapeutic option to prevent VG lesion development.

Inflammation and myointimal hyperplasia. Correlation with hemodynamic forces

ObjectivesThe aim of our study was to correlate flow dynamics and the release of inflammatory cytokines Interleukin 1, 2, 6, TNF (Tumour Necrosis Factor) alfa, both in vitro and in vivo. Materials and methodsEndothelial cells were exposed to laminar flow (6dyne/cm2) in an in vitro circulatory system and the release of Interleukin 1, 2, 6 and TNF alfa was quantified by ELISA. Interleukin 1, 2, 6 and TNF alfa release was also assessed in vein grafts implanted in the arterial circulation of Lewis rats. Arterial vein grafts were explanted at different time intervals from 3days to 12weeks after surgery. Vein grafts implanted in the arterial circulation for 4weeks, were re implanted in the venous circulation of syngenic Lewis rats, and the release of Interleukin 1, 2, 6 and TNF alfa, was assessed in an organ culture.Six vein grafts (4 occluded, 2 patent) implanted in humans as femorodistal bypass were examined for the presence of myointimal hyperplasia and perigraft inflammatory cells. ResultsIn vitro, endothelial cells exposed to laminar flow released an increased amount of Interleukin 1, 2, 6 and TNF alfa in comparison to endothelial cells not exposed to flow.In experimental vein grafts implanted in the arterial circulation there was an increased release of inflammatory cytokines associated to inflammatory changes in the adventitia. Once the vein grafts were re implanted in the venous circulation, the release of these cytokines diminished, while the inflammatory changes in the adventitia regressed. ConclusionsIncreased shear stress induces release of cytokines and inflammatory changes in the adventitia. These inflammatory changes can contribute to plaque progression and to un stable plaque. These findings support the use of anti-inflammatory therapy in patients prone to develop atherosclerosis and in those who had arterial reconstructive surgery.

Inhibition of intimal hyperplasia of the vein graft with degradable poly lactic-co-glycolic acid vascular external sheaths carrying slow-release bosentan.

Intimal hyperplasia is a major reason for the restenosis of vein grafts. A vascular external sheath was designed to inhibit intimal hyperplasia of vein grafts. Using the biodegradable material poly(lactic-co-glycolic acid) and fibrin, a high porosity external sheath carrying bosentan with a stratified internal spiral structure was made by grouping mould extraction and low-temperature deposition techniques. Bosentan, a dual endothelin A and B receptor antagonist, was carried in the new designed external sheath, and could be slowly released. Rabbits underwent carotid bypass with ipsilateral jugular vein without external sheath (control, Group C), with external sheath (Group S) or with external sheath carrying bosentan (Group S + B). Eight weeks after interposition, ultrasonography indicated that the change rate of the diameter of vein grafts of Group C (42.3 ± 5.8%) was much higher than that of Group S (18.5 ± 8.6%) or S + B (1.1 ± 13.2%) (P < 0.01, respectively). The change rate of peak velocity of Group S + B (-0.5 ± 9.8%) was lower than that of Group S (5.7 ± 7.4%) or C (25.6 ± 13.1%) (P < 0.01 vs Group C; P < 0.05 vs Group S). The sheaths degraded completely 9 weeks after interposition and the histological examination showed that the ratio of long/short diameter of vein grafts was much higher in Group C (P < 0.01, respectively). The ratio of the area of intima-media to the area of lumen and the ratio of thickness of intima to the thickness of media of Group S + B were significantly lower (P < 0.01 vs Group C; P < 0.05 vs Group S, respectively). Masson and Alcian Blue staining indicated that the remodelling of collagen and the distribution of proteoglycan in vein grafts of Group S + B were more similar to those of the artery. The degradable vascular external sheath with slow-release bosentan can inhibit hyperplasia of intima, and improve the shape of experimental vein grafts.

Abstract 146: Expression of Axl, a Receptor Tyrosine Kinase, Regulates Vein Graft Remodeling

Introduction: We recently found that expression of Axl, a receptor tyrosine kinase, in non-bone marrow derived cells is important for carotid thickening in response to low flood flow. We hypothesize that Axl plays a role in smooth muscle cell functions during vein graft remodeling. Methods and Results: A segment of inferior vena cava was harvested from male Axl wild type (Axl+/+) or knockout (Axl-/-) mice and anastomosed to a carotid artery in female Axl+/+ mice under isoflurane anesthesia. Ultrasound Vevo2100 system was used to evaluate successful engraftment of the veins. Initial assessments of the blood flow through the graft were done at 1week after the surgery. A 3-dimensional high-resolution imaging was performed in mice after 3weeks after surgery. We found similar mean blood flow velocity ~110mm/s in the vein grafts between Axl+/+ &gt;Axl+/+ and Axl-/- &gt;Axl+/+ mice. There were no differences in the length of the graft between the groups in reconstruction of 3D-Mode imaging. However, graft volumes had a tendency (p~0.1) to be lower in Axl-/- &gt;Axl+/+ vs. Axl+/+ &gt;Axl+/+ mice (5.3±0.2 vs. 5.8±0.4mm 3 ). We also observed ~20% reduction of graft wall thickness in Axl-/- &gt;Axl+/+ mice. Finally, pulsatility and resistive indexes of vein grafts were ~10% lower in Axl-/- &gt;Axl+/+ mice. Conclusions: This is the first study to demonstrate contribution of Axl to vein graft remodeling. Although, to confirm ultrasound differences between experimental groups vein graft wall changes are being evaluated by histology.

Tetrahydrobiopterin Determines Vascular Remodeling Through Enhanced Endothelial Cell Survival and Regeneration

Endothelial cell (EC) survival and regeneration are important determinants of the response to vascular injury that leads to neointimal hyperplasia and accelerated atherosclerosis. Nitric oxide (NO) is a key regulator of EC and endothelial progenitor cell function, but the pathophysiological mechanisms that regulate endothelial NO synthase in endothelial regeneration remain unclear. Endothelium-targeted overexpression of GTP cyclohydrolase (GCH) I increased levels of the endothelial NO synthase cofactor, tetrahydrobiopterin, in an EC-specific manner and reduced neointimal hyperplasia in experimental vein grafts in GCH/apolipoprotein E-knockout mice. These effects were mediated through enhanced donor-derived survival and recipient-derived repopulation of GCH transgenic ECs, revealed by tracking studies in Tie2-LacZ/GCH-Tg/apolipoprotein E-knockout recipient mice or donor grafts, respectively. Endothelial GCH overexpression increased endothelial NO synthase coupling and enhanced the proliferative capacity of ECs and circulating endothelial progenitor cell numbers after vascular injury. These observations indicate that endothelial tetrahydrobiopterin availability modulates neointimal hyperplasia after vascular injury via accelerated EC repopulation and growth. Targeting tetrahydrobiopterin-dependent endothelial NO synthase regulation in the endothelium is a rational therapeutic target to enhance endothelial regeneration and reduce neointimal hyperplasia in vascular injury states.

Modification of experimental vein grafts by homologous transplantation of skeletal myoblasts

Modification of experimental vein grafts by homologous transplantation of skeletal myoblasts

Retraction Note: Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts

Background Nanoparticles (NPs) possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded NPs can reduce neointima formation of vein graft disease in a rat model. Methods Poly(lactic-co-glycolic acid) (PLGA) NPs-containing rapamycin was prepared using an oil/water solvent evaporation technique. The size and morphology of the NP were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA NPs was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Excised rat jugular vein was treated ex vivo with blank NPs, or rapamycin-loaded NPs, and then interposed back into the carotid artery position using a cuff technique. Grafts were harvested for 21 days and subjected to morphometric analysis as well as immunohistochemical analysis and Western blotting. Results Rapamycin was efficiently loaded in PLGA NPs with an encapsulation efficiency of 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/mL significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts showed that the concentration of rapamycin in vein grafts at 3 weeks after grafting was 0.9 ± 0.1 μg/g. In grafted veins without treatment, intima-media thickness was 300.4 ± 181.5 μm at 21 days after grafting, whereas veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). Cell proliferation was measured by proliferating cell nuclear antigen immunohistochemistry staining. As expected, proliferating cell nuclear antigen index declined from 83.4% ± 7.4% to 66.2% ± 4.5% in vein grafts after 3 weeks (p = 0.002). Platelet endothelial cell adhesion molecule (PECAM-1/CD31) staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization at 21 days after grafting, with no significant effect of blank or rapamycin-loaded NPs group. Western blot analysis showed that treatment with rapamycin-loaded PLGA NPs markedly attenuated phosphorylation and activation of S6 kinase 1 phosphorylation and inactivation of 4E (eIF4E)-binding protein 1, both in vascular smooth muscular cells and vein grafts at 7 and 21 days after grafting. Conclusions We conclude that sustained-release rapamycin from rapamycin-loaded NPs inhibits vein graft thickening without affecting the endothelial cells in rat carotid vein-to-artery interposition grafts; thus, this may be a promising therapy for the treatment of vein graft disease.

Inhibitory effects of bortezomib on restenosis of rat experimental vein graft

Objective The anti-inflammatory and vaso-protective effects of bortezomib were analyzed in rat model to test whether it inhibits neointima formation in transplant-induced vasculopathy.Methods Seventy-six rats were subjected to autologous vein grafting surgery.Thereafter,all post-surgical rats were randomly divided into two groups and dosed intravenously with 0.15 mg/kg bortezomib or vehicle on the day 0,3,7,and 10.After 24 and 72 h,rats were humanely killed and vein grafts were processed for real-time reverse transcription-polymerase chain reaction (RT-PCR) to test the gene expression of cytokineinduced neutrophil chemoattractant-2β (CINC-2β),monocyte chemoattractant protein ( MCP)-1,interleukin (IL)-1,IL-6 and tumor necrosis factor (TNF)-α.Meanwhile,24-h vein grafts were processed for enzyme linked immunosorbent assay ( ELISA ) or neutrophil chemotaxis assay.Subsequently,rats were euthanized at 4th week after grafting and samples were processed for morphometric analysis.Results The expression of mRNA for chemokines ( CINC-2β,MCP-1 ) and cytokines ( IL-1,IL-6 and TNF-α) were markedly increased in the injured vessels at the first day after surgery and declined during the following three days,and protein levels of them were markedly increased at the same time [ CINC-2β (415.23 ±56.50) pg/mg; MCP-1 (9894.58 ±2795.62) pg/mg; IL-1 (3087.51 ±609.19) pg/mg; IL-6 (2225.27±206.06) pg/mg; TNF-α (402.31 ± 134.13) pg/mg] (P ＜0.05) accompanied with a marked increase in neutrophil migration toward homogenates of injured vessels as chemotactic index (CI) was (241.52 ±33.43 ) ％ ( P ＜ 0.05 ).Morphometric analysis revealed that the intima of grafts was thickened markedly as (70.93 ± 11.25) μm at 4th week after surgery (P ＜0.05).It was important that bortezomib significantly attenuated gene expression and protein levels in most of the inflammatory mediators except IL-1 [ CINC-2β(231.48 ±36.32) pg/mg; MCP-1 (5846.10 ± 1044.33) pg/mg; IL-6 (1023.22 ± 109.84) pg/mg;TNF-α (218.40 ± 38.06) pg/mg] (P ＜ 0.05 ),and simultaneously inhibited neutrophil chemotactic activity of the vessel homogenates as CI was ( 167.81 ± 30.00) ％ (P ＜0.05 ).And notably,bortezomib resulted in significant inhibition of intimal hyperplasia as (35.23 ±5.21 ) μm at 4th week compared with untreated controls (P ＜ 0.05).Conclusion Bortezomib could inhibit neointima formation by attenuating the inflammatory response in transplant-induced vasculopathy. Key words: Proteasome inhibitor; Bypass graft; Intimal hyperplasia; Restenosis

Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts

BackgroundNanoparticles possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded nanoparticles(NPs) can reduce neointima formation in a rat model of vein graft disease.MethodsPoly(lactic-co-glycolic acid) (PLGA) NPs containing rapamycin was prepared using an oil/water solvent evaporation technique. Nanoparticle size and morphology were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA (RPLGA) NPs was studied using MTT Assay. Excised rat jugular vein was treated ex vivo with blank-NPs, or rapamycin-loaded NPs, then interposed back into the carotid artery position using a cuff technique. Grafts were harvested at 21 days and underwent morphometric analysis as well as immunohistochemical analysis.ResultsRapamycin was efficiently loaded in PLGA nanoparticles with an encapsulation efficiency was 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/ml significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts shown that the concentration of rapamycin in vein grafts at 3 weeks after grafting were 0.9 ± 0.1 μg/g. In grafted veins without treatment intima-media thickness was 300.4 ±181.5 μm after grafting 21 days. Whereas, Veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). CD-31 staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization in 21 days vein grafts with no significant effect of blank or rapamycin-loaded NPs group.ConclusionsWe conclude that sustained-release rapamycin from rapymycin loaded NPs inhibits vein graft thickening without affecting the reendothelialization in rat carotid vein-to-artery interposition grafts and this may be a promising therapy for the treatment of vein graft disease.

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.

Inhibitory Effects of Calcitonin Gene-Related Peptides on Experimental Vein Graft Disease

Vein graft disease is a chronic inflammatory disease and limits the long-term clinical outcome of coronary revascularization. Because calcitonin gene-related peptide (CGRP) inhibits macrophage infiltration and inflammatory mediators, we hypothesized that transfected CGRP gene would inhibit macrophage infiltration and expression of inflammatory mediators in vein graft disease. Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of mosaic adeno-associated virus vectors containing CGRP gene (AAV2/1.CGRP) or Escherichia coli B-galactosidase gene (LacZ) or a saline solution and then interposed in the carotid artery. Expression of CGRP gene was identified by reverse transcription-polymerase chain reaction, and E. coli LacZ gene expression was identified by X-gal staining. Intima to media ratios were evaluated at postoperative 4 weeks. Macrophages were identified with CD68 antibody by immunocytochemistry. Inflammatory mediators were measured with real-time polymerase chain reaction. The CGRP and LacZ gene expression were positive at postoperative 4 weeks. The intima to media ratio was significantly inhibited in the AAV2/1.CGRP group. Macrophage infiltration and expression of inflammatory mediators including monocyte chemoattractant protein-1, tumor necrosis factor-alpha, inducible nitric oxide synthase, and matrix metalloproteinase-9 were also significantly inhibited in the AAV2/1.CGRP group. Transfection of AAV2/1.CGRP inhibited inflammatory mediator expression, macrophage infiltration, and neointimal hyperplasia in experimental vein graft disease.

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

The potassium channel opener levcromakalim causes expansive remodelling of experimental vein grafts

The potassium channel opener levcromakalim causes expansive remodelling of experimental vein grafts

Blockade of monocyte chemoattractant protein-1 by adenoviral gene transfer inhibits experimental vein graft neointimal formation

Clinical outcome of vascular bypass surgery using autologous vein graft is limited by neointimal formation associated with vein graft failure. Because inflammatory changes are one of the main pathologic features of vein graft failure, monocyte chemoattractant protein-1 (MCP-1) might therefore underlie in the mechanism of vein graft failure. There is no direct evidence, however, that shows the benefits of local anti-MCP-1 therapy as a novel molecular approach for prevention of vein graft failure. To block MCP-1, we used an N-terminal deletion mutant of the MCP-1 gene (7ND), which lacks the N-terminal amino acids 2 to 8, binds to its receptor CCR2, and blocks MCP-1-mediated monocyte chemotaxis. 7ND works as dominant-negative inhibitor of MCP-1. Autologous canine jugular vein grafts were transfected by incubating them ex vivo in a solution with or without adenovirus vectors containing 7ND gene or LacZ gene, and interposed into the carotid arteries. Adenovirus-mediated gene transfer of 7ND, but not LacZ gene transfer, significantly attenuated inflammation (monocyte infiltration per mm2 on day 7: 328+/-59, 220+/-11, 26+/-4 in control, LacZ, and 7ND groups, respectively, P<.05, n=4 each) and proliferation (appearance of proliferating cells per mm2 on day 7: 1005+/-186, 756+/-106, 252+/-27 in control, LacZ, and 7ND groups, P<.05, n=4 each) at 7 days after the operation and thus suppressed neointimal formation (neointimal area in mm2 on day 28: 1.63+/-0.51, 1.96+/-0.48, 0.68+/-0.10 in control, LacZ, and 7ND groups, P<.05, n=4 each). This strategy also attenuated upregulation of MCP-1 activities but did not affect endothelial regeneration process. Blockade of MCP-1 by adenoviral gene transfer of 7ND limits neointimal formation associated with vein graft failure in dogs. This study highlights the potential therapeutic benefit of local anti-MCP-1 therapy for prevention of neointimal formation associated with vein graft failure.

Heme Oxygenase-1

Schmid and coworkers1 were the first to report on the presence of heme oxygenase (HO) in liver microsomes capable of degrading heme to bilirubin, and this activity was subsequently dissociated from cytochrome P-450.2,3 HO catalyzes the first and rate-limiting step in the oxidative degradation of heme (Fe-protoporphyrin-IX) to carbon monoxide (CO), ferrous iron (Fe2+), and biliverdin-IX (Figure 1). The enzyme binds heme in a 1:1 molar complex, and HO-bound heme acts as prosthetic group and substrate. The reaction requires 3 molecules of molecular oxygen (O2) per heme molecule oxidized and reducing equivalents derived from nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide (reduced form) and transferred to the oxygenase via nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase. Regiospecific oxidation of heme is achieved in a stepwise reaction, with α-meso-hydroxyheme and verdoheme as intermediates, and the dissociation of CO followed by that of Fe2+.4 The release of biliverdin from HO is accelerated by biliverdin reductase, which reduces the green pigment to bilirubin-IX,4 which is then excreted into bile as the glucuronic acid conjugate. Figure 1. Oxidative metabolism of heme by HO and biliverdin reductase, giving rise to CO, iron, biliverdin, and bilirubin. Originally, the interest in HO was related to its well-established function in heme catabolism and the turnover of erythrocytes. For many years, CO and bilirubin were regarded as toxic waste byproducts of the HO reaction, but in 1987, a potential beneficial role of bilirubin was proposed5 based on the in vitro antioxidant activities of the pigment. Over the last decade, however, the interest in HO has shifted greatly from a metabolic to a protective function of the enzyme in a variety of conditions associated with cellular stress and pathologies, and this has been the subject of excellent recent reviews.6,7 The …

The potassium channel opener levcromakalim causes expansive remodelling of experimental vein grafts

Maintenance of luminal area is essential for the optimal performance of venous bypass grafts. However, injury and response to the arterial circulation evoke vascular remodelling that favors intimal hyperplasia, with luminal encroachment and inward remodelling. Potassium channel-opening drugs reduce tissue workload and peripheral vascular resistance and through these mechanisms could favor outward or expansive remodelling of vein grafts. We tested the hypothesis that levcromakalim, a potassium channel opener, would enhance expansive remodelling in vein grafts. A randomized, double-blind, placebo-controlled trial was conducted in 33 rats with vena cava-to-aorta bypass grafts. Drugs were administered via osmotic pump for 7 days after surgery. Half the cohort had bromodeoxyuridine (BrdU) infused at day 6. Morphometric analysis was conducted of pressure perfusion-fixed grafts harvested at 1 week and 4 weeks. At 1 week, lumen area was similar in both groups (1.82 +/- 0.39 mm(2) placebo vs 1.85 +/- 0.36 mm(2) levcromakalim), although medial cell density and BrdU staining were significantly increased in the placebo group. At 4 weeks, lumen area was unchanged in the placebo group (1.88 +/- 0.51 mm(2)) but had increased to 2.32 +/- 0.46 mm(2) in the levcromakalim group (P = .039 vs 1 week), with a very significant reduction in the intimal area (levcromakalim, 0.06 +/- 0.02 mm(2) vs placebo, 0.33 +/- 0.17 mm(2); P = .001). Early, short-term treatment with levcromakalim favors expansive remodelling of experimental vein grafts to mimic the effect of external stenting. This expansive remodelling was associated with a reduction in medial cell proliferation at 1 week. Critical limb ischemia can be treated by bypass surgery or angioplasty, but inward remodelling with restenosis is a common problem. There has been little previous experimental work to identify treatments associated with expansive remodelling, which would increase the chances of vessel patency. Here, in a randomized trial, we show that short-term treatment with a potassium channel opener (a class of drug that can be used to treat hypertension) results in strong, expansive remodelling, with increases the lumen area and graft size of experimental vein grafts by >25%.

Perivascular Treatment with Azathioprine Reduces Neointimal Hyperplasia in Experimental Vein Grafts

Azathioprine is an immunosuppressive and anti-inflammatory drug, and it has been shown to induce apoptosis in human T-lymphocytes. We investigated whether local treatment with azathioprine can inhibit neointimal hyperplasia in experimental vein grafts. C57BL/6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a cuff technique. In the treatment group azathioprine was perivascularly applied. The control group did not receive local treatment. Vein grafts were harvested at 1 and 2 weeks postoperatively and underwent morphometric analysis as well as immunohistochemical analysis for apoptosis (TUNEL). In grafted veins without treatment (controls), neointimal thickness was 10 microm (range, 6-29 microm), and 12 microm (range, 8-40 microm) at 1 and 2 weeks postoperatively, respectively. In azathioprine-treated grafts, the neointimal thickness was 2 microm (range, 1-5 microm) and 4 microm (range, 3-11 microm) at 1 and 2 weeks postoperatively, respectively. This reduction of neointimal thickness was significant at 1 week (P = .001) and 2 weeks (P = .016) postoperatively. Azathioprine-treated vein grafts showed an increased rate of apoptosis in the vascular wall as compared with controls (593 [range, 26-783] versus 45 [range, 0-106] apoptotic cells/mm(2) at 1 week, P = .063; and 656 [range, 327-1270] versus 19 [range, 0-79] apoptotic cells/mm(2) at 2 weeks, P = .016). We conclude that treatment of experimental vein grafts with azathioprine is associated with a reduction of neointimal hyperplasia and an increased apoptosis rate in the vascular wall. These results suggest that azathioprine may be useful for the prevention of vein graft disease after coronary artery bypass grafting.

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

Vein grafts respond to low flow and shear stress (tau(w)) by generating thicker walls and smaller lumens through the processes of neointimal hyperplasia and remodeling. Clinically, however, vein grafts with obviously low tau(w), such as those distal to high-grade proximal obstructions, are not infrequently found to be widely patent and pliable. One possible explanation for this phenomenon may be that vein grafts remodel more favorably in response to changes in shear that occur gradually over time compared to abruptly. This hypothesis was tested in an experimental animal model in this report. Two separate models of experimental vein graft failure were created, causing either immediate exposure to ultralow tau(w) (<1 dyne/cm2) or delayed exposure to ultralow tau(w). Under general anesthesia and using a sterile technique, the right external jugular (EJ) veins of 28 New Zealand white rabbits were surgically exposed and isolated. An end-to-side distal EJ/common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. For the immediate exposure group (n = 5), the EJ was suture-ligated just proximal to the thoracic inlet, distal to a small 10-50 microm venous tributary. This created a reversed vein segment immediately and abruptly exposed to high wall tension (2.0 +/- 0.3 x 10(4) dyne/cm) and ultralow tau(w) (0.15 +/- 0.08 dyne/cm2). For the delayed exposure group (n = 22), the EJ was ligated over a 0.035 guidewire, leaving a small aperture to sustain some measure of blood flow and tau(w). This predictably resulted in slightly less wall tension (1.4 +/- 0.2 x 10(4) dyne/cm) and higher tau(w) (0.68 +/- 0.21 dyne/cm2) than the immediate exposure group. During the first week, the small outflow aperture in the delayed exposure grafts thrombosed, eventually exposing them to the same low level of tau(w) as the immediate exposure grafts. Thus, the only difference in the two models was that delayed exposure grafts enjoyed a slower decline in tau(w) than immediate exposure grafts. Fourteen rabbits in the delayed exposure group were harvested over the first 7 days to define the patency curve of the restricted outflow channel. As expected, the small aperture had thrombosed in all animals by 7 days. The remaining 14 grafts were harvested after 4 weeks, and 13/14 remained patent. Examination of the hemodynamic parameters at the time of death confirmed that wall tension and tau(w) had equalized (wall tension 0.9 +/- 0.1 vs. 1.1 +/- 0.1 x 10(4) dyne/cm, tau(w) 0.45 +/- 0.12 vs. 0.30 +/- 0.08 dyne/cm2). Histological examination revealed less neointimal hyperplasia in the delayed exposure group compared to the immediate exposure group (wall thickness 266 +/- 16 vs. 180 +/- 24 microm, p = 0.025) as well as a slightly greater luminal diameter (0.30 +/- 0.02 vs. 0.40 +/- 0.02 cm, p = 0.038). The results of this experiment suggest that slow exposure to reduced tau(w) results in more favorable remodeling (less thickening) than abrupt exposure. This finding may explain the occasional clinical observation of a widely patent vein graft even in the face of proximal arterial obstruction and very low flow; the change in tau(w) presumably occurred slowly mitigating the remodeling response.