A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells.

BACKGROUND: Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. METHODS: Human saphenous vein adventitial cells and intimal/medial smooth muscle cells (SMCs) were derived from explants obtained at the time of lower extremity bypass operations. We determined the following in adventitial cells and SMCs as a function of the p27 SNP genotype: (1) p27 promoter activity, (2) p27 messenger (m)RNA and protein levels, and (3) growth and collagen gel contraction. Deoxyribonuclease I footprinting was also performed in adventitial cells and SMCs. RESULTS: p27 promoter activity, deoxyribonuclease I footprinting, p27 mRNA levels, and p27 protein levels demonstrated that the p27 SNP is functional in adventitial cells and SMCs. Both cell types with the graft failure protective AA genotype had more p27 mRNA and protein. As predicted because of higher levels of p27 protein, adventitial cells with the AA genotype grew slower than those of the CC genotype. Unexpectedly, SMCs did not show this genotype-dependent growth response. CONCLUSIONS: These results support the functionality of the p27 SNP in venous SMCs and adventitial cells, but an effect of the SNP on cell proliferation is limited to only adventitial cells. These data point to a potential role for adventitial cells in human vein graft failure and also suggest that SMCs express factors that interfere with the activity of p27.

Tissue Factor Activity in Dialysis Access Grafts.

BACKGROUND: Intimal hyperplasia at the venous anastomosis of dialysis grafts causes early failure. We developed a sheep model of arteriovenous prosthetic grafts that fail rapidly due to intimal hyperplasia with histologic features nearly identical to human access grafts. A prominent feature of lesion development in this model is formation of luminal thrombus that becomes organized into stenosing lesions by macrophage and myofibroblast infiltration. To better understand this process, we examined the presence and activity of tissue factor (TF) in this system. This protein is the physiological initiator of coagulation in vivo and is known to contribute to development of intimal hyperplasia after vascular injury. METHODS: Expanded polytetrafluorethylene (ePTFE) grafts were placed between the carotid artery and external jugular vein in sheep. Grafts were examined for luminal TF activity using a novel ex vivo assay. In a separate series of grafts, immunohistochemistry was used to localize smooth muscle cells, monocytes, and TF protein. RESULTS: At 2 days, luminal TF activity already was higher in the venous and arterial end of the graft than in the adventitia. This high level of activity persisted at 8 weeks. TF activity was higher in the venous end of the grafts than in the arterial end at 2 and 8 weeks (40% and 47% increase, n = 5, n = 3, respectively, P < 0.05). Immunohistochemistry revealed TF protein localized in regions with or adjacent to fibrin accumulation and often in regions close to the lumen. CONCLUSIONS: This study further examines the development of intimal hyperplasia in ePTFE dialysis access grafts. In this model, TF levels on the luminal surface were increased throughout the arteriovenous grafts and the adjacent vessels as early as 2 days after engraftment and for as long as 8 weeks thereafter. The highest levels of activity were found in the venous end of the graft, where hyperplasia is most robust. Increased activity of TF is associated with luminal thrombus, which provides a scaffolding for development of intimal hyperplasia. These findings present an opportunity to develop strategies to limit TF activity within the graft. Further studies using agents delivered locally or incorporated into the graft matrix to block the luminal activity of TF are warranted.

Scavenger receptor class A member 5 (SCARA5) and suprabasin (SBSN) are hub genes of coexpression network modules associated with peripheral vein graft patency.

OBJECTIVE: Approximately 30% of autogenous vein grafts develop luminal narrowing and fail because of intimal hyperplasia or negative remodeling. We previously found that vein graft cells from patients who later develop stenosis proliferate more in vitro in response to growth factors than cells from patients who maintain patent grafts. To discover novel determinants of vein graft outcome, we have analyzed gene expression profiles of these cells using a systems biology approach to cluster the genes into modules by their coexpression patterns and to correlate the results with growth data from our prior study and with new studies of migration and matrix remodeling. METHODS: RNA from 4-hour serum- or platelet-derived growth factor (PDGF)-BB-stimulated human saphenous vein cells obtained from the outer vein wall (20 cell lines) was used for microarray analysis of gene expression, followed by weighted gene coexpression network analysis. Cell migration in microchemotaxis chambers in response to PDGF-BB and cell-mediated collagen gel contraction in response to serum were also determined. Gene function was determined using short-interfering RNA to inhibit gene expression before subjecting cells to growth or collagen gel contraction assays. These cells were derived from samples of the vein grafts obtained at surgery, and the long-term fate of these bypass grafts was known. RESULTS: Neither migration nor cell-mediated collagen gel contraction showed a correlation with graft outcome. Although 1188 and 1340 genes were differentially expressed in response to treatment with serum and PDGF, respectively, no single gene was differentially expressed in cells isolated from patients whose grafts stenosed compared with those that remained patent. Network analysis revealed four unique groups of genes, which we term modules, associated with PDGF responses, and 20 unique modules associated with serum responses. The "yellow" and "skyblue" modules, from PDGF and serum analyses, respectively, correlated with later graft stenosis (P = .005 and P = .02, respectively). In response to PDGF, yellow was also associated with increased cell growth. For serum, skyblue was also associated with inhibition of collagen gel contraction. The hub genes for yellow and skyblue (ie, the gene most connected to other genes in the module), scavenger receptor class A member 5 (SCARA5) and suprabasin (SBSN), respectively, were tested for effects on proliferation and collagen contraction. Knockdown of SCARA5 increased proliferation by 29.9% ± 7.8% (P < .01), whereas knockdown of SBSN had no effect. Knockdown of SBSN increased collagen gel contraction by 24.2% ± 8.6% (P < .05), whereas knockdown of SCARA5 had no effect. CONCLUSIONS: Using weighted gene coexpression network analysis of cultured vein graft cell gene expression, we have discovered two small gene modules, which comprise 42 genes, that are associated with vein graft failure. Further experiments are needed to delineate the venous cells that express these genes in vivo and the roles these genes play in vein graft healing, starting with the module hub genes SCARA5 and SBSN, which have been shown to have modest effects on cell proliferation or collagen gel contraction.

Surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue.

OBJECTIVE: Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. METHODS: Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either (1) percentage of migration-positive explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry. RESULTS: There was significantly less cell migration from visibly blue compared with unstained outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9 ± 8.0 ng dye/explant compared with 2.1 ± 1.3 for nonblue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose dependently inhibited migration (IC50∼10 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 µg/mL, whereas the EC50 for death was between 1 and 10 µg/mL. CONCLUSIONS: Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts.

M2 Macrophage Polarization Mediates Anti-inflammatory Effects of Endothelial Nitric Oxide Signaling.

Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance.

Efficient gene transfer and durable transgene expression in grafted rabbit veins.

Venous bypass grafts are useful treatments for obstructive coronary artery disease. However, their usefulness is limited by accelerated atherosclerosis. Genetic engineering of venous bypass grafts that prevented atherosclerosis could improve long-term graft patency and clinical outcomes. We used a rabbit model of jugular vein-to-carotid interposition grafting to develop gene therapy for vein-graft atherosclerosis. Rabbit veins were easily transduced in situ with a first-generation adenoviral vector; however, most transgene expression (∼80%) was lost within 3 days after arterial grafting. This rapid loss of transgene expression was not prevented by transducing veins after grafting or by prolonged ex vivo transduction. However, delaying vein-graft transduction for 28 days (after the vein had adapted to the arterial circulation) prevented this early loss of transgene expression. We used the delayed transduction approach to test the durability of expression of a therapeutic transgene (apolipoprotein A-I) expressed from a helper-dependent adenoviral (HDAd) vector. HDAd DNA and apolipoprotein A-I mRNA were easily detectable in transduced vein grafts. Vector DNA and mRNA declined by 4 weeks, and then persisted stably for at least 6 months. Delaying transduction for 28 days after grafting permitted initiation of vein-graft neointimal growth and medial thickening before gene transfer. However, vein-graft lumen diameter was not compromised, because of gradual outward remodeling of grafted veins. Our data highlight the promise of HDAd-mediated gene therapy, delivered to arterialized vein grafts, for preventing vein-graft atherosclerosis.

Vasodilator-stimulated phosphoprotein protects against vascular inflammation and insulin resistance.

Among the pleotropic effects of endothelial nitric oxide (NO) is protection against vascular inflammation during high-fat diet (HFD) feeding. The current work investigated the role of the enzyme vasodilatory-stimulated phosphoprotein (VASP) as a downstream mediator of the anti-inflammatory effect of NO signaling in vascular tissue. Relative to mice fed a low-fat diet (LFD), levels of VASP Ser(239) phosphorylation, a marker of VASP activation, were dramatically reduced in aortic tissue of mice with obesity induced by consuming a HFD. As reported previously, the effect of the HFD was associated with increased aortic inflammation, as measured by increased NF-κB-dependent gene expression, and reduced vascular insulin sensitivity (including insulin-stimulated phosphorylation of eNOS and Akt). These effects of the HFD were recapitulated by VASP knockout, implying a physiological role for VASP to constrain inflammatory signaling and thereby maintain vascular insulin sensitivity. Conversely, overexpression of VASP in endothelial cells blocked inflammation and insulin resistance induced by palmitate. The finding that transplantation of bone marrow from VASP-deficient donors into normal recipients does not recapitulate the vascular effects of whole body VASP deficiency suggests that the protective effects of this enzyme are not mediated in immune or other bone marrow-derived cells. These studies implicate VASP as a downstream mediator of the NO/cGMP pathway that is both necessary and sufficient to protect against vascular inflammation and insulin resistance. As such, this work identifies VASP as a potential therapeutic target in the treatment of obesity-related vascular dysfunction.

Low levels of a natural IgM antibody are associated with vein graft stenosis and failure.

BACKGROUND: All humans have natural, protective antibodies directed against phosphorylcholine (PC) epitopes, a common inflammatory danger signal appearing at sites of cell injury, oxidative stress, and on bacterial capsules. In large human cohorts, low levels of anti-PC IgM were associated with a significantly increased risk of stroke or myocardial infarction. However, it is not known if these antibodies protect against the premature closure of arterial reconstructions. METHODS: A prospective, observational study of patients undergoing elective, infrainguinal, autogenous vein bypasses for atherosclerotic occlusive disease of the legs was conducted. Clinical data were recorded prospectively, and preoperative levels of anti-PC IgM measured with the CVDefine kit from Athera Biotechnologies (Solna, Sweden). The principal clinical end point was the loss of primary patency (loss of graft flow, or any intervention for stenosis). Patients were followed regularly by duplex ultrasound at 1, 3, 6, 12, 18 months, and yearly thereafter. RESULTS: Fifty-six patients were studied, for an average of 1.3 years. Indications for surgery were claudication (33.9%), ischemic rest pain (17.9%), and ischemia with ulceration or gangrene (48.2%). Seventeen (30.4%) patients experienced loss of primary patency (10 graft occlusions, seven surgical or endovascular revisions of graft stenoses). Kaplan-Meier survival analysis showed that the quartile of patients with the lowest anti-PC IgM levels had significantly worse primary graft patency (log-rank test, P = .0085). Uni- and multivariate Cox proportional hazards analysis revealed that the preoperative anti-PC IgM level was an important predictor of graft failure. Patients with IgM values in the lowest quartile had a 3.6-fold increased risk of graft failure (95% confidence interval: 1.1-12.1), even after accounting for other significant clinical or technical factors such as indication for surgery, site of distal anastomosis, or vein graft diameter. CONCLUSIONS: A naturally occurring IgM antibody directed against the proinflammatory epitope PC may be protective against vein graft stenosis and failure, through anti-inflammatory mechanisms. Measurement of this antibody may be a useful prognostic indicator, although larger studies of more diverse populations will be needed to confirm these results. The biological actions of anti-PC IgM suggest it may be useful in developing immunotherapies to improve bypass longevity.

Vasospasm as a cause for claudication in athletes with external iliac artery endofibrosis.

BACKGROUND: Exercise-induced external iliac artery endofibrosis (EIAE) is rare and has been described primarily in endurance male cyclists. Clinically, it presents as claudication during maximal exercise with quick resolution after exercise. Most patients have fibrotic changes within the external iliac artery (EIA). We describe our experience with EIAE and propose a hypothesis for the mechanism involved in the associated claudication. METHODS: This was a retrospective review of athletes who presented with symptomatic EIAE requiring operative repair between 2001 and 2010. Data collected included demographic information, initial presentation, type of exercise, repair, and long-term outcome. Diagnostic studies consisted of duplex evaluation, modified exercise treadmill test, and angiography. RESULTS: Eight women, presented with symptomatic EIAE. Two had bilateral EIAE. All were endurance athletes (three cyclists, one runner, and four were cyclists and runners). Median age at presentation was 42.5 years (range, 39-60 years). Median duration of symptoms was 5.5 years (range, 2-15 years). Diagnosis was confirmed with an exercise treadmill test modified to accommodate these patients' high level of conditioning and unmask the claudication. In the most recent two patients, marked EIA vasospasm was noted after exercise by duplex scanning. All patients were treated with EIA vein patch angioplasty. Follow-up ranged from 1 to 10 years. All had a normal result on the modified exercise treadmill test and resumed their athletic activities postoperatively. CONCLUSIONS: This series highlights a possible mechanism to explain the claudication associated with EIAE. Vasospasm may be more important than wall thickening for the reduction of blood flow during extreme exercise in affected athletes. Routine duplex ultrasound imaging to measure EIA diameter and flow velocities before and after maximal exercise is needed to confirm this phenomenon.

Inhibition of PDGF-B induction and cell growth by syndecan-1 involves the ubiquitin and SUMO-1 ligase, Topors.

Syndecans are receptors for soluble ligands, including heparin-binding growth factors, and matrix proteins. However, intracellular targets of syndecan-1 (Sdc-1)-mediated signaling are not fully understood. A yeast two-hybrid protein interaction screening of a mouse embryo library identified the ubiquitin and SUMO-1 E3 ligase, Topors, as a novel ligand of the Sdc-1 cytoplasmic domain (S1CD), a finding confirmed by ligand blotting and co-precipitation with Sdc-1 from cell lysates. Deletion mutagenesis identified an 18-amino acid sequence of Topors required for the interaction with the S1CD. By immunohistochemistry, Topors and Sdc-1 co-localized near the cell periphery in normal murine mammary gland (NMuMG) cells in vitro and in mouse embryonic epithelia in vivo. Finally, siRNA-mediated knockdown of Topors demonstrated that Topors is a growth promoter for murine arterial smooth muscle cells and is required for the inhibitory effect of Sdc-1 on cell growth and platelet-derived growth factor-B induction. These data suggest a novel mechanism for the inhibitory effects of Sdc-1 on cell growth that involves the interaction between the cytoplasmic domain of Sdc-1 and the SUMO-1 E3 ligase, Topors.

A link between smooth muscle cell death and extracellular matrix degradation during vascular atrophy.

OBJECTIVE: High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy. METHODS: DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries. RESULTS: Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction. CONCLUSION: Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

Syndecan-1: an inhibitor of arterial smooth muscle cell growth and intimal hyperplasia.

OBJECTIVE: Arterial injury induces smooth muscle cell (SMC) proliferation, migration, and intimal accumulation of cells and extracellular matrix. These processes are regulated by the administration of the glycosaminoglycans heparin and heparan sulfate, but little is known about the role of endogenous heparan sulfate proteoglycans in the vessel wall. We investigated the response to carotid injury of syndecan-1-null mice to assess the function of one of a conserved family of transmembrane heparan and chondroitin sulfate proteoglycans. METHODS AND RESULTS: Syndecan-1-null mice developed a large neointimal lesion after injury, whereas wild-type mice made little or none. This was accompanied by a significant increase in both medial and intimal SMC replication. Cultured syndecan-1-null SMCs showed a significant increase in proliferation in response to PDGF-BB, thrombin, FGF2, EGF, and serum. In response to thrombin, PDGF-BB, and serum syndecan-1-null SMCs expressed more PDGF-B chain message than did wild-type SMCs. Downregulation of PDGF-BB or PDGFRbeta inhibited thrombin-, PDGF-BB-, and serum-induced DNA synthesis in syndecan-1-null SMCs. CONCLUSIONS: These results suggest the possibility that syndecan-1 may limit intimal thickening in injured arteries by suppressing SMC activation through inhibition of SMC PDGF-B chain expression and PDGFRbeta activation.

Cell death-associated ADAMTS4 and versican degradation in vascular tissue.

High blood flow through baboon polytetrafluorethylene aorto-iliac grafts increases neointimal vascular smooth muscle cell (SMC) death, neointimal atrophy, and cleavage of versican to generate the DPEAAE neoepitope, a marker of ADAMTS-mediated proteolysis. In this study, we have determined the effect of high blood flow on transcript abundance in the neointima for ADAMTS1, -4, -5, -8, -9, -15, and -20. We found that after 24 hr of flow, the mRNA for ADAMTS4 was significantly increased, whereas that for the other family members was unchanged. Because vascular SMC death is markedly increased in the graft after 24 hr of high flow, we next examined the possibility that the ADAMTS4 induction and the cell death are causally related. The addition of Fas ligand to SMC cultures increased both ADAMTS4 mRNA and cell death approximately 5-fold, consistent with the idea that ADAMTS4-dependent cleavage of versican may be partly responsible for cell death and tissue atrophy under these conditions.

Proliferative capacity of vein graft smooth muscle cells and fibroblasts in vitro correlates with graft stenosis.

OBJECTIVE: About a quarter of peripheral vein grafts fail due in part to intimal hyperplasia. The proliferative capacity and response to growth inhibitors of medial smooth muscle cells and adventitial fibroblasts in vitro were studied to test the hypothesis that intrinsic differences in cells of vein grafts are associated with graft failure. METHODS: Cells were grown from explants of the medial and adventitial layers of samples of vein grafts obtained at the time of implantation. Vein graft patency and function were monitored over the first 12 months using ankle pressures and Duplex ultrasound to determine vein graft status. Cells were obtained from veins from 11 patients whose grafts remained patent (non-stenotic) and from seven patients whose grafts developed stenosis. Smooth muscle cells (SMCs) derived from media and fibroblasts derived from adventitia were growth arrested in serum-free medium and then stimulated with 1 muM sphingosine-1-phosphate (S1P), 10 nM thrombin, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml platelet-derived growth factor-BB (PDGF-BB), PDGF-BB plus S1P, or PDGF-BB plus thrombin for determination of incorporation of [(3)H]-thymidine into DNA. Cells receiving PDGF-BB or thrombin were also treated with or without 100 microg/ml heparin, which is a growth inhibitor. Cells receiving thrombin were also treated with or without 150 nM AG1478, an EGF receptor kinase inhibitor. RESULTS: SMCs and fibroblasts from veins of patients that developed stenosis responded more to the growth factors, such as PDGF-BB alone or in combination with thrombin or S1P, than cells from veins of patients that remained patent (P = .012). In addition, while PDGF-BB-mediated proliferation of fibroblasts from grafts that remained patent was inhibited by heparin (P < .03), PDGF-BB-mediated proliferation of fibroblasts from veins that developed stenosis was not (P > .5). CONCLUSION: Inherent differences in the proliferative response of vein graft cells to PDGF-BB and heparin may explain, in part, the variability among patients regarding long term patency of vein grafts.

Disparity in outcomes of surgical revascularization for limb salvage: race and gender are synergistic determinants of vein graft failure and limb loss.

BACKGROUND: Vein bypass surgery is an effective therapy for atherosclerotic occlusive disease in the coronary and peripheral circulations; however, long-term results are limited by progressive attrition of graft patency. Failure of vein bypass grafts in patients with critical limb ischemia results in morbidity, limb loss, and additional resource use. Although technical factors are known to be critical to the success of surgical revascularization, patient-specific risk factors are not well defined. In particular, the relationship of race/ethnicity and gender to the outcomes of peripheral bypass surgery has been controversial. METHODS AND RESULTS: We analyzed the Project of Ex Vivo Vein Graft Engineering via Transfection III (PREVENT III) randomized trial database, which included 1404 lower extremity vein graft operations performed exclusively for critical limb ischemia at 83 North American centers. Trial design included intensive ultrasound surveillance of the bypass graft and clinical follow-up to 1 year. Multivariable modeling (Cox proportional hazards and propensity score) was used to examine the relationships of demographic variables to clinical end points, including perioperative (30-day) events and 1-year outcomes (vein graft patency, limb salvage, and patient survival). Final propensity score models adjusted for 16 covariates (including type of institution, technical factors, selected comorbidities, and adjunctive medications) to examine the associations between race, gender, and outcomes. Among the 249 black patients enrolled in PREVENT III, 118 were women and 131 were men. Black men were at increased risk for early graft failure (hazard ratio [HR], 2.832 for 30-day failure; 95% confidence interval [CI], 1.393 to 5.759; P=0.0004), even when the analysis was restricted to exclude high-risk venous conduits. Black patients experienced reduced secondary patency (HR, 1.49; 95% CI, 1.08 to 2.06; P=0.016) and limb salvage (HR, 2.02; 95% CI, 1.27 to 3.20; P=0.003) at 1 year. Propensity score models demonstrate that black women were the most disadvantaged, with an increased risk for loss of graft patency (HR, 2.02 for secondary patency; 95% CI, 1.27 to 3.20; P=0.003) and major amputation (HR, 2.38; 95% CI, 1.18 to 4.83; P=0.016) at 1 year. Perioperative mortality and 1-year mortality were similar across race/gender groups. CONCLUSIONS: Black race and female gender are risk factors for adverse outcomes after vein bypass surgery for limb salvage. Graft failure and limb loss are more common events in black patients, with black women being a particularly high-risk group. These data suggest the possibility of an altered biological response to vein grafting in this population; however, further studies are needed to determine the mechanisms underlying these observed disparities in outcome.

Retrovirally mediated overexpression of glycosaminoglycan-deficient biglycan in arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointimae after vascular injury.

Galactosamine-containing glycosaminoglycans (GAGs), such as the chondroitin sulfate chains of the proteoglycan versican, have been shown to inhibit elastogenesis. Another proteoglycan that may influence elastogenesis is biglycan, which possesses two GAG chains. To assess the importance of these chains on elastogenesis in blood vessels, rat aortic smooth muscle cells were transduced with a GAG-deficient biglycan cDNA-containing retroviral vector (LmBSN). Control cells were transduced with either biglycan or empty vector. Transduced cells were characterized in vitro and then seeded into balloon-injured rat carotid arteries to determine the effects on neointimal structure. Cultured cells overexpressing LmBSN showed marked up-regulation of tropoelastin and fibulin-5 mRNAs, increased amounts of desmosine and insoluble elastin, and increased deposition of elastic fibers as compared with empty vector- and biglycan-transduced cells. Conversely, collagen alpha(1) synthesis and the deposition of collagen fibers were both markedly decreased in LmBSN cultures. In vivo, neointimae formed from cells that overexpressed LmBSN and showed increased deposits of elastin that aggregated into parallel nascent fibers, generally arranged circumferentially. Neointimae that formed from cells with biglycan or empty vector contained fewer and less aggregated deposits of elastin. These findings suggest that the GAG chains of biglycan serve as inhibitors of elastin synthesis and assembly, and that biglycan can act as an important modulator of the composition of the extracellular matrix of blood vessels.

Twentieth anniversary of American Vascular Association/Lifeline Foundation: a celebration.

The American Vascular Association/Lifeline Foundation is celebrating its 20th anniversary this year. This remarkable two-decade journey represents a cumulative effort by the leaders and members of the Society for Vascular Surgery (SVS). For the historical record, we would like to chart the sequence of events leading to various programs. In 1986, the Executive Council of SVS approved the formation of an Education/Research Foundation, from which the Lifeline Foundation evolved, with the mission to support the career development of young research-oriented vascular surgeons. Since that time, Lifeline has awarded 141 Student Fellowships, 21 Wylie Traveling Fellowships, 17 Mentored Clinical Scientist Development (K08) Awards, and three Mentored Patient-Oriented Research Career Development (K23) Awards. In 2001, the American Vascular Association (AVA) was established under the aegis of American Association for Vascular Surgery (formerly International Society for Cardiovascular Surgery-North American Chapter). In 2004, with the merger of the SVS and the American Association of Vascular Surgery into a single entity (SVS), Lifeline and the AVA merged into a single foundation, the AVA. As AVA/Lifeline is poised to launch a campaign for an endowment fund, we hope this report will let the members of the SVS know what has been accomplished, what we plan to do, and, most importantly, what we need to do in the future.

Effects of external wrapping and increased blood flow on atrophy of the baboon iliac artery.

OBJECTIVE: Increased blood flow causes neointimal atrophy, whereas relief of wall tension with an external wrap causes arterial medial atrophy. To study the effects of blood flow and wall tension separately and together, we applied tight or loose wraps on high-flow or normal-flow iliac arteries in baboons. METHOD: Baboon external iliac arteries were wrapped with loose-fitting and tight-fitting expanded polytetrafluoroethylene (ePTFE), leaving part unwrapped. A downstream arteriovenous fistula was constructed on one side to increase blood flow approximately twofold. The arteries were perfusion-fixed with 10% formalin after 4 (n = 5) and 28 days (n = 5). RESULTS: At 4 days, compared with the unwrapped artery, the loosely and tightly wrapped normal-flow artery showed significant medial atrophy (23% and 30%, respectively; P < .05). The tightly wrapped artery showed a loss of cells (27%; P = .02) but no change in cell density. At 28 days, the medial cross-sectional area was decreased by the tight wrap and loose wrap under normal (45% and 28%, respectively; P < .05) and high (43% and 29%, respectively; P < .05) flow. High flow did not alter the effect of wrapping nor did it affect the unwrapped medial area. At 28 days, the normal and high flow tightly wrapped media showed an insignificant loss of cells but had increased cell density (47% and 30%, respectively; P < .05), suggesting preferential loss of extracellular matrix. Decorin was expressed at the late time only in the tightly wrapped normal and high-flow media and was associated with tight packing of the collagen, as detected by picrosirius red staining. CONCLUSION: Loose-fitting and tight-fitting ePTFE wraps induced an inflammatory foreign body response that caused medial atrophy with loss of cells and extracellular matrix; the tight wrap was more effective. High blood flow did not prevent or augment medial atrophy. CLINICAL RELEVANCE: Research in arterial restenosis has focused on the biologic mechanisms and pharmacologic approaches to the prevention of intimal hyperplasia. An alternative therapeutic approach might be to induce atrophy of established intimal hyperplasia. We have previously reported that high blood flow induces neointimal regression in expanded polytetrafluoroethylene grafts in baboons. Here we provide another model of vascular atrophy induced by external wrapping. The similarity between baboons and humans in their vascular systems and individual genetic heterogeneity makes these experiments of great relevance. Up- or down-regulated genes common to both models might be key regulators of vascular atrophy and therefore suitable therapeutic targets for pharmacologic treatment of established lesions.

Induction of vascular atrophy as a novel approach to treating restenosis. A review.

Regardless of the type of arterial reconstruction, luminal narrowing (stenosis or restenosis) develops in approximately one third of the vessels. In the past, the focus of research has been on the mechanisms of stenosis (intimal hyperplasia, pathologic remodeling) and pharmacologic approaches to prevention. An alternative approach is to induce intimal atrophy after luminal narrowing has developed, thus limiting treatment to only those patients that develop a problem. This approach to treat established disease by reducing wall mass through induction of cell death and extracellular matrix removal would be particularly useful for treating stenosis in synthetic bypass grafts or stented vessels, in which intimal hyperplasia is the primary mechanism of stenosis. This approach may be applicable as well to other vascular proliferative disorders, such as pulmonary hypertension and chronic transplant arteriopathy. Proof of principle has been shown in experiments with antibodies to platelet-derived growth factor (PDGF) receptors that cause neointimal regression in baboon polytetrafluoroethylene (PTFE) grafts and with angiotensin-converting enzyme inhibitors that induce medial atrophy in hypertensive arteries. Possible molecular targets could include PDGF receptors, A20, and BMP4. Further studies are needed to determine the utility of such a therapeutic approach to vascular disease.

Technical factors affecting autogenous vein graft failure: observations from a large multicenter trial.

OBJECTIVE: The influence of operator-dependent variables on the outcomes of lower extremity bypass (LEB) surgery have primarily been reported in single-institution, retrospective studies. We utilized data from a prospective, multicenter trial to identify technical variables that were significantly associated with early and midterm results of autogenous LEB for limb salvage. METHODS: The PREVENT III trial database includes 1404 North American patients with critical limb ischemia (CLI) who underwent LEB using excised autogenous vein. The study protocol excluded claudicants and in situ reconstructions. Technical factors analyzed included vein diameter, conduit type, graft length, vein orientation, location of proximal and distal anastomoses, and performance of completion imaging. Univariate analysis was used to determine the effect of these factors on 30 day and 1-year outcomes. Multivariate Cox regression models evaluated the influence of these factors while adjusting for age, sex, race, tobacco, diabetes, dialysis-dependency, previous index limb bypass, and study drug (edifoligide) administration. The primary outcomes were primary patency (PP), primary assisted patency (PAP), and secondary patency (SP) assessed by Kaplan-Meier method. RESULTS: Univariate analysis revealed that vein diameter <3.5 mm and composite graft type were significantly associated with early (30 day) graft failure. At 1 year, multivariate analysis revealed that patency rates were negatively associated with diameter <3.5 mm (PP, PAP, SP), non-great saphenous vein (GSV) type (PP, SP), and graft lengths >50 cm (PP only). Limb salvage and survival at 1 year were not significantly impacted by technical variables. Employing a prespecified trial definition of high-risk conduits (diameter <3mm or nonsingle segment GSV; 24% of entire cohort) revealed that use of such conduits was associated with a 2.1-fold increased risk of 30 day graft failure (P < .05), as well as reduced PP, PAP, and SP at 1 year. Use of a high-risk conduit was also associated with an increased index length of stay (mean 9.37 vs 8.71 days, P = .03) and a greater number of reinterventions (mean 0.67 vs 0.42, P < .0001) over the ensuing year. CONCLUSIONS: In this large, multicenter cohort of patients undergoing LEB for CLI, vein diameter and conduit type were the dominant technical determinants of early and late graft failure. High-risk conduits and longer grafts may benefit from aggressive postoperative graft surveillance.