J.H.P. Lardenoye

Accelerated Atherosclerosis by Placement of a Perivascular Cuff and a Cholesterol-Rich Diet in ApoE*3Leiden Transgenic Mice

Intimal hyperplasia after vascular injury is usually studied in animal models with healthy, normocholesterolemic animals. Here, we assess the effect of diet-induced hypercholesterolemia on the induction of intimal hyperplasia in ApoE*3Leiden mice. A nonconstrictive polyethylene cuff was placed around the femoral artery of ApoE3*Leiden mice fed a highly cholesterol-rich diet, a mildly cholesterol-rich diet, or a chow diet for 4 weeks. Diets were continued after cuff placement until euthanization. At several time points (1 to 14 days), mice were euthanized and the intimal hyperplasia in the cuffed arteries was analyzed. In mice fed a chow diet, a 2- to 4-cell-layer-thick intima, predominantly consisting of alpha smooth muscle cell actin-positive cells, was observed after 14 days. A mildly cholesterol-rich diet (mean plasma-cholesterol level, 10.5 mmol/L) resulted in a 2.7-fold increase of total intimal area, and a highly cholesterol-rich diet (mean plasma cholesterol level 28. 6 mmol/L), in a 7.8-fold increase. In the high-cholesterol group, the intima consisted predominantly of lipid-loaded foam cells and alpha smooth muscle cell actin-positive cells. Foam cell accumulation could be observed by as early as 3 days, resulting in a near-total occlusion of the lumen after 14 days. Hypercholesterolemia resulted in a rapid, cholesterol-dependent induction of foam cell-rich intimal hyperplasia in cuffed femoral arteries of ApoE*3Leiden mice. In conclusion, the present data show that the combination of a local (cuff placement) and a systemic (hypercholesterolemic) risk factor of atherosclerosis results in a rapid induction (within 14 days) of atherosclerotic-like lesions in ApoE*3Leiden mice.

Adenoviral Expression of a Urokinase Receptor–Targeted Protease Inhibitor Inhibits Neointima Formation in Murine and Human Blood Vessels

Background —Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin–mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface–bound plasmin, which cannot be inhibited by its natural inhibitors, such as &agr;2-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. Methods and Results —The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5×109 pfu/mL) resulted in 87.5±3.8% (mean±SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9±2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. Conclusions —These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.

Vascular Endothelial Growth Factor Overexpression in Ischemic Skeletal Muscle Enhances Myoglobin Expression In Vivo

Therapeutic angiogenesis using vascular endothelial growth factor (VEGF) is considered a promising new therapy for patients with arterial obstructive disease. Clinical improvements observed consist of improved muscle function and regression of rest pain or angina. However, direct evidence for improved vascularization, as evaluated by angiography, is weak. In this study, we report an angiogenesis-independent effect of VEGF on ischemic skeletal muscle, ie, upregulation of myoglobin after VEGF treatment. Mice received intramuscular injection with adenoviral VEGF-A or either adenoviral LacZ or PBS as control, followed by surgical induction of acute hindlimb ischemia at day 3. At day 6, capillary density was increased in calf muscle of Ad.VEGF-treated versus control mice (P < 0.01). However, angiographic score of collateral arteries was unchanged between Ad.VEGF-treated and control mice. More interestingly, an increase in myoglobin was observed in Ad.VEGF-treated mice. Active myoglobin was 1.5-fold increased in calf muscle of Ad.VEGF-treated mice (P ≤0.01). In addition, the number of myoglobin-stained myofibers was 2.6-fold increased in Ad.VEGF-treated mice (P = 0.001). Furthermore, in ischemic muscle of 15 limb amputation patients, VEGF and myoglobin were coexpressed. Finally, in cultured C2C12 myotubes treated with rhVEGF, myoglobin mRNA was 2.8-fold raised as compared with PBS-treated cells (P = 0.02). This effect could be blocked with the VEGF receptor tyrosine kinase inhibitor SU5416. In conclusion, we show that VEGF upregulates myoglobin in ischemic muscle both in vitro and in vivo. Increased myoglobin expression in VEGF-treated muscle implies an improved muscle oxygenation, which may, at least partly, explain observed clinical improvements in VEGF-treated patients, in the absence of improved vascularization.

Accelerated Atherosclerosis and Calcification in Vein Grafts: A Study in APOE*3 Leiden Transgenic Mice

Abstract— Vein grafts fail due to development of intimal hyperplasia and accelerated atherosclerosis. Many murine genetic models in which genes are overexpressed, deleted, or mutated have been introduced recently. Therefore, mouse models are very well suited to dissect the relative contribution of different genes in the development of accelerated atherosclerosis. In the present study, we evaluated whether accelerated atherosclerosis in human vein grafts could be mimicked in hypercholesterolemic APOE*3 Leiden transgenic mice. Venous bypass grafting was performed in the carotid artery in APOE*3 Leiden mice fed either a standard chow diet or a high cholesterol–rich diet for 4 weeks. At several time points (0 hour to 28 days), mice were euthanized and the morphology of the vein grafts was analyzed. In normocholesterolemic mice, vein graft thickening up to 10-fold original thickness, predominantly consisting of &agr;-smooth muscle cell actin–positive cells, was observed after 28 days. In hypercholesterolemic mice, accelerated atherosclerosis with accumulation of lipid-loaded foam cells was observed within 7 days after surgery. This accelerated atherosclerosis progressed in time and resulted in significant increase in vein graft thickening up to 50 times original thickness with foam cell–rich lesions and calcification within 28 days after surgery. The atherosclerotic lesions observed in these murine grafts show high morphological resemblance with the atherosclerotic lesions observed in human vein grafts. This accelerated, diet-dependent induction of atherosclerotic-like lesions in murine vein grafts provides a valuable tool in evaluating the mechanisms of accelerated atherosclerosis and therapeutic interventions of vein graft disease.

The effect of implementation of an optimized care protocol on the outcome of arteriovenous hemodialysis access surgery

BACKGROUND The long-term patency of arteriovenous fistulas (AVFs) and arteriovenous grafts (AVGs) suffers from a high incidence of primary failure due to early thrombosis, myointimal hyperplasia at the venous access site, or failure to mature. A multidisciplinary meeting in vascular access surgery was initiated to optimize the timing, indication, type of intervention, and the logistics of AVFs/AVGs during the preoperative and postoperative period. This study evaluated the influence of the new optimized care protocol on the incidence of revisions (surgical and endovascular) and patency rates. METHODS This protocol for vascular access surgery of AVFs/AVGs for hemodialysis was introduced in January 2004. It was initiated with the presence of the vascular surgeons, nephrologists, interventional radiologists, dialysis nurses, and the ultrasound technicians. Every patient who needed an AVF/AVG because of long-term treatment of chronic renal failure or awaiting kidney transplantation, or who needed a revision of an AVF/AVG, was discussed. Two groups were compared. Group I patients were treated with an AVF/AVG before the introduction of the new protocol (2001 and 2002). Group II patients were treated with an AVF/AVG after the introduction of the new optimized care protocol (2004 and 2005). Both groups were followed up after 12 months. RESULTS During the study period, 146 AVFs/AVGs were attempted, and 111 postoperative revisions were performed to restore primary and secondary patency: 63 in group I (60 surgical, 3 radiology) and 48 in group II (23 surgical, 25 radiology). Significantly more segmental access replacements (P < 0.027) occurred in group I than in group II. Significantly fewer surgical revisions (P < 0.019) and more endovascular balloon angioplasties (P < 0.001) occurred in group II. Significantly higher cumulative primary and secondary patency rates of all AVFs/AVGs (P < 0.001), radial-cephalic direct wrist AVFs (P < 0.001), and brachial-cephalic forearm looped transposition AVGs (P < 0.001) were achieved in group II after follow-up. CONCLUSION The new protocol outlined in a bimonthly multidisciplinary meeting for vascular access surgery of AVFs/AVGs for hemodialysis resulted in more effective logistics according to preoperative diagnostics and operation. More importantly, a significant increase in endovascular balloon angioplasties and a significant decrease in surgical revisions was observed, resulting in less patient morbidity. Also, higher primary and secondary patency was achieved after the introduction of the new optimized care protocol.

Adenoviral Activin A Expression Prevents Intimal Hyperplasia in Human and Murine Blood Vessels by Maintaining the Contractile Smooth Muscle Cell Phenotype

Activin A alters the characteristics of human arterial smooth muscle cells (SMCs) toward a contractile, quiescent phenotype. We hypothesize that activin A may prevent SMC-rich neointimal hyperplasia. Here, we study the effect of adenovirus-mediated expression of activin A on neointima formation in vitro and in vivo. Human saphenous vein organ cultures, in which a neointima is formed spontaneously, were infected either with activin A- or lacZ-adenovirus. Activin A-overexpression reduces neointima formation by 78%, whereas no significant reduction was observed after control infection. In addition, the effect of activin A on neointima formation was assessed in vivo in mice with cuffed femoral arteries. In activin A adenovirus-infected mice (IV injection), neointimal hyperplasia is reduced by 77% compared with the SMC-rich neointima in mock-infected or in noninfected mice. Cultured human saphenous vein SMCs and murine aorta SMCs were incubated with activin A and an increased expression of SM22&agr; and SM &agr;-actin mRNA, and SM &agr;-actin protein was demonstrated. Laser-capture microdissection on sections of cuffed murine arteries and subsequent real-time RT-PCR established in vivo induction of SM &agr;-actin mRNA in the media of activin A–treated mice. In summary, activin A inhibits neointima formation in vitro and in vivo by preventing SMC dedifferentiation.

Inhibition of Accelerated Atherosclerosis in Vein Grafts by Placement of External Stent in ApoE*3-Leiden Transgenic Mice

Objective—Vein grafts fail because of the development of intimal hyperplasia and accelerated atherosclerosis. Placement of an external stent around vein grafts resulted in an inhibition of intimal hyperplasia in several animal studies. Here, we assess the effects of external stenting on accelerated atherosclerosis in early vein grafts in carotid arteries in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice. Methods and Results—Venous interposition grafting was performed in apolipoprotein E*3-Leiden mice fed standard chow or a highly cholesterol-rich diet for 4 weeks. After engraftment, external stents with different inner diameters (0.4 or 0.8 mm) were placed. In unstented vein grafts in hypercholesterolemic mice, thickening up to 50 times the original thickness, with foam cell–rich lesions, calcification, and necrosis, was observed within 28 days. The atherosclerotic lesions observed show high morphological resemblance to atherosclerotic lesions observed in human vein grafts. In stented vein grafts in hypercholesterolemic mice, no foam cell accumulation or accelerated atherosclerosis was observed. Compared with unstented vein grafts, stenting of vein grafts in a hypercholesterolemic environment resulted in a 94% reduction of vessel wall thickening. These effects were independent of stent size. Conclusions—Extravascular stent placement results in strong inhibition of accelerated vein graft atherosclerosis in hypercholesterolemic transgenic mice and thereby provides a perspective for therapeutic intervention in vein graft diseases.

Dutch experience with the fenestrated Anaconda endograft for short-neck infrarenal and juxtarenal abdominal aortic aneurysm repair

OBJECTIVE In the past decennium, the management of short-neck infrarenal and juxtarenal aortic aneurysms with fenestrated endovascular aneurysm repair (FEVAR) has been shown to be successful, with good early and midterm results. Recently, a new fenestrated device, the fenestrated Anaconda (Vascutek, Renfrewshire, Scotland), was introduced. The aim of this study was to present the current Dutch experience with this device. METHODS A prospectively held database of patients treated with the fenestrated Anaconda endograft was analyzed. Decision to treat was based on current international guidelines. Indications for FEVAR included an abdominal aortic aneurysm (AAA) with unsuitable neck anatomy for EVAR. Planning was performed on computed tomography angiography images using a three-dimensional workstation. RESULTS Between May 2011 and September 2013, 25 patients were treated in eight institutions for juxtarenal (n = 23) and short-neck AAA (n = 2). Median AAA size was 61 mm (59-68.5 mm). All procedures except one were performed with bifurcated devices. A total of 56 fenestrations were incorporated, and 53 (94.6%) were successfully cannulated and stented. One patient died of bowel ischemia caused by occlusion of the superior mesenteric artery. On completion angiography, three type I endoleaks and seven type II endoleaks were observed. At 1 month of follow-up, all endoleaks had spontaneously resolved. Median follow-up was 11 months (range, 1-29 months). There were no aneurysm ruptures or aneurysm-related deaths and no reinterventions to date. Primary patency at 1 month of cannulated and stented target vessels was 96%. CONCLUSIONS Initial and short-term results of FEVAR using the fenestrated Anaconda endograft are promising, with acceptable technical success and short-term complication rates. Growing experience and long-term results are needed to support these findings.

Functional status as a prognostic factor for primary revascularization for critical limb ischemia

BACKGROUND Lower extremity arterial revascularization (LEAR) is the gold-standard for critical lower limb ischemia (CLI). The goal of this study was twofold. First, we evaluated the long-term functional status of patients undergoing primary LEAR for CLI. Second, prognostic factors of long-term functional status and survival after primary LEAR for CLI were assessed. METHODS All primary LEAR procedures were analyzed. Patients were stratified by preoperative functional status: ambulatory (group I) vs nonambulatory (group II). Patients were followed-up after 3 and 6 years. Adverse events (AEs) were categorized according to predefined standards: minor, surgical, failed revascularization, and systemic. Associated patient demographic/clinical data were analyzed using univariate and multivariate methods. RESULTS There were 106 LEAR patients (group I: n = 42, 40% vs group II: n = 64, 60%). Group II patients were significantly older (75 vs 62 years; P = .00), were classified ASA 3-4 more frequently (78% vs 52%; P < .02), had more cardiac disease (n = 42, 66% vs n = 10, 24%; P = .00), renal disease (n = 26, 41% vs n = 7, 17%; P = .00), diabetes (n = 36, 56% vs n = 8, 19%; P = .00), hypertension (n = 47, 73% vs n = 13, 31%; P = .00) and severe CLI (n = 42, 66% vs n = 18, 38%; P < .01). Group II patients had a higher incidence of death (65.6% vs 14.3%; P = .00), minor AEs (n = 38, 26% vs n = 10, 22%; P = .00), surgical AEs (n = 48, 33% vs n = 12, 26%; P < .02) and systemic AEs (n = 24, 86% vs n = 4, 9%; P < .02). Also more unplanned reinterventions occurred in group II (n = 148, 76% vs n = 47, 24%; P = .00). Nonambulatory status was a multivariate independent predictor of nonambulatory status after LEAR during 6 years follow-up (odds ration [OR[: 21.47; 95% confidence interval [CI]: 2.76-166.77; P = .00). Pulmonary disease (OR: 7.49; 95% CI: 2.17-25.80; P = .00), not prescribing beta-blockers (OR: 4.67; 95% CI: 1.28-17.03; P < .02), nonambulatory status (OR: 22.99; 95% CI: 6.27-84.24; P = .00), and systemic AEs (OR: 9.66; 95% CI: 1.84-50.57; P < .01) were independent predictors of death. Functional status was not improved in group II after long-term follow-up. CONCLUSION Nonambulatory patients suffer from extensive comorbid conditions. They are accompanied with an increased occurrence of AEs, unplanned reinterventions, and poor long-term survival rates. Successful LEAR did not improve their functional status after 6 years. This emphasizes that attempts for limb salvage must be carefully considered in these patients.

In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI.

Injury-induced neointimal development results from migration and proliferation of vascular smooth muscle cells (SMC). Cell migration requires controlled proteolytic degradation of extracellular matrix surrounding the cell. Plasmin is a major contributor to this process by degrading various matrix proteins directly, or indirectly by activating matrix metalloproteinases. This makes it an attractive target for inhibition by gene transfer. An adenoviral vector, Ad.ATF.BPTI, was constructed encoding a hybrid protein, which consists of the aminoterminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) linked to bovine pancreas trypsin inhibitor (BPTI), a potent inhibitor of plasmin. This hybrid protein binds to the u-PA receptor, thereby inhibiting plasmin activity at the cell surface, and was found to be a potent inhibitor of cell migration in vitro. Local infection with Ad.ATF.BPTI of balloon-injured rat carotid artery resulted in detectable expression of ATF.BPTI mRNA and protein in the vessel wall. Morphometric analysis of arterial cross-sections revealed that delivery of Ad.ATF.BPTI to the carotid artery wall at the time of balloon injury inhibited neointima formation by 53% (P < 0.01) at 14 days and 19% (P = NS) at 28 days after injury when compared with control vector-infected arteries. Intima/media ratios were decreased by 60% (P < 0.01) and 35% (P < 0.05) at 14 and 28 days, respectively, when compared with control vector-infected arteries. Furthermore, a small but significant increase in medial area was found in the Ad.ATF.BPTI-treated arteries at 28 days (P < 0.05). These results show that local infection of the vessel wall with Ad.ATF.BPTI reduces neointima formation, presumably by inhibiting SMC migration, thereby offering a novel therapeutic approach to inhibiting neointima development.