Author Correction: 3D-printed vascular networks direct therapeutic angiogenesis in ischaemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blockade of vascular endothelial growth factor receptor 2 inhibits intraplaque haemorrhage by normalization of plaque neovessels.

BACKGROUND: Plaque angiogenesis is associated with atherosclerotic lesion growth, plaque instability and negative clinical outcome. Plaque angiogenesis is a natural occurring process to fulfil the increasing demand of oxygen and nourishment of the vessel wall. However, inadequate formed, immature plaque neovessels are leaky and cause intraplaque haemorrhage. OBJECTIVE: Blockade of VEGFR2 normalizes the unbridled process of plaque neovessel formation and induces maturation of nascent vessels resulting in prevention of intraplaque haemorrhage and influx of inflammatory cells into the plaque and subsequently increases plaque stability. METHODS AND RESULTS: In human carotid and vein graft atherosclerotic lesions, leaky plaque neovessels and intraplaque haemorrhage co-localize with VEGF/VEGFR2 and angiopoietins. Using hypercholesterolaemic ApoE3*Leiden mice that received a donor caval vein interposition in the carotid artery, we demonstrate that atherosclerotic vein graft lesions at t28 are associated with hypoxia, Hif1α and Sdf1 up-regulation. Local VEGF administration results in increased plaque angiogenesis. VEGFR2 blockade in this model results in a significant 44% decrease in intraplaque haemorrhage and 80% less extravasated erythrocytes compared to controls. VEGFR2 blockade in vivo results in a 32% of reduction in vein graft size and more stable lesions with significantly reduced macrophage content (30%), and increased collagen (54%) and smooth muscle cell content (123%). Significant decreased VEGF, angiopoietin-2 and increased Connexin 40 expression levels demonstrate increased plaque neovessel maturation in the vein grafts. VEGFR2 blockade in an aortic ring assay showed increased pericyte coverage of the capillary sprouts. CONCLUSION: Inhibition of intraplaque haemorrhage by controlling neovessels maturation holds promise to improve plaque stability.

3D-printed vascular networks direct therapeutic angiogenesis in ischaemia.

Arterial bypass grafts remain the gold standard for the treatment of end-stage ischaemic disease. Yet patients unable to tolerate the cardiovascular stress of arterial surgery or those with unreconstructable disease would benefit from grafts that are able to induce therapeutic angiogenesis. Here, we introduce an approach whereby implantation of 3D-printed grafts containing endothelial-cell-lined lumens induces spontaneous, geometrically guided generation of collateral circulation in ischaemic settings. In rodent models of hind-limb ischaemia and myocardial infarction, we demonstrate that the vascular patches rescue perfusion of distal tissues, preventing capillary loss, muscle atrophy and loss of function. Inhibiting anastomoses between the construct and the host's local capillary beds, or implanting constructs with unpatterned endothelial cells, abrogates reperfusion. Our 3D-printed grafts constitute an efficient and scalable approach to engineer vascular patches able to guide rapid therapeutic angiogenesis and perfusion for the treatment of ischaemic diseases.

Altered rheology of lymphocytes in the diabetic mouse.

AIMS/HYPOTHESIS: Clinical complications associated with diabetes may be related to altered physical properties of leucocytes. We used micropipette techniques to examine leucocyte rheology (specifically lymphocyte rheology) in the non-obese diabetic (NOD) mouse model of diabetes mellitus. We hypothesised that diabetes affects lymphocyte rheology, and specifically that lymphocyte membranes from diabetic mammals have a higher cortical tension than those from non-diabetic mammals. METHODS: Lymphocytes were isolated from diabetic and control mice. Lymphocyte deformation and activation were assessed with a micropipette apparatus. Cellular activation was assessed visually. Projection length into the micropipette during aspiration was used to calculate the viscosity of the cell. Recovery length following expulsion from the micropipette was used to derive the recovery time constant, which is the ratio of cortical tension : viscosity (T(o)/mu) for each cell. The cell cortical/surface tension was calculated from this ratio. RESULTS: Of 692 control lymphocytes, 29% were spontaneously activated compared with 39% of 624 diabetic cells (p<0.06) and 31.5% of 315 non-diabetic NOD cells (p=0.14). Viscosity values for diabetic lymphocytes were equivalent to those for control cells (1345.12+/-1420.97 Pa.s vs 996.84+/-585.07 Pa.s, p=0.13). The average T(o)/micro value for diabetic lymphocytes (35.4+/-16.5x10(-6) cm/s) was significantly higher than that for control cells (24.8+/-11.3x10(-6) cm/s, p<0.03) and cells from non-diabetic NOD mice (26.3+/-9.0x10(-6) cm/s, p<0.005). The mean cortical tension values for diabetic and control cells were 4.7+/-2.3x10(-4) N/m and 2.8+/-0.7x10(-4) N/m respectively (p<0.003). CONCLUSIONS/INTERPRETATION: Lymphocytes from diabetic mice tend to spontaneously activate. They have an equivalent cytoplasmic viscosity but a larger recovery time constant compared with cells from control mice. The results suggest that diabetic lymphocytes are stiffer than control cells.

The outcome in the United States after thoracoabdominal aortic aneurysm repair, renal artery bypass, and mesenteric revascularization.

OBJECTIVES: The purpose of this study was to determine outcome and identify predictors of death after thoracoabdominal aortic aneurysm (TAA) repair, renal artery bypass (RAB), and revascularization for chronic mesenteric ischemia (CMI). PATIENTS AND METHODS: In this retrospective analysis, data were obtained from the Nationwide Inpatient Sample, a 20% all-payer stratified sample of hospitals in the United States during 1993 to 1997. Patients were identified by the presence of a diagnostic or procedure code from the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM). The main outcomes we examined were death, ICD-9-CM -based complications, length of stay, hospital charges, and disposition. A multivariate model was constructed to predict death. RESULTS: A total of 2934 patients were identified (TAA, 540; RAB, 2058; CMI, 336) in the database. The mean age was comparable (TAA, 69 +/- 9 years; RAB, 66 +/- 12 years; CMI, 66 +/- 11 years), but the breakdown between the sexes varied by procedure (male: TAA, 53%; RAB, 55%; CMI, 24%). The mortality rate (TAA, 20.3%; RAB, 7.1%; CMI, 14.7%), complication rate (TAA, 62.2%; RAB, 37.4%; CMI, 44.6%), and the percentage of patients discharged to another institution (TAA, 21.2%; RAB, 9.3%; CMI, 12.0%) were clinically significant for all procedures. The mortality rate for RAB was greater when performed concomitant with an aortic reconstruction (4.4% vs 8.3%). All three procedures were resource intensive as reflected by the median length of stay (TAA, 14 days; RAB, 9 days; CMI, 14 days) and median hospital charges (TAA, $64,493; RAB, $36,830; CMI, $47,390). The multivariate model identified several variables for each procedure that had an impact on the predicted mortality rate (TAA, 14%-76%; RAB, < 1%-46%; CMI, < 2%-87%). CONCLUSIONS: The operative mortality rates across the United States for patients undergoing TAA repair and RAB are greater than commonly reported in the literature and mandate reexamining the treatment strategies for these complex vascular problems.

External-beam radiation therapy for improved dialysis access patency: feasibility and early safety.

Rectenwald, J. E., Pretus, H. A., Seeger, J. M., Huber, T. S., Mendenhall, N. P., Zlotecki, R. A., Palta, J. R., Li, Z. F., Hook, S. Y., Sarac, T. P., Welborn, M. B., Klingman, N. V., Abouhamze, Z. S. and Ozaki, C. K. External-Beam Radiation Therapy for Improved Dialysis Access Patency: Feasibility and Early Safety. Radiat. Res. 156, 53-60 (2001).Prosthetic dialysis access grafts fail secondary to neointimal hyperplasia at the venous anastomosis. We hypothesized that postoperative single-fraction external-beam radiation therapy to the venous anastomosis of hemodialysis grafts can be used safely in an effort to improve access patency. Dogs (n = 8) underwent placement of expanded polytetrafluoroethylene grafts from the right carotid artery to the left jugular vein. Five dogs received single-fraction external-beam photon irradiation (8 Gy) to the venous anastomosis after surgery. Controls were not irradiated. Shunt angiograms were completed 3 and 6 months postoperatively. Anastomoses, mid-graft, and the surrounding tissues were analyzed. Immunohistochemistry for smooth muscle cell alpha-actin, proliferating cellular nuclear antigen (PCNA), and apoptosis was performed. Incisions healed well, though all animals developed wound seromas. One control suffered graft thrombosis 4 months postoperatively. Angiography/histology confirmed severe neointimal hyperplasia at the venous anastomosis. The remaining seven dogs developed similar amounts of neointimal hyperplasia. PCNA studies showed no accelerated fibroproliferative response at irradiated anastomoses compared to controls. Skin incisions and soft tissues over irradiated anastomoses revealed no radiation-induced changes or increase in apoptosis. Thus we conclude that postoperative single-fraction external-beam irradiation of the venous anastomosis of a prosthetic arteriovenous graft that mimics the situation in humans is feasible and safe with regard to early wound healing.

Experience in the United States with intact abdominal aortic aneurysm repair.

OBJECTIVES: The purpose of this study was to determine the current outcome in the United States and to identify predictors of mortality and "bad outcome" after open, intact abdominal aortic aneurysm (AAA) repair. METHODS: In a retrospective analysis, data were obtained from the Nationwide Inpatient Sample during 1994-1996. The Nationwide Inpatient Sample is a 20% all-payer stratified sample of nonfederal United States hospitals. Patients older than 49 years were identified by the presence of primary diagnostic (441.4-intact AAA) and procedure (38.44-resection of abdominal aorta with replacement) codes of the International Classification of Diseases, Ninth Revision (ICD-9 ). In-hospital mortality rate, discharge disposition, bad outcome (death or discharge to an institution), complications (ICD-9 postoperative codes), length of stay, and charges were determined. The mortality rate and bad outcome were analyzed by the use of patient demographics (age, sex, race), patient comorbidities (ICD-9 diagnostic codes), calendar year, and hospital characteristics (size, location, teaching status) with univariate and multivariate analyses. RESULTS: We identified 16,450 intact AAAs repairs during the study years. The mean patient age was 72 +/- 7 (+/- SD) years, and most patients were male (79.7%) and white (94.6%). Most repairs were performed at large (67.3%), urban (92.5%), and nonteaching (66.7%) institutions. The in-hospital mortality rate was 4.2%, the overall complication rate was 32.4%, and 91.2% of patients were discharged home, whereas the bad outcome rate was 12.6%. The median length of stay was 8 days (mean, 10.0 +/- 8.1), and median hospital charges were $28,052 (mean, $35,681 +/- $33,006) in 1996 dollars. Multivariate analysis showed that the mortality rate (P <.05) increased with age (70-79 years, 1.8 odds ratio [OR] [95% CI, 1.4-2.3], > 79 years, 3.8 OR [95% CI, 2.9-4.9]), sex (female, 1.6 OR [95% CI, 1.3-1.9]), cerebral vascular occlusive disease (1.8 OR [95% CI, 1.3-2.5]), preoperative renal insufficiency (9.5 OR [95% CI, 7.7-11.7]), and more than three comorbidities (11.2 OR [95% CI, 3.6-35.4]). Multivariate analysis also showed that bad outcome was associated with the same variables in addition to hospital size (small/medium), year of procedure (1996), chronic obstructive pulmonary disease, and two to three comorbidities. CONCLUSIONS: Outcome after open repair of intact AAA across the United States is quite good. Older, sicker patients may benefit from nonoperative treatment or the potentially lower risk endovascular approaches.

Direct evidence for cytokine involvement in neointimal hyperplasia.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) are proximal inflammatory cytokines that stimulate expression of adhesion molecules and induce synthesis of other proinflammatory cytokines. In addition, TNF-alpha and IL-1 influence vascular smooth muscle cell migration and proliferation in vitro. In view of the inflammatory nature of neointimal hyperplasia (NIH), we tested the hypothesis that endogenous TNF-alpha and IL-1 modulate low shear stress-induced NIH. METHODS AND RESULTS: Mice underwent unilateral common carotid artery (CCA) ligation. Low shear stress in the patent ligated CCA has previously been shown to result in remodeling and NIH. Reverse transcriptase-polymerase chain reaction for TNF-alpha and IL-1alpha mRNA demonstrated both TNF-alpha and IL-1alpha mRNA in ligated CCAs, whereas normal and sham-operated CCAs had none. Mice lacking functional TNF-alpha (TNF-/-) developed 14-fold less neointimal area than WT controls (P:<0.05). p80 IL-1 type I receptor knockout (IL-1RI-/-) mice tended to develop less (7-fold, P:>0.05) neointimal area than WT controls. Furthermore, no IL-1alpha mRNA expression was detected in CCAs from TNF-/- mice; however, TNF-alpha mRNA expression was found in the IL-1RI-/- mice. Mice that overexpress membrane-bound TNF-alpha but produce no soluble TNF-alpha display an accentuated fibroproliferative response to low shear stress (P:<0.05). CONCLUSIONS: These results directly demonstrate that TNF-alpha and IL-1 modulate NIH induced by low shear stress. NIH can proceed by way of soluble TNF-alpha-independent mechanisms. Specific anti-TNF-alpha and anti-IL-1 therapies may lessen NIH.

Long-term outcome after treatment of aortic graft infection with staged extra-anatomic bypass grafting and aortic graft removal.

OBJECTIVE: The purpose of this study was to determine long-term outcome in patients with infected prosthetic aortic grafts who were treated with extra-anatomic bypass grafting and aortic graft removal. METHODS: Between January 1989 and July 1999, 36 patients were treated for aortic graft infection with extra-anatomic bypass grafting and aortic graft removal. Extra-anatomic bypass graft types were axillofemoral femoral (5), axillofemoral (26; bilateral in 20), axillopopliteal (3; bilateral in 1) and axillofemoral/axillopopliteal (2). The mean follow-up was 32.3 +/- 4. 8 months. RESULTS: Four patients (11%) died in the postoperative period, and two patients died during follow-up as a direct consequence of extra-anatomic bypass grafting and aortic graft removal (one died 7 months after extra-anatomic bypass graft failure, one died 36 months after aortic stump disruption). One additional patient died 72 months after failure of a subsequent aortic reconstruction, so that the overall treatment-related mortality was 19%, whereas overall survival by means of life table analysis was 56% at 5 years. No amputations were required in the postoperative period, but four patients (11%) required amputation during follow-up. Twelve patients (35%) had extra-anatomic bypass graft failure during follow-up, and six patients underwent secondary aortic reconstruction (thoracobifemoral [2], iliofemoral [2], femorofemoral [2]). However, with the exclusion of patients undergoing axillopopliteal grafts (primary patency 0% at 7 months), only seven patients (25%) had extra-anatomic bypass graft failure, and only two patients required amputation (one after extra-anatomic bypass graft removal for infection, one after failure of a secondary aortic reconstruction). Furthermore, primary and secondary patency rates by means of life table analysis were 75% and 100% at 41 months for axillofemoral femoral grafts and 64% and 100% at 60 months for axillofemoral grafts. Only one patient required extra-anatomic bypass graft removal for recurrent infection, and only one late aortic stump disruption occurred. CONCLUSIONS: Staged extra-anatomic bypass grafting (with axillofemoral bypass graft) and aortic graft removal for treatment of aortic graft infection are associated with acceptable early and long-term outcomes and should remain a primary approach in selected patients with this grave problem.

Use of superficial femoral vein for hemodialysis arteriovenous access.

Maintaining hemodialysis access in the expanding number of patients with end-stage renal disease is a difficult and challenging problem. Published guidelines outline the initial recommendations for hemodialysis access; however, there is little consensus about the most appropriate options for the subset of patients with repeated access failures and/or unsuitable veins. Two case reports are presented describing the use of composite saphenous-superficial femoral vein autogenous accesses placed in the upper and lower extremities. The function of the autogenous accesses appeared to be similar to a mature arteriovenous fistula in the short-term, although further longitudinal studies are required. The superficial femoral vein may be a useful hemodialysis access conduit for patients with limited access options.

Surgical decision making for carotid endarterectomy and contemporary magnetic resonance angiography.

BACKGROUND: Benefit from carotid endarterectomy (CEA) centers on patient selection and percent stenosis as determined by cerebral angiography. However, angiography remains expensive and poses risks. Validated carotid duplex ultrasonography has proven to be an accurate tool for selecting patients for CEA. However, the role of another noninvasive test-magnetic resonance angiography (MRA)-remains uncertain. Because of recent advances in MRA hardware and software, we hypothesized that clinically appropriate patients could be accurately selected for CEA based on MRA alone. METHODS: Fifty-four carotid arteries in 29 patients (with and without symptoms) underwent both three-dimensional time-of-flight MRA (1.5 Tesla) with multiple overlapping thin slab acquisition and biplanar intra-arterial digital subtraction angiography. All patients undergoing both tests over a 24-month period were included. The majority of these patients did not undergo carotid duplex ultrasound owing to the clinical practice of the hospital's neurosurgery service. Staff radiologists interpreted each study. The accuracy of patient selection based on MRA was calculated using angiography as the standard (NASCET method). Since operative thresholds vary depending on clinical history, we considered four commonly used ranges of percent stenosis for CEA. RESULTS: Patient selection accuracy of MRA alone was low, but increased as percent stenosis increased. Out of 10 occluded arteries by angiography, 5 were interpreted as patent with stenosis (70% to 99%) by MRA. One patent artery was misread as occluded on MRA. CONCLUSION: Reliance solely on contemporary MRA for surgical decision making cannot be justified in view of low accuracy, which leads to high rates of error in patient selection for CEA.

Impact of race on the treatment for peripheral arterial occlusive disease.

PURPOSE: The purpose of this study was to determine the impact of race on the treatment of peripheral artery occlusive disease (PAOD) and to examine the role of access to care and disease distribution on the observed racial disparity. METHODS: The study was performed as a retrospective analysis of hospital discharge abstracts from 1992 to 1995 in 202 non-federal, acute-care hospitals in the state of Florida. The subjects were patients older than 44 years of age who underwent major lower extremity amputation or revascularization (bypass grafting or angioplasty) for PAOD. The main outcome measures were incidence of intervention, incidence per demographic group, multivariate predictors of amputation versus revascularization, multivariate predictors of amputation versus revascularization among those patients with access to sophisticated care (hospital with arteriographic capabilities), and multivariate predictors of surgical bypass graft type (aortoiliac vs infrainguinal). RESULTS: A total of 51,819 procedures (9.1 per 10,000 population) were performed for PAOD during the study period and included 15,579 major lower extremity amputations (30.1%) and 36,240 revascularizations (69.9%). Although the incidence of a procedure for PAOD was comparable between African Americans and whites (9.0 vs 9.6 per 10, 000 demographic group), the incidence of amputation (5.0 vs 2.5 per 10,000 demographic group) was higher and the incidence of revascularization (4.0 vs 7.1 per 10,000 demographic group) was lower among African Americans. Furthermore, multivariate analysis results showed that African Americans (odds ratio, 3.79; 95% confidence interval [CI], 3.34 to 4.30) were significantly more likely than whites to undergo amputation as opposed to revascularization. The secondary multivariate analyses results revealed that African Americans (odds ratio, 2.29; 95% CI, 1.58 to 3. 33) were more likely to undergo amputation among those patients (n = 9193) who underwent arteriography during the procedural admission and to undergo infrainguinal bypass grafting (odds ratio, 2.00; 95% CI, 1.48 to 2.71) among those patients (n = 27,796) who underwent surgical bypass grafting. CONCLUSION: There is a marked racial disparity in the treatment of patients with PAOD that may be caused in part by differences in the severity of disease or disease distribution.

Potential predictors of outcome in patients with tissue loss who undergo infrainguinal vein bypass grafting.

PURPOSE: Aggressive attempts at limb salvage in patients with ischemic tissue loss are justified by favorable initial results in most patients. The identification of patients whose conditions will not benefit from attempted revascularization remains difficult. METHODS: This study was designed as a retrospective review of prospectively collected clinical data. The subjects were 210 consecutive patients who underwent infrainguinal vein bypass grafting for ischemic tissue loss in the setting of an academic medical center. Bypass grafting was to the popliteal artery in 56 patients, to the infrapopliteal arteries in 131 patients, and to the pedal arteries in 23 patients. The follow-up examination was complete in 209 of 210 patients. One hundred twenty-five patients underwent blinded review of duplex scan venous mapping and arteriography to determine simplified vein and run-off scores. The outcome measures were the influence of risk factors, venous conduit, and runoff on mortality, limb loss, and graft failure at the 6-month follow-up examination. RESULTS: One hundred seventy patients (81%) were alive and had limb salvage. Nineteen patients (9.1%) died, with need for a simultaneous inflow procedure and end-stage renal disease being most commonly associated with mortality. Thirty-three patients (15.8%) had undergone amputation: 18 after graft failure, and 15 for progressive tissue loss despite a patent graft. Amputation was significantly more common in patients with diabetes (P =.05) and with poor runoff scores (poor runoff, 44.4% vs good runoff, 7.4%; P <.01). Amputation despite a patent graft also correlated with runoff (poor runoff, 41.7% vs good runoff, 4.3%; P <.01). Twenty-five patients had graft failure without amputation, so that only 145 patients (69.4%) were alive, had limb salvage, and had a patent graft. Run-off score was the strongest predictor of outcome, with 70% of patients with poor run-off scores having death, amputation, or graft failure. CONCLUSION: Aggressive use of infrainguinal vein bypass grafting in patients with ischemic tissue loss results in a high rate of initial limb salvage but significant morbidity and mortality. Arteriographically determined runoff scores appear to potentially identify patients at high risk for a poor initial outcome and may provide a method of selecting patients for primary amputation.

Warfarin improves the outcome of infrainguinal vein bypass grafting at high risk for failure.

OBJECTIVE: Patients with marginal venous conduit, poor arterial runoff, and prior failed bypass grafts are at high risk for infrainguinal graft occlusion and limb loss. We sought to evaluate the effects of anticoagulation therapy after autogenous vein infrainguinal revascularization on duration of patency, limb salvage rates, and complication rates in this subset of patients. METHODS: This randomized prospective trial was performed in a university tertiary care hospital and in a Veterans Affairs Hospital. Fifty-six patients who were at high risk for graft failure were randomized to receive aspirin (24 patients, 27 bypass grafts) or aspirin and warfarin (WAR; 32 patients, 37 bypass grafts). All patients received 325 mg of aspirin each day, and the patients who were randomized to warfarin underwent anticoagulation therapy with heparin immediately after surgery and then were started on warfarin therapy to maintain an international normalized ratio between 2 and 3. Perioperative blood transfusions and complications were compared with the Student t test or with the chi2 test. Graft patency rates, limb salvage rates, and survival rates were compared with the Kaplan-Meier method and the log-rank test. RESULTS: Sixty-one of the 64 bypass grafts were performed for rest pain or tissue loss, and 3 were performed for short-distance claudication. There were no differences between the groups in ages, indications, bypass graft types, risk classifications (ie, conduit, runoff, or graft failure), or comorbid conditions (except diabetes mellitus). The cumulative 5-year survival rate was similar between the groups. The incidence rate of postoperative hematoma (32% vs 3.7%; P = .004) was greater in the WAR group, but no differences were seen between the WAR group and the aspirin group in the number of packed red blood cells transfused, in the incidence rate of overall nonhemorrhagic wound complications, or in the overall complication rate (62% vs 52%). The immediate postoperative primary graft patency rates (97.3% vs 85.2%) and limb salvage rates (100% vs 88.9%) were higher in the WAR group as compared with the aspirin group. Furthermore, the cumulative 3-year primary, primary assisted, and secondary patency rates were significantly greater in the WAR group versus the aspirin group (74% vs 51%, P = .04; 77% vs 56%, P = .05; 81% vs 56%, P = .02) and cumulative limb salvage rates were higher in the WAR group (81% vs 31%, P = .01). CONCLUSIONS: Perioperative anticoagulation therapy with heparin increases the incidence rate of wound hematomas, but long-term anticoagulation therapy with warfarin improves the patency rate of autogenous vein infrainguinal bypass grafts and the limb salvage rate for patients at high risk for graft failure.

Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice.

BACKGROUND: Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. METHODS AND RESULTS: We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). CONCLUSIONS: PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.

Bypass grafting for chronic mesenteric ischemia.

A critical analysis of the literature suggests that there is no clearly superior technique for mesenteric revascularization and that the choice of operation must be individualized. Bypass grafting using either an antegrade or retrograde technique with prosthetic or autogenous conduits should produce excellent long-term results for most patients with this complex surgical problem. In most situations multiple vessel revascularizations are preferred. Surgeons caring for such patients must have the ability to utilize all available techniques to ensure optimal outcomes.

Platelet activation by healing ePTFE grafts.

The purpose of this study was to examine the effect of the in vivo maturing ePTFE graft surface on platelet activation. Ten canines were randomized to receive either a carotid to infrarenal aorta ePTFE graft or sham operation. Animals were sampled at specific time points up to 3 months postoperatively. Whole blood platelet aggregometry (arachidonic acid, ADP, and collagen agonists) and ATP secretion (in response to arachidonic acid, ADP, collagen, and thrombin) were measured. Additionally, complete hematologic analysis and histology were performed. With time, graft animals showed significantly more decrease in platelet aggregation in response to ADP compared to sham animals (P = .023). The total amount of ATP per platelet was not different, as demonstrated by equivalent ATP release per platelet in response to thrombin. Over the first week, grafted dogs developed a decrease in systemic platelet count of 50% (P < .001) that persisted over the 3-month follow-up period. With time, overall regression model slopes of graft and sham platelet count data were not statistically different (P = .29). Histologically, the grafts demonstrated limited cellular ingrowth at both anastomoses, with fibrin matrix along the remainder of the blood-biomaterial interface. These data suggest that, similar to Dacron, exposure to an ePTFE surface results in significant changes in platelet biology, and these platelet-ePTFE interactions persist even after the graft has formed a mature pseudointima. The pseudointima appears to be the primary determinant of the blood-biomaterial interaction.

Isolation of genes differentially expressed at the downstream anastomosis of prosthetic arterial grafts with use of mRNA differential display.

PURPOSE: Downstream anastomotic intimal hyperplasia in prosthetic arterial grafts remains a major cause of delayed graft failure. The new method of messenger RNA (mRNA) differential display was used to screen numerous genes to gain insight into the molecular mechanisms of intimal hyperplasia. METHODS: Fifty-centimeter-long 8 mm expanded polytetrafluoroethylene grafts were placed in four mongrel dogs from the carotid artery to the distal abdominal aorta. At 3 months the distal anastomoses and adjacent normal aortas were harvested; a portion was taken for histologic examination, and total RNA was isolated from the remainder. Differential mRNA display was used to identify candidate cDNA clones whose expression differed in anastomotic intimal hyperplasia as compared with adjacent unaffected aorta. The clones were sequenced, and national gene databases were searched. Northern blot analysis confirmed alteration of gene expression. RESULTS: Approximately 5000 mRNA species were screened, and 11 candidate clones were obtained. DNA sequence revealed homology of five clones to known gene sequences. Homologous genes included an interferon-gamma-induced human gene, (IGUP I-5111), alpha-1 protease inhibitor gene, human retinoblastoma susceptibility gene, and human creatine kinase gene (two clones). Northern blot analysis revealed altered gene expression in 4 of 11, nonregulation in 1 of 11, and undetectable signals in 6 of 11. Expression of the clone representing IGUP I-5111 in the segment of intimal hyperplasia was found to be decreased over threefold to only 31% +/- 16.4% SE of the level seen in normal aorta. CONCLUSIONS: The technique of mRNA differential display has identified differences in gene expression in an in vivo model of anastomotic intimal hyperplasia. Expression of RNA with homology to an interferon-gamma-induced human gene was consistently decreased within the hyperplastic region at the downstream polytetrafluoroethylene arterial anastomosis.

Covalent linkage of streptokinase to recombinant hirudin: a novel thrombolytic agent with antithrombotic properties.

In a continuing effort to create an agent which has both thrombolytic and antithrombotic properties, streptokinase (SK) was covalently bound to the potent antithrombin agent recombinant hirudin (rHir). Linkage of SK to 125I-rHir was accomplished via heterobifunctional crosslinkers in an average molar ratio of 1:1. The 125I-rHir-SK complex was purified from starting components by anion exchange and gel filtration chromatography. The major band containing covalently bound 125I-rHir had a molecular weight of 53 kDa as determined by SDS-PAGE and autoradiography. Biologic activity of each component was then assayed utilizing the chromogenic substrate for each compound. Complex bound 125I-rHir exhibited a 1.2 fold decrease in thrombin inhibition when compared to concentrations of 125I-rHir greater than 3.13 nM. Complex bound 125I-SK, replacing the 125I label on rHir, displayed a 7.9-fold loss in plasminogen activation when compared to 125I-SK. These chromogenic assay results were not adversely altered in the presence of the converse compound's substrate. The 125I-SK-rHir complex (examined at various concentrations) also demonstrated a 0.17- to 17-fold greater affinity for thrombin immobilized onto Sepharose beads as compared to 125I-SK. These findings indicate the rHir-SK complex maintained both thrombolytic and antithrombin properties while also obtaining affinity for immobilized thrombin.

In vivo testing of an infection-resistant vascular graft material.

Present prosthetic arterial conduits continue to suffer the clinically and economically catastrophic complication of infection. We recently described a novel technique for binding quinolone antibiotics to Dacron based on principles of textile chemistry. This thermofixation procedure ("pad/heat") utilizes the limited fibrophilic characteristics of the quinolone antibiotic ciprofloxacin (Cipro) to permit pad/heat application and allowed controlled, sustained release from Dacron in several in vitro assays. The objective of this study was to test this infection-resistant prosthetic vascular graft material in an in vivo model. Dacron segments (1 cm2, either plain, dipped into antibiotic immediately prior to implantation, or Cipro pad/heat treated) were implanted in the dorsal subcutaneous tissue of the rabbit and directly contaminated with 10(6) Staphylococcus aureus. After 1 week, the samples were sterily harvested. Wounds were blindly graded on a scale from 1 (no evidence of infection, good tissue incorporation) to 4 (suppurative infection extending outside of the graft pocket, no gross tissue incorporation). Plain Dacron was easily infected in this model (mean grade 3.1 +/- 0.6, 92% culture positive). Notably, however, a significant (P < 0.05) wound grade difference between the dipped (2.3 +/- 1.0) and pad/heat (1.4 +/- 0.6) samples was demonstrated. Determination of adherent bacteria present on the implanted Dacron pieces by sonication and culture studies again revealed a significant difference between the dipped (56% culture positive) and pad/heat (12% culture) groups (P < 0.025). Histologic studies confirmed good tissue incorporation of the pad/heat samples. This project opens new avenues in the development of infection-resistant biomaterials.