Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness

OBJECTIVE: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. METHODS: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm2 shear stress and then for an additional 3 days with 1 or 25 dyne/cm2 shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with beta-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. RESULTS: The pretreatment with 25 dyne/cm2 but not with 0 or 1 dyne/cm2 shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that beta-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained alpha-smooth muscle actin were present. The thickness of this neointima averaged 41 +/- 12 micrometer and 60 +/- 23 micrometer in endothelial cell-seeded grafts that were pretreated with 25 dyne/cm2 shear stress and 1 dyne/cm2 shear stress, respectively, and 158 +/- 46 micrometer in grafts that were not seeded with endothelial cells. CONCLUSION: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness.     

Performance of small diameter synthetic vascular prostheses with confluent autologous endothelial cell linings

Autologous grafts are superior to their synthetic counter-parts for grafting arteries smaller than 6-mm diameter both in terms of acute thrombogenicity and chronic intimal hyperplasia. Endothelial cell (EC) coating of the blood contacting surface may reduce thrombogenicity of synthetic small diameter vascular prostheses. In this study, the survival of EC monolayers on synthetic 4-mm diameter arterial prostheses over short-term implantations (< or = 6 weeks) was examined. Graft types examined were expanded polytetra-fluoroethylene (ePTFE) and microporous polyurethane (PU). Lumenal coverage with ECs was achieved by culturing ovine ECs on prostheses treated by either physical adsorption or covalent binding of ovine fibronectin (Fn). An ovine carotid interposition model was used to examine the performance of EC coated ePTFE and microporous PU over implantation periods of 1, 3, and 6 weeks. Outcomes assessed at the end of each experiment were graft patency, area covered by ECs, and thrombus free surface area (TFSA). Fn concentration, cell density at the time of coating and prostacyclin production in vitro were similar for both graft types. Occlusion occurred more frequently in unseeded grafts compared with EC coated grafts over 3 and 6 week implantation periods; however, the difference was not significant (p = 0.099). In prostheses precoated with ECs, approximately 40-60% of the surface area remained covered with endothelial-like cells following the first postoperative week. Recovery of EC layers occurred rapidly thereafter with 80-90% coverage at 3 weeks. TFSA remained low in comparison to EC cover in these prostheses until between 3 and 6 weeks postoperatively, suggesting a lag phase in recovery of EC function of seeded cells. In contrast, EC cover of unseeded prostheses only achieved 10-30% at 3 weeks, primarily by pannus EC ingrowth from the adjacent artery. TFSA of unseeded grafts increased in direct proportion to EC cover over time suggesting that there was no lag phase in function of these ingrowing cells.     

Urokinase expression in transduced endothelial cells

Increased thromboresistance through the release of lytic agents by endothelial cells may improve the patency of endothelial lined prosthetic grafts. We have evaluated the expression of urokinase from cells transduced with a retrovirus containing the gene for a human preprourokinase. Endothelial cells were enzymatically harvested from canine external jugular vein in nine animals and grown to confluence in culture. One-third of these cells served as controls, and the remaining two-thirds were transduced via incubation with an LXSN-type retroviral vector carrying the urokinase gene and a neomycin resistance gene. Successfully transduced cells were selected by incubation with 400 micrograms/mL G418 and pure cultures grown to confluence. Supernatants from confluent control and experimental cell cultures after 48 hours in defined, serum-free medium were assayed for human urokinase concentration and overall enzyme activity. ELISA quantitation of concentration using mouse antihuman urokinase antibody showed 0.15 +/- 0.11 ng/mL/hr/10(6) cells in the transduced cell supernatant; no measurable concentration was found in the control cells. (P < 0.01) Overall (human plus canine) enzyme activity of urokinase was determined using an indirect spectrophotometric assay based on plasminogen activation (ploug U/mL). Transduced cells showed activities of 0.12 at 10 days and 0.45 at confluence; control cell activity was 0.0 and 0.15, respectively. (P < 0.05) These data show that endothelial cells can be transduced with a urokinase expressing gene that increases the release of this thrombolytic agent. Lining small diameter prosthetic grafts with these cells may improve their thromboresistance and long-term patency.     

Thiamine deficiency-induced disruptions in the diurnal rhythm and regulation of body temperature in the rat

A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells. Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining. In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced. However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells. The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.     

Contributions of the most ancient medical schools to cardiology

Endothelial seeding of vascular grafts has been shown to decrease graft thrombogenicity and prolong longevity when implanted in vivo. Previous studies have utilized anatomic grafts to study endothelialization and healing. Anatomic thoracoabdominal grafts do not allow for sequential biopsy for evaluation of individual grafts nor do they approximate the environment for long bypass grafts used in limb salvage. This study evaluated the use of an extra-anatomic aortic bypass graft to assess the healing of endothelial cell seeded expanded polytetrafluoroethylene (ePTFE). Radionuclide angiography was used to evaluate graft patency and quantify blood flow through the graft. Dogs underwent placement of an extra-anatomic 60 cm long, 8 mm internal diameter, graft seeded with autologous endothelium. Grafts were biopsied from 2 weeks up to 1 year. Radionuclide studies were performed postimplantation and following each graft biopsy. Graft placement and biopsies were well tolerated in all dogs. Biopsied segments of graft allowed for sequential studies of the healing of implanted grafts by scanning electron and light microscopy. Flow through the implanted graft was close to 50% of the total caudal abdominal aortic flow. No significant difference in graft flow was noted either between animals or over time.     

Improving endothelial cell adhesion to vascular graft surfaces: clinical need and strategies

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to existing therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p < 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm(-2). HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mnm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.     

The prevention and treatment of postoperative thrombosis of the deep veins of the lower extremities (II. Treatment)

Defining the most appropriate conditions for strengthening the retention of endothelial cells (ECs) by small-diameter prosthetic endothelialized grafts is indispensable to their clinical application. The incubation time after seeding is one of the most important factors in EC retention. The effects of different postincubation times (0, 2, 4, 8, 16, 24, and 36 hr) on EC monolayers on two different types of graft, fibronectin-coated expanded polytetrafluoroethylene (ePTFE) and collagen-coated knitted Dacron grafts (4 mm x 5 cm) were examined. In situ counting of ECs on the grafts was performed by light microscopy. The percentage cell retention was calculated by dividing the cell counts for grafts exposed to pulsatile flow for 90 min by those for control grafts. To characterize the EC coverage of the grafts, scanning electron microscopy was also performed. The average cell density of control grafts ranged from 5.59 +/- 1.1 to 6.69 +/- 1.5 x 10(4) cells/cm2 and did not differ according to the kind of graft or incubation time. The knitted Dacron grafts showed the maximal cell retention (88 +/- 5%) after incubation for 8 hr, whereas ePTFE grafts did so after 24 hr (83 +/- 6%). Scanning electron microscopic examination after incubation for 8 hr revealed that the density of human ECs on the surfaces of ePTFE and Dacron grafts differed, although there was no morphological difference between the ECs on the two types of graft. Knitted Dacron grafts achieved a high percentage retention in a shorter time than ePTFE grafts.     

Frequency dependent force generation correlates with sarcoplasmic calcium ATPase activity in human myocardium

The purpose of this study was to implement and evaluate a clinical protocol for following longitudinally the luminal responses of microvessel cell seeded expanded polytetrafluoroethylene (ePTFE) vascular grafts implanted for hemodialysis access. Half of the patients enrolled in the study were randomized to receive grafts that were "seeded" with transplanted microvessel cells derived from autologous subcutaneous fat; the other half of the patients received nonseeded grafts. The patients agreed to scheduled biopsies of their grafts at three postoperative times. All biopsy samples were evaluated by routine histologic and electron microscopy techniques. Three men and six women were enrolled in the study. All operative procedures were tolerated well. However, only two of the nine patients agreed to 1-year postimplantation biopsies; one of these patients had been randomized to receive a "nonseeded" ePTFE graft and one randomized to receive a "seeded" graft. The "seeded" graft at 3 months showed endothelial cells on the luminal surface as well as some intimal thickening. By 20 months, the same "seeded" graft showed significant concentric intimal thickening and by 24 months, this "seeded" graft thrombosed. The "nonseeded" graft at 16 months had irregular areas of intimal thickening which were quite patchy in nature. The flow contacting surface of the "nonseeded" graft remained thin. The intima of the "seeded" graft was twice as thick as that of the "nonseeded" graft. The methodologies implemented in the study design were appropriate. Biopsy samples were obtained without complication and were easily processed for analysis. Patient compliance with the biopsy protocol was problematic however. The study was terminated because of the development of significant concentric intimal hyperplasia in a "seeded" graft.     

Prophylactic balloon angioplasty fails to prolong the patency of expanded polytetrafluoroethylene arteriovenous grafts: results of a prospective randomized study

PURPOSE: Maintenance of hemodialysis access grafts represents an enormous social and clinical problem. Current grafts and graft salvage techniques are inadequate. Consequently, there has been increasing interest in the use of minimally invasive catheter techniques to prophylactically treat stenoses in functioning arteriovenous grafts. Prophylactic balloon angioplasty has been widely suggested as prolonging assisted primary patency. We have performed a prospective randomized trial to compare patients who underwent percutaneous transluminal angioplasty (PTA) for graft stenoses > 50% with a control group that received no intervention. Our hypothesis was that to be efficacious a minimal benefit of 20% prolongation in patency would be necessary. METHODS: Color flow duplex scanning was used to detect > 50% stenoses in functioning expanded polytetrafluoroethylene grafts. Patients were then subjected to confirmatory angiographic evaluation. Those who had angiographic stenoses > 50% were randomized to balloon angioplasty or observation. Patients were followed-up with duplex scanning every 2 months. Statistical analysis was performed using the Kaplan-Meier technique. Although demographically the patient groups were well matched, there were more prior interventions and concurrent central stenoses in the treatment group. Outcomes were graft thrombosis, graft dysfunction that precluded dialysis, and six or more PTA procedures within 18 months. RESULTS: In the treatment and observation groups, the 6-month patency rates were 69% +/- 7% and 70% +/- 7%, respectively. The 12-month patency rates for the treatment and observation groups were 51% +/- 6% and 47% +/- 4%, respectively. There was no significant difference between these two groups (p = 0.97), with an 80% confidence limit for detection of a difference greater than 20%. CONCLUSIONS: This study demonstrates that a generic approach of PTA to treat all polytetrafluoroethylene grafts with stenoses > 50% does not prolong patency and cannot be supported.     

Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector

Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.01 to ensure 1 proviral copy/genome, were selected with either G418 for neo expression or colchicine for MDR1 expression. The titer determined on HeLa cells with G418 selection was eight-fold higher than that with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based viral titer values for CTAC cells. The transduced HeLa cells, with one intact proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold higher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-glycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This could explain the difference in viral titers obtained on CTAC cells but not that obtained on HeLa cells. The vector-mediated increase in expression of P-gp was about 20-fold higher in CTAC cells as compared with HeLa cells. These results indicated suppression of expression of vector-derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigate further the possible reasons for this difference, genomic DNA was isolated from the G418-resistant individual colonies of infected cells and analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detected aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberrant forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA sequencing across the 2-kb and the 0.7-kb deletion junction suggests cryptic splicing in the producer cell line as the origin of these deletions. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via donor (codon 113) and acceptor (codon 773). The 0.7-kb deletion corresponds to splicing via the same donor and a different acceptor (codon 344). When transduced HeLa cells were selected at a higher concentration of G418 (3 mg/ml), the aberrant forms were detected at an increased frequency of about 50% of colonies tested. These results indicate that vector-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bicistronic Mo-MuLV vector, may provide an advantage for expression of the second transgene in HeLa cells.     

Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.     

Thrombogenesis of different cell types seeded on vascular grafts and studied under blood-flow conditions

BACKGROUND: Small-diameter vascular grafts tend to have an early and high occlusion rate. Cell seeding on the luminal surfaces of small-diameter vascular prostheses may provide an antithrombotic lining and improve both the short-term and the long-term patency rates. We studied the net results of procoagulant and anticoagulant properties of seeded grafts under blood-flow conditions, and we compared the different available types of donor cells. METHODS: Monolayers of liposuction-derived cultured human microvascular endothelial cells (MVECs), human adult endothelial cells (HAECs), human umbilical vein endothelial cells (HUVECs), and human mesothelial cells (MCs) that had been seeded on expanded polytetrafluoroethylene (ePTFE) grafts were perfused with marginally anticoagulated blood (20 U/mL low molecular weight heparin; shear rate, 400/s, 10 minutes) or with non-anticoagulated blood (shear rate, 100/s, 5 minutes). The thrombin and fibrin generation in time was studied with the measurement of the plasma levels of prothrombin fragment 1 and 2 (F 1+2) and of fibrinopeptide A (FPA). The plain ePTFE graft was taken as a control. RESULTS: When the seeded MCs were perfused with recirculating anticoagulated blood, a linear generation of F 1+2 in time was seen, with high levels of F 1+2 and FPA after 10 minutes (4.38 nmol/L and 362 ng/mL, respectively). Allopurinol was added, and the MCs generated less F 1+2 than the HAECs (0.7 nmol/L vs 1.86 nmol/L; P <.05). No fibrin formation was seen. The MVECs generated low amounts of F 1+2 (0.7 nmol/L; 10 minutes), and the HUVECs and the plain ePTFE graft generated the lowest amounts of F 1+2 (0.26 and 0.25 nmol/L, respectively). When the MCs were perfused with non-anticoagulated blood, high amounts of thrombin and fibrin were generated immediately and constantly and could not be decreased with allopurinol. The perfusion of the plain ePTFE graft showed a dramatic increase in F 1+2 and FPA levels towards the end of the experiments. The seeded HAECs, HUVECs, and MVECs inhibited this increase. These results were confirmed by means of scanning electron microscopy. CONCLUSION: Vascular prostheses that are seeded with cultured MCs are highly procoagulant. Standard ePTFE graft prostheses also initiate coagulation, which supports the idea of cell seeding. The endothelial cells, of which the MVECs are the most readily available, seem to preserve their anticoagulant properties after being seeded on vascular grafts.     

Reconstruction of the superior vena cava: benefits of postoperative surveillance and secondary endovascular interventions

PURPOSE: Superior vena cava (SVC) reconstructions are rarely performed; therefore the need for surveillance and the results of secondary interventions are unknown. METHODS: During a 14-year period 19 patients (11 male, 8 female; mean age 41.9 years, range 8 to 69 years) underwent SVC reconstruction for symptomatic nonmalignant disease. Causes included mediastinal fibrosis (n = 12), indwelling foreign bodies (n = 4), idiopathic thrombosis (n = 2), and antithrombin III deficiency (n = 1). Spiral saphenous vein graft (n = 14), polytetrafluoroethylene (n = 4), or human allograft (n = 1) was implanted. RESULTS: No early death or pulmonary embolism occurred. Four early graft stenoses or thromboses (spiral saphenous vein graft, n = 2, polytetrafluoroethylene, n = 2) required thrombectomy, with success in three. During a mean follow-up of 49.5 months (range, 4.7 to 137 months), 95 imaging studies were performed (average, five per patient; range, one to 10 studies). Venography detected mild or moderate graft stenosis in seven patients; two progressed to severe stenosis. Two additional grafts developed early into severe stenosis. Four of 19 grafts occluded during follow-up (two polytetrafluoroethylene, two spiral saphenous vein graft). Computed tomography failed to identify stenosis in two grafts, magnetic resonance imaging failed to confirm one stenosis and one graft occlusion, and duplex scanning was inconclusive on graft patency in 10 patients. Angioplasty was performed in all four patients with severe stenosis, with simultaneous placement of Wallstents in two. One of the Wallstents occluded at 9 months. Repeat percutaneous transluminal angioplasty was necessary in two patients, with placement of Palmaz stents in one. Only one graft occlusion and one severe graft stenosis occurred beyond 1 year. The primary, primary-assisted, and secondary patency rates were 61%, 78%, and 83% at 1 year and 53%, 70%, and 74% at 5 years, respectively. CONCLUSION: Long-term secondary patency rates justify SVC grafting for benign disease. Postoperative surveillance with contrast venography is indicated in the first year to detect graft problems. Endovascular techniques may salvage and improve the patency of SVC grafts.     

Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo

A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2-dependent NK-92 and IL-2-independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 and neor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/10(6) cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.     

Percutaneous angioplasty for infrainguinal graft-related stenoses

OBJECTIVE: To assess the success of percutaneous transluminal angioplasty (PTA) in treating infrainguinal graft-related stenoses. DESIGN: Retrospective analysis of stenoses undergoing PTA over 6 years. MATERIALS: Fifty-seven stenoses in 42 grafts. METHODS: Site, length and type of stenoses recorded. Follow-up till discharge, graft occlusion or death. RESULTS: PTA was successful in 48/57 stenoses in 36 grafts (G), with a poor result in seven. Further PTA was required in seven stenoses (7 G). One graft occluded at PTA and one stenosis was inaccessible. Overall graft (G) patency (median 13 months) was 82% (1 year patency 84%). Of 48 successful PTAs (37 G), 36 remained patent (28 G), eight (4 G) occluded and four were lost to follow-up (4 G). Fourteen of thirty-six stenoses which remained patent required further intervention (seven PTA, six jump grafts, one vein patch). The four occlusions were associated with small veins (two), multiple stenoses (one) and a PTFE graft which occluded 10 days following PTA. Of the seven PTAs with a poor angiographic result, five remained patent, three after further intervention. CONCLUSION: PTA is the best treatment for localised stenoses. Stenoses > 2 cm or multiple (three or more) stenoses are best treated surgically. Follow-up is essential, as 20% require further intervention.     

Surgical treatment of threatened reversed infrainguinal vein grafts

PURPOSE: Current information concerning the results of surgical revision of threatened infrainguinal vein grafts is largely limited to in situ conduits. Infrainguinal grafts may be threatened by intrinsic graft lesions or significant stenosis in the adjacent inflow or outflow arteries. To assess the results of operative revision of infrainguinal reversed vein grafts, we reviewed our experience with surgical revision of threatened infrainguinal reversed vein grafts identified through a program of postoperative clinical and vascular laboratory graft surveillance. METHODS: All patients who underwent surgical revision of a threatened but patent infrainguinal reversed vein graft from January 1987 through April 1993 were identified through review of our vascular registry. Data were analyzed for type of vein used, date of original reversed vein graft, clinical and vascular laboratory findings leading to reversed vein graft revision, results of preoperative angiography, patient risk factors, operative techniques and complications, and long-term assisted primary graft patency and limb salvage. RESULTS: Ninety-six patients with 100 infrainguinal reversed vein grafts (69) femoral-popliteal, 31 femoral-tibial) underwent 117 surgical vein graft revisions or inflow procedures during the study period. Eighty-one percent of the original reversed vein grafts consisted of a single segment of greater saphenous vein. All revised grafts had at least a 50% stenosis in the graft itself or the proximal or distal artery. A single revision was performed in 85 grafts, two revisions in 13 grafts, and three revisions in two grafts. There were nine (8%) isolated inflow procedures, eight (7%) vein patch angioplasties, 62 (53%) interposition vein grafts, and 29 (25%) vein graft extensions to a new distal anastomotic site. The remaining nine (8%) procedures consisted of combinations of the above. Median time to primary graft revision after initial graft implantation was 15 months (range 2 days to 316 months). Mean time to secondary revision after primary revision was 21 months. Operative mortality was 0.9%. Cumulative assisted primary patency of the original grafts revised for stenotic lesions was 99%, 96%, and 92% at 1, 3, and 5 years, respectively. Limb salvage was 99%, 97%, and 97% at 1, 3, and 5 years, respectively. CONCLUSIONS: Although surgical revision of reversed vein graft requires much use of alternative vein sources, these procedures can be performed with minimum mortality and provide excellent assisted primary graft patency and limb salvage.     

Cell adhesion and short-term patency in human endothelium preseeded 1.5-mm polytetrafluoroethylene vascular grafts: an experimental study

It has been shown that endothelialization improves short-term patency of 1.5-mm expanded polytetrafluoroethylene vascular grafts. A model for endothelialization of 1.5-mm expanded polytetrafluoroethylene vascular grafts with human endothelial cells is described. In this model, the adherence of endothelial cells was increased significantly in grafts coated with serum proteins and collagen. By means of this protocol, graft patency was tested after implantation in two animal models: the rat aorta and the rabbit common carotid artery. Anastomosis was performed with a 3M Precise Microvascular Anastomotic System. In both animal models, no significant loss of endothelial cells in the precoated grafts (rat, n = 8) were noted 1 hour after blood flow restoration. All uncoated grafts showed significant endothelial cell loss. In the rabbit model, all nonendothelialized grafts (n = 8) clotted 5 to 25 minutes after flow restoration. Seven (n = 8) endothelialized grafts showed no clotting during 1 hour's observation: one clotted immediately for a patency rate of 87.5 percent. These results indicate that endothelialization of 1.5-mm grafts is practical. Furthermore, adhesion of endothelial cells to the graft walls is not affected by short-term, pulsatile, high-pressure blood flow.     

A review of Lewy body disease, an emerging concept of cortical dementia

Small-diameter vascular grafts rapidly fail after implantation, due to occlusion caused by thrombosis. This problem cannot be overcome using medication. A promising improvement of graft patency is the seeding of endothelial cells (EC) on the luminal surface of the vascular graft. Conjugates of albumin and heparin, which were developed to obtain nonthrombogenic coatings, could form an ideal coating for vascular grafts. Besides presenting anticoagulant function, heparin will bind proteins with cell adhesive properties, thus facilitating adherence of EC to the graft surface. EC were able to grow to confluency on CO(2) gas plasma-treated polystyrene (PS-CO(2)) coated with albumin-heparin conjugate. CO(2) gas plasma treatment resulted in the introduction of functional groups at the surface (e.g., hydroxyl, aldehyde, carboxylic acid, and epoxide groups). Addition of albumin-heparin conjugate to the functionalized surface in an aqueous solution with pH 8.2 yielded a stable monolayer of covalently bound conjugate. The number of cells adhering and proliferating on this surface was comparable to the number of cells on fibronectin-coated PS-CO(2). However, the structure and size of EC proliferating on surface-immobilized albumin-heparin was more irregular. Long-term adherence might be improved by adding fibronectin to the albumin-heparin surface, either as a mixture with albumin-heparin or in a separate incubation step.     

Effects of omental wrap on performance of small-caliber high-porosity expanded polytetrafluoroethylene grafts

The purpose of this study was to evaluate the effects of omental wrap on the performance of small-caliber expanded polytetrafluoroethylene (ePTFE) grafts, with a special reference to transmural capillary ingrowth. High-porosity ePTFE grafts with a fibril length of 60 micrometer, an internal diameter of 4 mm, and 40 mm in length were implanted into the canine bilateral carotid arteries. The grafts on the right side were wrapped in an omental pedicle flap (omentum-wrap grafts), while those on the left were not (nonwrap grafts). The grafts were retrieved at intervals of 4 and 12 weeks. At 4 weeks, the patency rates of the omentum-wrap grafts and nonwrap grafts were almost the same (5/6 vs 6/6). At 12 weeks, patency tended to be higher in the omentum-wrap grafts than in the nonwrap grafts (3/5 vs 1/5). At 4 weeks, the thrombus-free surface score (TFS) was significantly higher in the omentum-wrap grafts than in the nonwrap grafts (44.8% vs 30.2%, P < 0.05). At 12 weeks, the TFS tended to be higher in the omentum-wrap grafts than in the nonwrap grafts (84.6% vs 62.4%). At 4 weeks, both the capillary transsectional area score (CTS) and capillary density were significantly higher in the omentum-wrap grafts than in the nonwrap grafts (CTS, 3.3% vs 1.1%, P < 0.05; capillary density, 196.0/mm2 vs 83.3/mm2, P < 0.05). At 12 weeks, both CTS and capillary density tended to be higher in the omentum-wrap grafts than in the nonwrap grafts (CTS, 1.1% vs 0%; capillary density, 69.4/mm2 vs 0/mm2). At 4 weeks, in the omentum-wrap grafts, extracellular matrices and cells in the interstices of the graft were stained with an antibody against VEGF. In conclusion, the omental wrap enhances transmural capillary ingrowth and thereby promotes endothelialization in small-caliber high-porosity ePTFE grafts. VEGF appears to play an active role in transmural capillary ingrowth enhanced by omental wrap.