Dual cell seeding and the use of zymogen tissue plasminogen activator to improve cell retention on polytetrafluoroethylene grafts.

OBJECTIVE: The purpose of this study was to enhance the retention of seeded endothelial cells (EC) on prosthetic vascular grafts. Dual-layer EC and smooth muscle cell (SMC) seeding and gene transfer of a zymogen tissue plasminogen activator gene (tPA) into seeded EC were studied. METHODS: Polytetrafluoroethylene (PTFE) grafts were precoated with fibronectin, seeded with SMC followed by EC a day later, and then, 24 hours later, exposed to an in vitro flow system for 1 hour. Cell retention rates were determined for grafts seeded with EC only, a dual layer of EC on top of SMC, EC transduced with wild-type tPA, and EC transduced with zymogen tPA. RESULTS: Seeding efficiency of PTFE pretreated with fibronectin was 260 +/- 8 cell/mm(2). After exposure to flow, only 39% +/- 14% of the EC were retained when EC were seeded alone, whereas 73% +/- 22% of EC remained on grafts when EC were seeded on top of SMC (P <.001, n = 10). The enzyme activity of a mutant zymogen tPA in absence of fibrin was 14 +/- 1 IU/mL, which is 3.6-fold lower than that in the presence of fibrin (50 +/- 19 IU/mL), whereas fibrin has no effect on the wild-type tPA activity. EC expressing a high level of wild-type tPA had a lower retention rate (37%) when compared with normal EC (45%). EC expressing the mutant zymogen tPA had an improved retention rate (54%, P =.001, n = 10) in absence of fibrin, whereas its retention rate was reduced to 43% when the cells were exposed to fibrin. CONCLUSION: SMC seeded between EC and PTFE improves EC retention in vitro. Transduction of zymogen tPA increases thrombolytic ability of seeded cells with less adverse impact on cell retention than wild-type tPA.

Staged thoracic and abdominal aortic aneurysm repair using stent graft technology and surgery in a patient with acute renal failure.

A 52-year-old male presented with severe hypertension and acute renal failure. Carbon dioxide (CO(2)) angiography identified a saccular thoracic aortic aneurysm, right renal artery stenosis, left renal artery occlusion, an infrarenal aortic aneurysm, celiac artery, and inferior mesenteric artery (IMA) orificial stenoses. Via an anterior retroperitoneal approach, bilateral renal artery thromboendarterectomy, infrarenal aortic aneurysmectomy, and IMA reimplantation were performed. The patient's tortuous iliac arteries were straightened to permit future passage of a thoracic stent graft by mobilizing the aortic bifurcation and anastomosing it to a Dacron graft within 4 cm of the renal vessels. Two weeks later, a stent graft was placed via a femoral incision utilizing CO(2) angiography, successfully excluding the saccular thoracic aneurysm. Recovery from both procedures was quick, with rapid return of renal function, and alleviation of the hypertension. At 8 months follow-up, his renal arteries and aorta are patent.

Human macrovascular endothelial cells: optimization of culture conditions.

The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 microg/ml) and endothelial cell growth supplement (ECGS) (50 microg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 microg/ml heparin, and 50 microg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.

High efficiency in vitro gene transfer into vascular tissues using a pseudotyped retroviral vector without pseudotransduction.

Murine leukemia virus (MuLV)-derived retroviral vectors have had limited application in vascular gene therapy because of low transduction efficiency of vascular tissues, both in vitro and in vivo. In this study, we compared the gene transfer efficiency of two retroviral vectors: amphotropic MuLV and a MuLV vector pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. Target vascular tissues included human endothelial cells (EC), smooth muscle cells (SMC) and saphenous veins (SV). Transduction efficiency of human EC and SMC was significantly higher for VSV-G pseudotyped MuLV vector (90%) than for Amphotropic MuLV (20%). Luminal surface en face analysis of transduced cultured SV showed a six- to 10-fold greater transduction efficiency with VSV-G pseudotyped MuLV. The tissue plasminogen activator (tPA) gene was transduced into EC using each vector. Four days following transduction, a 12-fold higher tPA antigen concentration and a 38-fold higher tPA enzymatic activity was measured from cells transduced with the VSV-G pseudotyped vectors as compared with the amphotropic MuLV. There was no detectable pseudotransduction (protein transfer) associated with the VSV-G MuLV vector. Both AZT inhibition of reverse transcriptase and cell division arrest by gamma irradiation inhibited transduction, indicating that viral transduction correlated with RNA reverse transcription and cell proliferation. MuLV pseudotyped with the VSV-G envelope glycoprotein is an effective retroviral vector for vascular gene therapy.

Genetic engineering of stent grafts with a highly efficient pseudotyped retroviral vector.

PURPOSE: The purpose of this study was first to compare the gene transfer efficiency of amphotrophic murine leukemia viral vector (ampho-MuLV) with the efficiency of MuLV pseudotyped with the vesicular stomatitis virus G glycoprotein (VSVG-MuLV) in tissue of vascular origin. The second purpose of this study was to determine cell retention after the implantation of genetically engineered stent grafts. METHODS: Gene transfer efficiency was ascertained with the b-galactosidase assay. The target tissues included endothelial cells (ECs), smooth muscle cells (SMCs), and human saphenous veins (HSVs). Polyurethane stent grafts were suffused with lac Z-transduced ECs and SMCs that were harvested from porcine jugular vein. The grafts were implanted into the iliac artery of each pig whose jugular vein had been harvested. Cell retention was analyzed at 1 and 4 weeks with X-Gal staining. RESULTS: VSVG-MuLV transduction efficiency exceeded that of ampho-MuLV in human ECs (VSVG-MuLV, n = 24, 89% +/- 6%; ampho-MuLV, n = 18, 14% +/- 6%; P <. 001), human SMCs (VSVG-MuLV, n = 5, 92% +/- 3%; ampho-MuLV, n = 4, 17% +/- 2%; P <.001), pig ECs (VSVG-MuLV, n = 4, 81% +/- 2%; ampho-MuLV, n = 4, 13% +/- 3%; P <.001), and pig SMCs (VSVG-MuLV, n = 5, 89% +/- 3%; ampho-MuLV, n = 4, 16% +/- 1%; P <.001). As much as a 10-fold higher transduction efficiency was observed with VSVG-MuLV in HSVs. After the stent graft implantation, the engineered cells were retained and proliferated on the stent membrane, with ingrowth into the underlying intima. CONCLUSION: VSVG-MuLV significantly increased the gene transfer efficiency in vascular SMCs and ECs and in organ-cultured HSVs. The cells were retained and proliferated on stent grafts for the short term in the pig.

Combined carotid endarterectomy and coronary artery bypass grafting in asymptomatic carotid artery stenosis.

The role of combined carotid endarterectomy (CEA) and coronary artery bypass grafting (CABG) in patients with severe asymptomatic carotid artery disease and concurrent symptomatic coronary artery disease is controversial. The objective of this report is to investigate the safety of combined CEA/CABG. The medical records of 30 patients who underwent combined CEA/CABG for coexistent asymptomatic carotid and symptomatic coronary artery occlusive disease were reviewed. All patients were scheduled for either elective or urgent myocardial revascularization due to their symptomatic coronary artery disease. Color-flow duplex scanning identified internal carotid artery stenosis of 80 to 99 per cent in 28 patients (93%) and 50 to 79 per cent in 2 patients (7%). Seventeen patients (57%) were male. The mean age was 64 +/- 10 years (range, 42-84 years). Contralateral internal carotid artery occlusion was present in four patients. Severe left main coronary artery disease was present in 12 patients (40%) and 7 patients (23%) had an ejection fraction of less than 50 per cent. There were no perioperative deaths or strokes. One patient suffered a myocardial infarction on postoperative day 1. This study demonstrates the safety of combined CEA/CABG for coexistent coronary and asymptomatic carotid disease. Using this surgical approach for critical coexistent disease may minimize the incidence of perioperative cerebrovascular complications in patients undergoing CABG.