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Circulation. 1996;93:1439-1446

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*Genes and Gene Therapy

(Circulation. 1996;93:1439-1446.)
© 1996 American Heart Association, Inc.

Articles

Seeding of Vascular Grafts With Genetically Modified Endothelial Cells

Secretion of Recombinant TPA Results in Decreased Seeded Cell Retention In Vitro and In Vivo

Peter F. Dunn, MD; Kurt D. Newman, MD; Michael Jones, MD; Izumi Yamada, MD; Vafa Shayani, MD; Renu Virmani, MD; David A. Dichek, MD

From the Molecular Hematology Branch (P.F.D., K.D.N., V.S., D.A.D.) and the Laboratory of Animal Medicine and Surgery (M.J., I.Y.), National Heart, Lung, and Blood Institute, Bethesda, Md; Department of Surgery, Children's National Medical Center, Washington, DC (K.D.N.); Armed Forces Institute of Pathology, Washington, DC (R.V.); and Gladstone Institute of Cardiovascular Disease, San Francisco, Calif (D.A.D.).

Correspondence to David A. Dichek, MD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100. E-mail david dichek@quickmail.ucsf.edu.


*    Abstract
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Background Seeding of small-diameter vascular grafts with endothelial cells (ECs) genetically engineered to secrete fibrinolytic or antithrombotic proteins offers the potential to improve graft patency rates.

Methods and Results Sheep venous ECs were transduced with a retroviral vector encoding human tissue plasminogen activator (TPA). The ECs were seeded onto 4-mm-ID synthetic (Dacron) grafts. Retention of the seeded ECs was measured 2 hours after placement of the seeded grafts both in vitro in a nonpulsatile flow system and in vivo (in sheep) as femoral and carotid interposition grafts. On exposure to flow in vitro, ECs transduced with TPA were retained at a significantly lower rate (median, 67%) than either untransduced ECs (81%) or ECs transduced with a control retroviral vector producing ß-galactosidase (ß-Gal) (80%) (P<.05 for TPA versus either control). On implantation in vivo, ECs transduced with TPA were retained at a very low rate (median, 0%), significantly less than the retention of ECs transduced with the ß-Gal vector (32%; P<.00001). Decreased in vivo retention of ECs transduced with TPA correlated modestly with increased in vitro cellular passage level (r2=.48; P<.0001) but not with in vivo blood flow rate (P=.45). Addition of the protease inhibitor aprotinin to the cell culture and graft perfusion media resulted in a significant (P<.05) increase in in vitro retention of ECs transduced with TPA.

Conclusions Increased TPA expression significantly decreases seeded EC adherence in vitro and in vivo. Gene therapy strategies for decreasing graft thrombosis may require expression of antithrombotic molecules that lack proteolytic activity.


Key Words: carotid arteries • cells • plasminogen activators • viruses


*    Introduction
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 *Introduction
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Small-diameter (<=4 mm) prosthetic grafts are inferior to autologous artery and vein in both infrainguinal and coronary arterial revascularization procedures.1 2 3 4 Autologous saphenous vein and internal mammary artery are therefore the conduits of choice for infrainguinal and coronary bypass grafting, respectively. However, the limited availability of both of these autologous vessels mandates a continued search for a clinically acceptable small-diameter prosthetic graft.

A theoretical solution to the problem of small-diameter prosthetic graft failure is the establishment of an EC monolayer on the luminal graft surface before implantation, a technique termed "seeding."5 Initial animal studies with seeded grafts were encouraging6 7 ; however, confirmation of the ability of seeded ECs to improve small-diameter graft patency in humans has been lacking. Recently, Zilla et al8 reported 32-month patency rates in human lower-extremity bypass grafts of 85% for seeded grafts compared with 55% for unseeded grafts. However, as the grafts used in that study were 6 mm in diameter, the ability of EC seeding to improve the patency rates of small-diameter grafts remains unproven.

We and others have proposed gene therapy as a means of improving the outcome of EC seeding.9 10 11 12 In previous in vitro studies,13 14 we demonstrated that ECs could be genetically modified to secrete large amounts of TPA. These genetically enhanced ECs could be seeded onto grafts at densities sufficient to produce relatively large amounts of TPA in vitro.15 We hypothesized that these large amounts of TPA might prevent small-diameter graft thrombosis in vivo. Other groups10 11 showed that ECs transduced with marker genes and seeded onto synthetic grafts could survive for at least 5 weeks after implantation in dogs. Those authors also suggested that seeded TPA-transduced cells might eventually be used to improve graft patency. No study, however, has taken the next step of investigating whether ECs genetically engineered to secrete TPA will survive and continue to express TPA in vivo.

In the present study, we seeded ECs engineered to secrete recombinant human TPA onto 4-mm-diameter synthetic grafts and implanted the grafts in vivo in sheep arteries. We measured the retention and viability of the seeded ECs after implantation and investigated factors contributing to the loss of the seeded ECs in vivo.


*    Methods
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 *Methods
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Endothelial Cell Harvest and Transduction
All animal protocols were approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. ECs were harvested from eight adult sheep, each weighing 25 to 40 kg. General anesthesia and lateral saphenous vein removal, as well as EC harvest, culture, and characterization, were performed essentially as described previously.15 Cultured ECs were passaged at a 1:2 ratio and were used between passages 5 and 14. In individual experiments, the potential contribution of passage level to experimental results was minimized by the use of cells of identical or very similar passage level. Grafts implanted for 2 hours in vivo were seeded with nonautologous ECs. Grafts implanted for 1 week in vivo were seeded with autologous ECs.

ECs were transduced with either of two retroviral vectors: LtSN16 or LBgSN,17 both derived from LXSN.18 LtSN contains a cDNA for human TPA and LBgSN contains the Escherichia coli ß-Gal gene, both under the transcriptional control of the viral long-terminal repeat promoter. The vectors are otherwise identical. For clarity, the vectors are referred to herein as LtSN(TPA) and LBgSN(ß-Gal). Retroviral vector stocks used in these experiments had titers of 1 to 5x105 G418-resistant colonies per milliliter. All retroviral supernatants used were helper-virus negative by either S+/L-19 or marker rescue assays.20

Seeding of Vascular Grafts
Four-millimeter-ID collagen-impregnated synthetic (Dacron) vascular grafts were supplied by Meadox Medicals, Inc; both 5- and 10-cm lengths were used. Grafts were coated with fibronectin (Becton Dickinson Labware) and seeded with ECs.15 All grafts were seeded at a density of 8x105 cells/cm2. After the 2-hour seeding period, the grafts were placed in fresh medium overnight at 37°C under 5% CO2. In certain experiments, the plasmin inhibitor aprotinin (Sigma Chemical Co) was added to the graft culture medium at a concentration of 1 antithrombin U/mL. After overnight incubation, media conditioned by the grafts were collected for measurement of TPA antigen (see below), and the grafts were then used for flow studies.

In Vitro Flow Studies
A nonpulsatile flow circuit was used for studies of seeded EC retention in vitro.15 In experiments that tested the effect of aprotinin on cell retention, aprotinin was added to the perfusion medium at 1 antithrombin U/mL. Pressure (driven by gravity) for all experiments was maintained at 95 to 100 mm Hg; flow ranged between 80 and 150 mL/min.

For each in vitro flow experiment, groups of 5-cm graft segments were seeded with either untransduced, LtSN(TPA)-transduced, or LBgSN(ß-Gal)-transduced cells. For experiments with LtSN(TPA)-transduced ECs, after seeding and overnight incubation, one of each group of grafts was placed in tissue culture medium as a non–flow-exposed control. The culture medium was collected for this non–flow-exposed graft according to the same schedule used to collect media from the flow-exposed grafts (see below). The other seeded grafts of each group were loaded into the flow circuit for 2 hours, then removed and incubated overnight. Media conditioned by both flow-exposed and non–flow-exposed control grafts were collected and assayed for TPA antigen by ELISA. The grafts were then sonicated15 and the graft sonicates assayed for DNA content by fluorometric assay (see below). For experiments with LBgSN(ß-Gal)-transduced ECs, a similar protocol of control and flow-exposed grafts was followed, except that ß-Gal activity21 was measured after overnight incubation and graft sonication (see below). For grafts seeded with untransduced ECs, only DNA content was measured, as no recombinant protein was present.

In Vivo Flow Studies
Grafts were transported to and from the operating room in transport medium consisting of M-199, 20% fetal calf serum, and 2x concentrations of penicillin, streptomycin, and amphotericin (see above). The grafts were placed in a tissue-culture incubator in the operating room until implantation.

After an overnight fast, adult sheep (weight, 30 to 40 kg) were premedicated with diazepam (10 mg IM) as well as sodium pentobarbital (500 to 600 mg IV) and intubated. Anesthesia was maintained with inhaled 1% to 2% isoflurane. Gentamicin (3 mg/kg IV), procaine penicillin (900 000 U IM), and aspirin (600 mg PO) were given preoperatively. Before the incision was made, succinylcholine chloride (1 mg/kg IV) was administered for skeletal muscle paralysis.

We implanted the seeded segments as bilateral interposition grafts in the common femoral or common carotid arteries. The periadventitial tissues were infiltrated with papaverine (30 mg/mL, 0.5 to 1 mL) and lidocaine hydrochloride (10 mg/mL, 0.5 to 1 mL) to prevent vasospasm. All visible branches were ligated and divided. Bovine lung sodium heparin (300 U/kg IV) was injected for systemic anticoagulation. The vessels were encircled with silicone elastomer tourniquets; end-to-side anastomoses 10 mm in length were performed between the grafts and the arteries with running 5-0 monofilament polypropylene suture. Air was evacuated from the vessels and flow reestablished. The arteries were ligated just distal to the proximal anastomosis and just proximal to the distal anastomosis. The intervening segment of artery was transected, converting the anastomosis to a functional end-to-end anastomosis. Grafts were irrigated at regular intervals with the transport medium (see above) before establishment of flow. For studies comparing retention of LtSN(TPA)-transduced versus LBgSN(ß-Gal)-transduced cells, a graft seeded with each cell type was implanted into each animal (left and right sides). A non–flow-exposed, seeded, control graft was transported to the operating room, cut to match the implanted graft, and irrigated at the same time points as the implanted graft. Once flow was established for the first implanted graft, the control graft was returned to the incubator in transport medium. Heparin was readministered 2 hours after the first dose.

Carotid or femoral blood flow was measured before implantation and immediately after implantation. An electromagnetic flow probe (model EP406, Carolina Medical Electronics) was placed snugly around the skeletonized common carotid or common femoral artery distal to the graft. This flow probe was connected to a flowmeter (model FM501, Carolina Medical Electronics) interfaced with a physiological recorder (model ES 2000, Gould, Inc). Preimplantation flows in the femoral artery were 160±70 mL/min, significantly less than the mean preimplantation flows in the carotid artery of 325±108 mL/min (P<.001). Postimplantation flows were 153±60 mL/min for the femoral artery and 236±47 mL/min for the carotid artery (P<.007).

Grafts were harvested after either 2 hours or 1 week of exposure to flow. For 1-week harvests, anesthesia and surgical approach were as described above. After graft removal, the animals were killed by IV potassium chloride administration. The grafts were placed in fresh tissue-culture medium for overnight incubation at 37°C under 5% CO2. The next morning, the media were collected for TPA antigen measurement, and the grafts were sonicated. Assays for secreted TPA, intracellular ß-Gal, or vector-specific DNA were performed.

Separate in vivo experiments were performed to determine the recovery rate of a known quantity of TPA antigen added to tissue-culture media used for overnight incubation of in vivo flow-exposed grafts. Grafts seeded with untransduced ECs were implanted into sheep carotid arteries for 2 hours, harvested, and incubated overnight in tissue culture media containing TPA (1 ng/mL). The media were assayed for TPA antigen by ELISA, and the result was compared with results obtained by assaying the following specimens for TPA antigen: (1) media with TPA (1 ng/mL) not placed in contact with any graft; (2) media with TPA (1 ng/mL) incubated with an uncoated, unseeded graft; (3) media with TPA (1 ng/mL) incubated with a fibronectin-coated graft; and (4) media with TPA (1 ng/mL) incubated with a fibronectin-coated, EC-seeded but non–flow-exposed graft.

TPA, ß-Gal, and DNA Measurements
The concentration of human TPA antigen in conditioned media was determined by ELISA as previously described.15 Sheep TPA is nonreactive in this assay,13 giving specificity for the recombinant human protein. We did not measure TPA enzymatic activity in the present study; however, we have reported previously that sheep ECs transduced with human TPA have an enhanced ability to activate plasminogen.14

For measurement of DNA and ß-Gal, grafts were cut into 1-cm lengths and sonicated while submerged in sonication buffer consisting of 20 mmol/L Tris, pH 7.4, 2 mol/L sodium chloride, and 10 mmol/L EDTA. Sonication was performed for 15 seconds on each 1-cm segment by use of the continuous duty cycle of a desktop sonicating device (Heat Systems-Ultrasonics, Inc). The sonicate was used for assay of either ß-Gal or total cellular DNA, or for PCR amplification and Southern blotting to detect vector-specific sequences. Control experiments established that both the ß-Gal activity assay and PCR amplification protocol could be performed satisfactorily with cell sonicates (data not shown).

ß-Gal activity was measured in cell extracts with a luminometer by use of a modification of a previously described method.21 The activity assay gave a linear response to levels of the ß-Gal standard ranging from 1.5 to 1500 µU; the amount of light emission at 1.5 µU was approximately 5 to 7x background values. All calculated ß-Gal values were within the assay standard curve.

The DNA content of graft sonicates was determined by fluorometry.15 The value of fluorescence was converted to cell number by the use of standard curves constructed from sonicated pellets of known cell number.15 22 Readings obtained from assays of all unknown samples were within the calibrated linear range of the fluorometer.

Identification of Vector Sequences by PCR Amplification and Southern Blotting
One microliter of sonicate from either in vivo exposed grafts or cell pellet standards (3x104 to 1x106 cells) was added to 19 µL of Gene Releaser (Bioventures, Inc), overlaid with 50 µL of mineral oil, and subjected to thermal cycling. To minimize nonspecific priming, the reaction mixture was held at 80°C while PCR reagents were added. Oligonucleotide primers were used that specifically amplify a 790-bp segment of the neomycin phosphotransferase gene.23 Primers (final concentration, 0.5 µmol/L), dNTPs (each at a final concentration of 200 µmol/L), and Taq polymerase (Perkin Elmer) at 2.5 U/100 µL were added. After an initial hold at 94°C for 2 minutes, the amplification was carried out for 30 cycles: 94°C (30 seconds), 60°C (30 seconds), 72°C (1 minute). After the 30th cycle, products were extended at 72°C for 5 minutes.

PCR products were separated in a 2% agarose gel, followed by Southern blotting according to standard protocols.24 The blot was hybridized overnight to a 1023-bp cDNA probe (EcoRI-Nru I fragment from pLNSX)23 that was labeled by the random-priming method to a specific activity of 5x109 cpm/µg. Final washes (two) of the blot were performed with 0.2xSSC, 0.1% SDS at 65°C for 30 minutes each. Washed blots were imaged with a blot analyzer (Betascope 603, Betagen) and autoradiographed with Kodak XAR film at -70°C. We do not consider this PCR-based assay to be strictly quantitative. However, by using both negative controls (without vector DNA) as well as sonicates of grafts known to have large numbers of adherent cells, we established that this PCR assay was capable of detecting both the absence and presence of large numbers of seeded cells (data not shown). In addition, supportive of the semiquantitative nature of the PCR assay, standard curves generated by counting radioactivity from specific bands amplified from sonicated cell pellets containing known numbers of transduced ECs (3x104 to 1x106) were generally linear (typical r2=.81; data not shown).

Scanning Electron Microscopy
Certain graft segments were fixed, cut open, and examined by SEM. For those grafts exposed to flow in vivo, at the end of 2 hours of flow, intra-arterial cannulas were placed proximally and distally to the graft, all intervening branches were ligated, and the graft was flushed with Ringer's lactate until the effluent was clear ({approx}250 mL). The graft was then perfusion-fixed in situ with 250 mL of a 2% glutaraldehyde, 0.1 mol/L cacodylic acid solution at a perfusion pressure of 100 mm Hg. Grafts were harvested and stored in the glutaraldehyde/cacodylic acid solution. Just before scanning, the grafts were dehydrated and critical point–dried with liquid carbon dioxide, coated with gold palladium in a plasma coater, and visualized with a Zeiss DSEM 960A scanning electron microscope. Non–flow-exposed grafts were simply immersed in the fixative and otherwise processed exactly as the flow-exposed grafts.

To assess EC coverage, grafts were cut longitudinally into two segments, and five areas of each segment were viewed at low (x200) and high (x600) power. The five areas corresponded to the midportion of each segment and one sample from each quadrant of the segment.

Statistical Analysis
Since retention data were not normally distributed, group data are presented as median and range. Blood flow rates are presented as mean±SD. Comparison of cell passage versus retention was performed by use of Spearman's rank correlation. For comparison of retention values and flow values between graft segments seeded with LBgSN(ß-Gal)- and LtSN(TPA)-transduced ECs and inserted into the same animal, Wilcoxon signed-rank tests were used. For comparison of retention of in vitro and in vivo flow-exposed grafts implanted into different animals, Wilcoxon rank sum tests or Kruskal-Wallis tests were used. Statistical significance was assumed at a value of P<.05.25


*    Results
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 *Results
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Summary of Graft Implantations
A total of 80 grafts were implanted into 40 sheep as carotid or femoral artery interposition grafts (TableDown). Seventy grafts of 5-cm length were implanted for 2 hours: 56 grafts were seeded with LtSN(TPA)-transduced ECs; 12 grafts were seeded with LBgSN(ß-Gal)-transduced ECs; and 2 grafts were seeded with untransduced ECs. Six grafts of 5-cm length were implanted for 1 week; all of these 1-week grafts were seeded with autologous LtSN(TPA)-transduced ECs. Four grafts of 10-cm length were seeded with LtSN(TPA)-transduced ECs and implanted for 2 hours. Seventy-nine of 80 grafts were patent when removed, and no animal had clinical evidence of graft or wound infection.


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  		Table 1. Seeded Vascular Grafts Implanted In Vivo

Retention of LtSN(TPA)-Transduced ECs After In Vivo Implantation
Forty-four seeded grafts were implanted into either the femoral or carotid arteries of 22 sheep. Four explanted grafts were excluded from analysis because of bacterial contamination in tissue culture after explantation. Of the 40 remaining grafts used for analysis, 12 were implanted in the carotid arteries and 28 in the femoral arteries.

We measured the retention of viable LtSN(TPA)-transduced ECs by measuring TPA antigen secretion from explanted grafts after exposure to 2 hours of flow. Median TPA secretion from all non–flow-exposed grafts was 3.8 ng/cm2 per 24 hours (0.5 to 9.2 ng/cm2 per 24 hours; n=18). Median TPA secretion from all flow-exposed control grafts was 0.5 ng/cm2 per 24 hours (0.0 to 5.6 ng/cm2 per 24 hours; n=40). Because of variability in the absolute levels of EC TPA secretion from different lines of cells used in individual experiments, the percent retention of the LtSN(TPA)-transduced ECs was most appropriately calculated not by comparing these overall values but rather by dividing the TPA secretion of individual flow-exposed grafts by the TPA secretion measured from the control graft included in each experiment. According to this method, the overall median retention of LtSN(TPA)-transduced ECs to the 40 grafts was 6% (0% to 75%).

Given the wide range of EC retention values in the individual experiments, we analyzed the retention data retrospectively to determine whether EC retention was greater in the lower-flow femoral grafts. There was no difference between retention of cells on grafts implanted in the femoral arteries (6.3% [0% to 75%]; n=28) and in the carotid arteries (9% [0% to 40%]; n=12; P=.45).

We next investigated other reasons for the low retention of seeded LtSN(TPA)-transduced cells. Since the properties of cultured ECs, potentially including adhesiveness, may change with extended in vitro culture,26 27 we analyzed the data from the retention studies to test the hypothesis that seeded EC retention in vivo was correlated with EC passage level. There was a moderate but significant negative correlation between EC passage level in vitro and seeded EC retention in vivo (r2=.48; P<.0001; Fig 1Down).



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  		Figure 1. In vivo retention of seeded ECs. Saphenous vein ECs were transduced with a retroviral vector expressing human TPA. At passage levels varying from 5 to 14, the transduced cells were seeded onto synthetic grafts that were implanted in vivo as carotid or femoral interposition grafts. Cell passage level is the number of in vitro passages between cell harvest and the reimplantation of progeny of the harvested cells in vivo. Percentage retention of seeded cells for each graft was calculated from measurements of TPA antigen secretion from each seeded graft both before and after implantation (see "Methods"). Each triangle represents an individual graft; heights of the bars represent the median retention for cells of a given passage level. All TPA assays were performed in duplicate.

We also considered whether the relatively low retention of LtSN(TPA)-transduced ECs in vivo was a result of the extensive surgical manipulation that is inherent in implantation of a short (5-cm) graft segment. We tested this hypothesis by implanting four 10-cm-long grafts seeded with LtSN(TPA)-transduced ECs into sheep carotid arteries for 2 hours. The retention of the seeded ECs on these grafts was low (1.5% [0% to 8%]), not significantly different from the retention of seeded ECs on 5-cm graft segments (P=.15).

We next considered that although retention of seeded ECs might be low at 2 hours, graft coverage with seeded ECs might improve at later time points because of proliferation of the seeded ECs. This hypothesis was suggested by kinetic studies of seeded EC retention that demonstrated that loss of seeded ECs is greatest during the first 30 minutes after implantation, followed by a steady but slower rate of loss through 24 hours and negligible loss thereafter.28 Six 5-cm grafts were seeded with autologous LtSN(TPA)-transduced ECs and placed bilaterally in the femoral arteries of three sheep. After 1 week of flow, five of six graft segments were patent. No TPA antigen was detected in media conditioned by any of the explanted grafts. Given the lower limit of the assay to detect approximately 0.05 ng/mL of TPA and the measured secretion rate of the seeded ECs of 100 ng/106 cells per 24 hours, failure to detect any TPA secretion corresponds to an upper limit of retention of <1% of the seeded ECs.

In summary, our initial in vivo experiments of both 2 hours' and 7 days' duration demonstrated surprisingly low retention of seeded LtSN(TPA)-transduced ECs; investigation of potential causes of the low retention yielded only increasing passage level as a modestly contributing factor.

Comparison of Retention of LtSN(TPA)-Transduced, LBgSN(ß-Gal)-Transduced, and Untransduced ECs In Vitro
We next performed an in vitro experiment to investigate whether either transduction per se11 12 or transduction specifically with the LtSN(TPA)-expressing vector might be responsible for the low retention of seeded ECs. We compared retention of LtSN(TPA)-transduced, LBgSN(ß-Gal)-transduced, and untransduced ECs in an in vitro flow system. Because of the inclusion of untransduced ECs, cell retention could be calculated for all groups of ECs only by measuring DNA, not by measuring recombinant protein. In each experimental group, the range of retention values was narrow, demonstrating the reproducibility of both the seeding and quantification techniques (Fig 2Down). The percentages of retention of the seeded ECs after 2 hours of flow were 81% (72% to 87%) for untransduced ECs, 80% (71% to 83%) for LBgSN(ß-Gal)-transduced ECs, and 67% (60% to 70%) for LtSN(TPA)-transduced ECs (Fig 2Down). There was no significant difference in the retention of untransduced and LBgSN(ß-Gal)-transduced ECs (P=.37); however, the LtSN(TPA)-transduced ECs were retained at a significantly lower (by {approx}20%) rate than either the untransduced or the LBgSN-transduced ECs (P<.05 for both comparisons). Therefore, whereas retroviral transduction alone did not affect EC retention in in vitro flow, expression of TPA significantly reduced seeded EC retention.



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  		Figure 2. In vitro retention of ECs: role of transduction per se. ECs, either untransduced or transduced with one of two retroviral vectors, were seeded onto synthetic grafts that were placed into an in vitro nonpulsatile flow system. Transduced cells carried either the LBgSN vector (expressing ß-Gal) or the LtSN vector (expressing human TPA). Cell retention for all grafts was determined by assaying graft sonicates for total DNA content and comparing these values with DNA values measured from sonicated control grafts that were not exposed to flow. Each triangle represents an individual graft; heights of the bars represent the median retention for each cell type. LBgSN(ß-Gal)-transduced cells were retained at essentially the same rate as untransduced cells, whereas LtSN(TPA)-transduced cells were retained at a significantly lower level than either untransduced or LBgSN(ß-Gal)-transduced cells. DNA assays were performed in duplicate and the means used to calculate cell retention.

Comparison of Retention of LtSN(TPA)-Transduced and LBgSN(ß-Gal)-Transduced ECs In Vitro and In Vivo
We further investigated the effect of TPA secretion on seeded EC retention in a series of in vitro and in vivo experiments performed with only the LtSN(TPA)- and LBgSN(ß-Gal)-transduced ECs. Untransduced ECs were omitted because of the lack of a quantitative method by which retention of viable untransduced ECs could be measured in vivo.

To optimize our quantitative methods, we began with an in vitro experiment to compare retention of LtSN(TPA)- and LBgSN(ß-Gal)-transduced ECs, using both DNA and protein assays. Again, within-group results were all within a narrow range, with both the DNA and protein assays (Fig 3Down). The retention of LtSN(TPA)-seeded cells was 65% (60% to 80%) by DNA assay (n=10), {approx}20% below the retention of LBgSN(ß-Gal)-seeded cells (80% [65% to 89%]; n=10; P<.002). These data, which were produced with use of three different lines of transduced cells, essentially confirmed the results obtained in the previous in vitro experiment (Fig 2Up).



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  		Figure 3. In vitro retention of transduced ECs: role of TPA production. ECs were transduced either with the LBgSN vector (expressing ß-Gal) or with the LtSN vector (expressing human TPA). Transduced ECs were seeded onto synthetic grafts that were placed into an in vitro nonpulsatile flow system. Cell retention for all grafts was determined with two independent assays: (1) by assaying graft sonicates for total DNA content ("DNA assay") and (2) by assaying each graft for recombinant protein production (either intracellular ß-Gal or secreted TPA ["Enzyme Assay"]; see "Methods"). The results of the two assays are presented separately. Each triangle represents an individual graft; heights of the bars represent the median retention for each cell type in each of the two assays. LtSN(TPA)-transduced cells were retained at a lower rate than LBgSN(ß-Gal)-transduced cells; this difference was statistically significant with the DNA assay only. Cell retention values for each graft were calculated with the mean of duplicate DNA or protein assays.

We also measured the retention of seeded ECs in these experiments using recombinant protein assays for TPA secretion and ß-Gal activity (Fig 3Up). TPA secretion was 3.3 (3.2 to 7.5) ng/cm2 per 24 hours for the non–flow-exposed control grafts (n=4) and 1.4 (0.8 to 3.4) ng/cm2 per 24 hours for the flow-exposed grafts (n=10). The median ß-Gal activity for all non–flow-exposed control grafts (n=4) was 11 (4.4 to 16) mU/cm2 and 4.9 (1.6 to 9.5) mU/cm2 for all flow-exposed grafts (n=10). The percentage of seeded EC retention calculated from these protein data (ie, the mean of all retention rates in the individual experiments) was 45% (25% to 62%) for the LtSN(TPA)-transduced ECs and 52% (37% to 61%) for LBgSN(ß-Gal)-transduced ECs. This {approx}15% decrease in retention of LtSN(TPA)-transduced ECs was not statistically significant (P=.13).

We next compared the retention of LtSN(TPA)-transduced ECs with LBgSN(ß-Gal)-transduced ECs in vivo. Nine grafts seeded with each cell type were implanted bilaterally in sheep common carotid arteries, LtSN(TPA)-transduced ECs on one side and LBgSN(ß-Gal)-transduced ECs of the same harvest and identical or very similar passage level on the contralateral side. Arterial blood flow rates did not differ between the LtSN(TPA)-seeded grafts and the LBgSN(ß-Gal)-seeded grafts either preimplantation or postimplantation (data not shown).

Use of recombinant protein assays to measure in vivo EC retention yielded highly reproducible within-group results (Fig 4Down). The ß-Gal activity for the non–flow-exposed control grafts was 4.7 (1.3 to 8.4) mU/cm2 (n=5); for the flow-exposed grafts, ß-Gal activity was 1.4 (0.4 to 2.5) mU/cm2 (n=9). The median retention for the flow-exposed grafts was 32% (22% to 36%). Notably, no ß-Gal activity was detectable in the sonicates of explanted LtSN(TPA)-seeded grafts, confirming that endogenous ß-Gal activity was not a confounding variable in this assay. TPA secretion from LtSN(TPA)-seeded, non–flow-exposed control grafts was 1.2 (1.1 to 14) ng/cm2 per 24 hours (n=5). For the corresponding flow-exposed grafts, TPA secretion was 0.1 (0 to 5.3) ng/cm2 per 24 hours (n=9). The median retention for the flow-exposed grafts was 0% (0% to 11%). Thus, the in vivo retention rate of ECs secreting TPA was significantly less than the retention rate of ECs transduced with the ß-Gal vector (P<.0001).



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  		Figure 4. In vivo retention of transduced ECs: role of TPA production. ECs were transduced either with the LBgSN vector (expressing ß-Gal) or the LtSN vector (expressing human TPA). Percent cell retention for each explanted graft was calculated on the basis of measurements of either TPA antigen secretion (LtSN grafts) or intracellular ß-Gal activity (LBgSN grafts; see "Methods"). Each triangle represents an individual graft; heights of the bars represent the median retention for each cell type. LtSN(TPA)-transduced cells were retained at a significantly lower rate in vivo. Cell retention values were calculated from the means of duplicate protein assays performed on each sample.

We considered that the apparently low retention of TPA-secreting cells might be an artifactual finding caused by our use of an assay based on detection of TPA secretion to calculate cell retention. However, in experiments designed to address this possibility, essentially equivalent amounts of TPA were recovered after overnight incubation of human TPA in a media-filled tube both in the presence and absence of an explanted graft (data not shown).

We considered a second possible reason why our use of TPA secretion values to compute the retention of LtSN(TPA)-transduced cells might result in falsely low calculated retention rates. Although the seeded cells might be retained and viable, TPA secretion might be specifically downregulated after implantation in vivo. We felt that this was an unlikely possibility because of the reported upregulation of TPA expression in ECs exposed to increased flow in vitro.29 Nevertheless, we investigated whether seeded LtSN(TPA)-transduced cells were largely retained on the grafts by assaying the graft sonicates for vector-specific DNA sequences. PCR Southern blot analysis was performed for all in vivo implanted grafts seeded with LtSN(TPA)-transduced ECs that were negative for TPA secretion (n=15). In all cases, the predicted band (790 bp) was either absent or was present with an intensity below the lowest point on the standard curve (3x104 transduced ECs; 3% of seeded ECs). In contrast to these results, amplification of DNA from non–flow-exposed control grafts, in vitro flow-exposed grafts, and in vivo flow-exposed grafts with measurable seeded EC retention by TPA ELISA routinely revealed bands of intensities that were within the range of the assay standard curve (3x104 to 1x106 transduced ECs). These data are only semiquantitative because of a reliance on PCR amplification of very small quantities of DNA. Nevertheless, these data support a conclusion that failure to detect TPA secretion from an explanted graft seeded with LtSN(TPA)-transduced cells is due to loss of the seeded cells rather than downregulation of TPA secretion.

To investigate further the mechanism by which LtSN(TPA)-transduced ECs detached from the graft surface, eight graft segments were seeded with LtSN(TPA)-transduced ECs. Four of the eight segments were cultured and exposed to flow in the presence of the plasmin inhibitor aprotinin. EC retention was significantly improved by the addition of aprotinin (77% versus 57% by DNA assay; 41% versus 23% by TPA ELISA; P<.05 for the results of each of the assays), consistent with the hypothesis that EC detachment is due to enhanced plasmin-mediated proteolysis.

To obtain a visual correlate of the above quantitative data, SEM was performed on four grafts seeded with LtSN(TPA)-transduced ECs (three flow-exposed, one control) and four grafts seeded with LBgSN(ß-Gal)-transduced ECs (three flow-exposed, one control). Images of the two control grafts demonstrated near-confluent monolayers of ECs, validating the ability of our seeding protocol to produce complete graft coverage. The six flow-exposed grafts appeared to have less graft surface area covered with ECs than did the control grafts. Difficulty in identifying and counting individual ECs with high confidence precluded use of the SEM images to perform quantitative comparisons between individual grafts (data not shown).


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major findings of this study are as follows: (1) retention of genetically modified ECs seeded onto vascular grafts may be quantitatively and reproducibly assessed by measuring transgene product in explanted grafts; (2) retroviral transduction per se does not decrease EC retention in vitro; (3) ECs transduced with a TPA-expressing retroviral vector are retained only at a very low level on grafts implanted in vivo; and (4) the origin of the low retention of seeded, TPA-expressing ECs in vivo is multifactorial and includes both the in vitro age of the ECs and the expression of recombinant TPA.

Graft seeding as a potential means of improving small-diameter graft performance was introduced nearly 20 years ago5 ; however, the failure of graft seeding to yield major improvements in clinical outcomes despite 2 decades of effort has provoked comments as to whether graft seeding will ever enter routine clinical practice.30 31 Coincident with the development of this pessimism, the potential for graft seeding to improve graft patency rates was revived by the relatively recent demonstration that ECs could be genetically engineered before being seeded onto a vascular graft.10 The advent of techniques to genetically modify ECs combined with a recognition of the role of ECs in mediating local fibrinolysis32 and the identification of TPA as an effective fibrinolytic agent in humans33 suggested to us13 and to others11 34 that seeded ECs might be genetically modified to secrete TPA, thereby preventing occlusive thrombosis in seeded small-diameter vascular grafts. The present study is the first to seed grafts with TPA-transduced ECs and implant these grafts in vivo.

Our major finding in attempting to implant ECs expressing the potentially therapeutic TPA gene was that these ECs detached from the graft surface at an unexpectedly high rate, far higher than the rate of detachment of ECs transduced with a control vector expressing ß-Gal. We examined several potential mechanisms for this observation. First, we excluded the possibility that the apparent loss was artifactual (see "Results"). We also considered two factors that have been associated with loss of seeded ECs in vivo: flow rate35 36 and leukocyte adhesion to graft surfaces with immune-mediated displacement of ECs.37 In agreement with previous reports,37 38 39 40 EC retention was not affected by flow rates in the present study. Increased leukocyte adhesion to grafts seeded with LtSN(TPA)-transduced versus LBgSN(ß-Gal)-transduced ECs was not excluded systematically; however, an examination of several implanted grafts with SEM did not reveal an increased number of leukocytes on grafts seeded with LtSN(TPA)-transduced versus LBgSN(ß-Gal)-transduced ECs (not shown). Furthermore, TPA-expressing ECs were also retained at a significantly lower rate in the in vitro flow system, in which there are no leukocytes. For these reasons, we believe it is unlikely that immune mechanisms are responsible for the increased loss of TPA-transduced cells.

The presence of increased proteolytic activity in LtSN(TPA)-transduced ECs is a more likely mechanism for the increased loss of TPA-transduced ECs from the graft surface. The involvement of plasminogen activators and plasmin in mediating cell adhesion to extracellular matrix in vitro is well established.41 42 In accordance with this, others37 have suggested that serum proteolytic activity is a major factor responsible for the loss of seeded ECs in vivo. Plasmin, a relatively nonspecific serum protease, may initiate digestion of the extracellular matrix with consequent detachment of seeded ECs. In support of this hypothesis, {epsilon}-aminocaproic acid (an inhibitor of plasmin activity) improved the retention of seeded ECs on grafts implanted into dogs.37 Our results using aprotinin are in agreement with these data and lend support to the hypothesis that plasminogen activators can modulate cell adherence to matrix both in vitro and in vivo. Unfortunately, the ability of LtSN(TPA)-transduced ECs to lyse fibrin clots43 may carry with it a decreased ability to remain adherent to a graft. A last potential mechanism for the increased loss of LtSN(TPA)-transduced cells is a synergistic effect of retrovirus infection11 12 and TPA expression. Although our in vitro data (Fig 2Up) indicate no direct contribution of infection with the LBgSN retrovirus to EC loss, future studies with other vector systems will be required to exclude any retrovirus-specific contribution to the loss of seeded ECs.

One interpretation of our results is that antithrombotic and fibrinolytic gene therapy cannot be applied to the problem of graft thrombosis. We believe such an interpretation is premature. First, selection of a graft matrix component, such as laminin, that is relatively more resistant to proteinase digestion than fibronectin44 45 46 might improve EC retention. Incorporation of a plasmin inhibitor such as aprotinin into the graft matrix might also increase EC retention,35 although the presence of aprotinin might also mitigate any local increases in EC thrombolytic activity. If, however, use of TPA-transduced ECs to enhance antithrombotic activity carries with it an obligatory enhancement of EC loss, then it is possible that TPA is not an optimal therapeutic protein for use in enhancing graft patency. Other proteins besides TPA that might be used to decrease vascular graft thrombosis include the factor Xa inhibitor antistasin47 and the thrombin inhibitor hirudin,48 both of which are antithrombotic proteins that lack proteolytic activity. An additional molecular strategy by which plasminogen activators could be used to decrease thrombosis while preserving cell retention would involve specific localization of recombinant plasminogen activators to the apical (ie, luminal) EC surface.49 In this manner, luminal (antithrombotic) activity would be increased while abluminal (matrix degrading) activity would not be affected.

If we assume that the problem of enhanced seeded EC loss consequent to the expression of wild-type TPA can be circumvented through appropriate choices of matrix and transgene, other practical concerns remain to be addressed in graft seeding work. Cells of the lowest possible passage should be used, which mandates the development of more efficient EC harvest and transduction protocols.50 In addition, the long-term survival of significant numbers of seeded cells must be ensured. If implanted ECs assume the quiescent phenotype characteristic of vascular endothelium (mitotic rate 1:102 to 1:104 per day51 ), ECs present at a density of 1x105 ECs/cm2 would persist for years. Methods must be found to ensure that this low rate of seeded EC turnover is achieved. Finally, even if seeded ECs remain on the graft for prolonged periods of time, continued expression of the inserted therapeutic gene must also be ensured. Shutdown of transgene expression after implantation of transduced cells in vivo has been a challenging and potentially cell type–specific problem.52

In summary, major issues remain before the clinical use of vascular grafts seeded with genetically modified ECs. Failure of ECs transduced with TPA (the primary candidate gene for local graft delivery11 14 15 34 ) to remain adherent in vivo is a setback. However, the need for an acceptable small-diameter prosthetic graft should continue to drive research in this area.


*    Selected Abbreviations and Acronyms
 
bp = base pair
EC = endothelial cell
ß-Gal = ß-galactosidase
LBgSN(ß-Gal) = LBgSN vector expressing ß-galactosidase
LtSN(TPA) = LtSN vector expressing human tissue plasminogen activator
PCR = polymerase chain reaction
SEM = scanning electron microscopy
TPA = tissue plasminogen activator


*    Acknowledgments
 
This study was supported by the Division of Intramural Research of the National Heart, Lung, and Blood Institute. We thank Meadox Medicals, Inc for supplying the synthetic vascular grafts. The authors gratefully acknowledge the members of the Laboratory of Animal Medicine and Surgery for expert assistance with animal protocols, Marie Jenkins for assistance with SEM, and Genetic Therapy, Inc, for the LBgSN vector and for assistance with the helper-virus assays.

Received September 19, 1995; revision received October 27, 1995; accepted November 9, 1995.


*    References
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 up arrowAbstract
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 *References
 

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