This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dichek, D. A. Articles by Harker, L. A. Search for Related Content PubMed PubMed Citation Articles by Dichek, D. A. Articles by Harker, L. A.

(Circulation. 1996;93:301-309.)
© 1996 American Heart Association, Inc.

Articles

Enhanced In Vivo Antithrombotic Effects of Endothelial Cells Expressing Recombinant Plasminogen Activators Transduced With Retroviral Vectors

David A. Dichek, MD; Johanna Anderson; Andrew B. Kelly, DVM; Stephen R. Hanson, PhD; Laurence A. Harker, MD

From the Division of Hematology and Oncology, Department of Medicine, and Yerkes Regional Primate Research Center, Emory University School of Medicine, Atlanta, Ga; and the Molecular Hematology Branch (D.A.D.), National Heart, Lung, and Blood Institute, Bethesda, Md.

Correspondence to Laurence A. Harker, MD, Division of Hematology and Oncology, Emory University School of Medicine, PO Drawer AR, Atlanta, GA 30322.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The effects of regulating endothelial cell (EC) plasminogen activator production on thrombus accumulation in vivo are incompletely understood. By overexpressing plasminogen activators in ECs via gene transfer, the hypothesis was tested that increased levels of plasminogen activators inhibit the accumulation of thrombus in vivo.

Methods and Results Cultured baboon ECs transduced with human cDNAs for wild-type tissue plasminogen activator (TPA) or for glycosylphosphatidylinositol-anchored urokinase-type plasminogen activator (a-UPA) were seeded onto collagen-coated segments of vascular graft (collagen segments) and exposed overnight to flow using an in vitro perfusion circuit. The antigenic levels of TPA and UPA each increased 10-fold in the media perfusing the corresponding transduced ECs compared with untransduced ECs (P.05 in both cases). In baboons the antithrombotic effects of TPA-transduced or a-UPA–transduced ECs were measured as ¹¹¹In-platelet deposition and ¹²⁵I-fibrin accumulation on collagen segments bearing sparsely attached ECs (transduced versus untransduced) interposed in exteriorized arteriovenous femoral shunts. Platelet-rich thrombus formed on the collagen segments with fibrin-rich thrombus propagated distally. The presence of TPA-transduced or a-UPA–transduced ECs on collagen segments at a density of 25 000 ECs/cm² decreased ¹¹¹In-platelet deposition and ¹²⁵I-fibrin accumulation on collagen surfaces compared with untransduced ECs present at equivalent density (P<.05 for platelet deposition with TPA-transduced ECs and P<.05 for platelet deposition on the propagated tail, as well as fibrin accumulation on the graft with a-UPA–transduced ECs). The systemic levels of fibrinopeptide A, thrombin-antithrombin complex, D-dimer, and both local and systemic levels of TPA and UPA were not increased by transduced ECs compared with untransduced ECs. The focal antithrombotic effects of transduced ECs appear to be due to local enhancement of thrombolysis.

Conclusions ECs transduced with recombinant TPA and a-UPA enhance local antithrombotic activity in vivo. This strategy of attaching transduced ECs overexpressing plasminogen activators may be therapeutically useful by preventing thrombo-occlusive failure of implanted cardiovascular devices or mechanically denuded vessels.

Key Words: plasminogen activators • thrombosis • endothelium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Vascular endothelium impedes thrombus accumulation by binding, synthesizing, and regulating antithrombotic molecules¹ including thrombomodulin,² protein S³ , prostacyclin,^{4 5} nitric oxide,^{6 7 8} and TPA.^{9 10 11} Increasing the production of antithrombotic molecules by manipulating EC phenotype offers a useful strategy for preventing thrombosis.

The development of techniques for transferring and expressing genes at high efficiency in ECs^{12 13} permits testing the hypothesis that increased EC expression of specific antithrombotic or fibrinolytic genes reduces thrombus formation in vivo. Previous publications report the attachment of retroviral-transduced ECs onto denuded arterial surfaces^{14 15} or prosthetic devices deployed in vitro^{13 16 17} and in vivo.¹⁸ Human plasminogen activator genes also have been transferred and overexpressed in ovine, bovine, and baboon ECs in vitro.^{19 20 21 22} In the present study, the antithrombotic effects of ECs transduced with plasminogen activator genes were assessed in a quantitative model of thrombosis in baboons.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Endothelial Cell Culture and Assays for Recombinant Gene Expression
Jugular vein ECs from a juvenile male baboon, either untransduced or transduced with the retroviral vectors LtSN, expressing wild-type human TPA, or LUK+ASN, expressing a-UPA,²⁰ were maintained in culture and propagated as previously described.²² The properties of these ECs have been characterized extensively in vitro, with findings suggesting that the LtSN-transduced and LUK+ASN-transduced ECs are suitable for in vivo experiments designed to increase local fibrinolysis.²² While the urokinase cDNA in the LUK+ASN vector was modified to incorporate a GPI-anchoring signal, this membrane anchor does not provide an absolute level of cell surface localization in either the present system or in those previously described by others.²³ Therefore a-UPA would be expected to accumulate on the surface of LUK+ASN-transduced cells, in conditioned medium in vitro,^{20 22} and secreted into the bloodstream from LUK+ASN-transduced cells implanted in vivo.

In the present experiments, the continued production of high levels of recombinant TPA and UPA antigen by the transduced cells was confirmed by collecting conditioned medium from an in vitro perfusion apparatus containing segments of collagen-coated vascular graft to which transduced ECs were attached (see below). Medium was assayed for TPA and UPA antigen with ELISAs. These ELISAs detect both human and baboon plasminogen activators and appear to do so with similar efficiency (American Diagnostica).²² Baboon plasma from in vivo experiments was assayed for TPA and UPA antigen with the same ELISAs used for in vitro experiments.

Labeling of Endothelial Cells
Confluent cell cultures in T25 flasks were washed free of proteins with 37°C DPBS. The flasks were incubated for 10 minutes with 5 mL ¹¹¹In-oxine containing 1x10⁷ cpm/mL in RPMI-1640–HEPES medium.^{24 25} After withdrawing excess unbound ¹¹¹In-oxine, each flask was washed once with warm DPBS and incubated overnight in 5 mL of complete culture medium. This procedure ensured the functional recovery of the labeled cells and achieved consistent labeling of approximately 1 to 2 cpm/cell at the time cells were used in experimental protocols.

Preparation of Thrombogenic Graft Segments
To assemble thrombogenic segments of vascular graft material,^{26 27} Gore-Tex vascular grafts (WL Gore Inc), 5-µm pore, 4-mm internal diameter, were threaded over 4-mm Teflon rods and stabilized in configuration and diameter by encasement in sleeves of "heat-shrink" Teflon tubing (Small Parts, Inc). The encased grafts were then cut into 1-cm lengths and connected with 12-cm-long and 7-cm-long segments of silicone rubber tubing, 4-mm internal diameter, abutted at each end and encompassed in heat-shrink Teflon. Care was taken to avoid junctional irregularities in the flow surface. These graft segments were autoclaved at 250°F for 25 minutes and maintained sterile thereafter. Before their use in experiments, the luminal surfaces of the graft segments were wetted by instilling 95% ethanol into the circuit for 20 minutes, followed by flushing with sterile water (500 mL) to remove all ethanol from the system.

The resultant segments of Gore-Tex graft were coated with collagen to create a smooth thrombogenic luminal surface. To accomplish this, the circuit was charged with an acidic buffer (Horme buffer, pH 2) followed by the addition of 100 µL of 1 mg/mL equine collagen solution (Horme AG) into the lumen of each 1-cm-long graft segment. The segments then were incubated at room temperature for 2 hours, followed by air drying for 1 hour, using sterile air flowing through the tubing. Subsequently, the collagen solution (100 µL) was again introduced into the graft lumen and incubated overnight, followed by air drying. These collagen-coated segments then were incubated with human fibronectin (Biomedical Technologies Inc; 20 µg/100 µL) for 1 hour, with subsequent final air drying. In the experiments described, the completed sterile collagen segments were prewarmed to 37°C before introducing cultured ECs.

Endothelial Cell Attachment to Collagen Segments
To increase the probability of discriminating between the antithrombotic effects of plasminogen activators overexpressed by transduced ECs versus plasminogen activators constitutively expressed by untransduced ECs, we compared direct measurements of platelet and fibrin accumulation on collagen segments bearing ECs at subconfluent densities. Subconfluent endothelial coverage permitted thrombus to form on exposed intervening collagen while assuring direct contact of EC-secreted plasminogen activators with forming thrombus.

¹¹¹In-labeled ECs were washed twice with 0.2 mg/mL EDTA in PBS and removed from the culture flask by trypsin digestion (0.05% trypsin in EDTA). Trypsin was inactivated by adding FCS. The labeled EC suspension was diluted to 50 mL with DPBS, centrifuged at 200g for 15 minutes, then gently resuspended in DPBS at 10⁵ cells/100 µL. The specific activity of the cell suspension was determined using gamma scintillation counting (Packard Instrument Co) and electronic cell counting (Serono/Baker). The lumen of each graft segment was filled with 100 µL cell suspension per 1 cm of graft using a long, 16-gauge needle. Grafts were maintained at 37°C and rotated 90° each minute for 30 minutes to obtain an even distribution of attached ECs. Unattached cells then were removed by flushing the graft lumen with 1 mL DPBS. This procedure produced a graft surface bearing ECs attached at a density of approximately 25 000 cells/cm² (Fig 1). This density of attached ECs was shown to be suitable in a series of preliminary experiments using untransduced ECs. In these studies, sensitivity was compromised by either increasing EC density to 50 000/cm², in which case there was reciprocal reduction in baseline thrombus formation, or decreasing EC density to 10 000/cm², in which case there was no detectable thrombolysis (data not shown).

View larger version (186K): [in this window] [in a new window]   Figure 1. Scanning electron microscopy of graft surfaces. A, Collagen-coated luminal surface of vascular graft; B, collagen-coated graft surface with baboon ECs attached at 25 000 cells/cm2; C, thrombus formation on a luminal collagen surface seeded with untransduced ECs, after 60-minute exposure to flowing native blood (40 mL/min); and D, thrombus formation on a luminal collagen surface seeded with ECs transduced with the LtSN (TPA-expressing) vector. The antithrombotic benefit of the recombinant TPA expression is evident (compared with C). Original magnification for A through D, x250.

The graft segments bearing attached ECs were incorporated into an in vitro perfusion circuit maintained at 37°C (Fig 2). This system consisted of a microcarrier spinner flask, 100-mL size without the stir-paddles (Dow-Corning, Inc), containing approximately 30 mL of culture medium that was equilibrated with 5% CO₂ in air. This gas mixture was humidified and injected through a 0.22-µm pore-sterilizing filter via the spinner flask portal. Inflow and outflow of culture medium was performed through the two sidearms of the flask via silicone rubber tubing at a flow rate of 15 mL/min by roller pump, beginning 2 hours before interposing the cell-attached graft segments and continuing for 16 hours thereafter.

View larger version (46K): [in this window] [in a new window]   Figure 2. Drawing of in vitro perfusion apparatus for attaching cultured ECs onto collagen-coated segments of vascular graft. EC-bearing segments (approximately 25 000 cells/cm2) were incorporated into the perfusion circuit and perfused at 37°C at a flow rate of 15 mL/min for 16 hours. Typically three to five segments were inserted into the circuit, although only one is shown here (as inset photomicrograph).

Three to five graft segments seeded with aliquots of the same type of ECs (untransduced, TPA-transduced, or a-UPA–transduced) were perfused in series and imaged together with a standard of ¹¹¹In-labeled ECs of known concentration. After calculating the density of attached ECs on each segment, segments bearing approximately 25 000 attached ECs/cm² were selected for subsequent studies in vivo; the remaining segments were discarded. In this way, comparisons of different types of ECs were performed at equivalent EC density. Before dismantling the perfusion apparatus and removing the segments, samples of the perfusate were collected for assaying TPA or UPA antigen. The perfusate then was displaced by DPBS, and the grafts were maintained at 37°C with warmed gel packs during transport and manipulation before incorporation into exteriorized AV shunts.

Preparation of Animals for In Vivo Studies
Normal male juvenile baboons (Papio anubis) weighing 9 to 11.5 kg were used in these experiments. All procedures were approved by the Institutional Animal Care and Use Committee in compliance with the National Institutes of Health guidelines (Guide For the Care and Use of Laboratory Animals, 1985), Public Health Service policy, the Animal Welfare Act, and related university policies. The baboons were observed to be disease free for at least 3 months before in vivo studies were performed.

For surgical procedures, animals were given ketamine hydrochloride (20 mg/kg IM) for induction, 1% halothane by endotracheal tube for anesthetic maintenance, and buprenorphine (0.01 mg/kg every 8 hours as needed) for postoperative analgesia. Thereafter, ketamine hydrochloride (5 to 20 mg/kg IM) was used for subsequent short-term immobilization in performing experimental procedures.

Chronic exteriorized AV access shunts were surgically placed between the femoral artery and vein to permit interposition of thrombogenic graft segments and blood sampling. The shunts were composed of silicone rubber tubing, 3.0-mm internal diameter (Silastic, Dow Corning Corp). The arterial and venous arms of the shunt were connected with 1-cm-long, blunt-edge PTFE tubing (2.8-mm inner diameter). These chronic AV shunts do not detectably activate platelets or fibrinogen.^{28 29 30}

The thrombogenic collagen segments^{24 26 31} were interposed into exteriorized AV shunts of awake animals, and blood flows in the AV shunts were maintained at 40 mL/min by measuring flow with a C-clamp–type ultrasonic flow probe interfaced with a Transonic T206 blood flow analyzer. Initially, experiments were performed with unseeded collagen segments. Subsequently, a series of paired morning/afternoon experiments was performed comparing collagen segments bearing LtSN-transduced versus untransduced ECs. Paired segments were placed sequentially in the same baboon (one segment in the morning and another in the afternoon), but with the order of the segments varied among animals. Analogous experiments compared collagen segments bearing LUK+ASN-transduced versus untransduced ECs.

Assays of Blood and Plasma Hemostatic and Fibrinolytic Factors
Blood counts and hematocrits were measured on whole blood collected in EDTA (2 mg/mL) with the use of a Serono/Baker model 9000 whole blood analyzer. Plasma fibrinogen concentrations were measured as thrombin-clottable protein using the method described previously.^{30 31 32}

Blood samples were taken before and after each experiment and evaluated for platelet counts, plasma levels of fibrinogen, TPA, UPA, FPA, platelet factor 4, ß-thromboglobulin, TAT, and fibrinolytic product D-dimer.²⁷ These blood tests of fibrinolytic and hemostatic function were performed on plasma samples obtained before placing the thrombogenic devices (t=0 minutes, baseline), and repeat tests were performed on blood samples drawn at the end of the experiments (t=60 minutes). At 60 minutes, samples were also obtained proximal and distal to the devices for assaying TPA and UPA antigen.

In Vivo Measurements of Thrombus Accumulation
Thrombus accumulation on collagen segments was quantified by measuring the deposition of platelets and fibrin. Autologous baboon platelets were labeled with 1 mCi ¹¹¹In (¹¹¹In-oxine, as previously described²⁶ ) and reinjected at least 1 hour before incorporation of thrombogenic devices in the AV shunts. Labeling efficiencies averaged 90%. ¹¹¹In-labeled platelets were functionally normal.^{26 28} Baboon fibrinogen was purified by ß-alanine precipitation and labeled with ¹²⁵I with use of the ICI method as described previously^{30 33} ; labeling efficiency averaged 70%, yielding >90% clottable fibrinogen. A 5-µCi dose of ¹²⁵I-labeled fibrinogen was injected intravenously 10 minutes before interposing the device in the AV shunt.

Images of the collagen segments, including proximal and distal segments of the AV shunts, were acquired with a General Electric 400T MaxiCamera and stored and analyzed with a Medical Data Systems A³ image processing system (Medtronic) interfaced with the camera.²⁶ The low energy peak (172 KeV) of ¹¹¹In was imaged with a 10% energy window. Dynamic images were acquired at 5-minute intervals. Immediately after each dynamic study, standards were imaged, including a syringe containing 5.0 mL of whole blood (blood standard) and an identical thrombogenic device filled with static autologous blood (device standard). The imaging routines and isotopic detection protocols for these shunt studies used procedures reported previously.^{24 28 31 32} Studies of individual grafts in awake animals were terminated after 60 minutes by agreement with the Animal Care and Use Committee because previous studies showed that thrombus formation in this model generally reached a steady state at this time.^{26 32 33}

The thrombus consisted of two regions of interest that were analyzed separately, that is, the platelet-rich thrombus on collagen segments, analyzed over 1 cm (8x10-pixel region of interest), and the propagated fibrin-rich tail, analyzed over a length of 10 cm (80x10-pixel). The total number of deposited platelets in each region (labeled plus unlabeled platelets) was calculated, as described previously.³² All blood radioactivity measurements were corrected for the small fraction of nonplatelet isotope in each experiment. ¹¹¹In-platelet emissions were counted to measure thrombus formation. For this measurement, emissions from both the graft and the fibrin-rich tail regions of the experimental device were counted. Deposited ¹¹¹In-labeled platelet activity was calculated by subtracting the device standard activity from each region of interest.

To measure fibrin in platelet- and fibrin-rich thrombi, after completion of the experiment, the device was thoroughly washed with isotonic saline solution. The vascular graft thrombus then was divided from the propagated tail for separate counting of ¹²⁵I emissions. These emissions were counted at least 30 days after the study to allow for the decay of ¹¹¹In activity (half-life, 2.8 days). Total fibrin deposition was calculated by dividing the deposited ¹²⁵I-fibrin activity (counts per minute) by the clottable fibrinogen activity (counts per minute per milliliter) and multiplying by the plasma fibrinogen level (milligrams per milliliter).

Statistical Analysis
Data are presented as mean±1 SD unless indicated otherwise. Significance was generally determined with use of the Student's t test, unpaired, two-tailed analysis, except for data not normally distributed, when nonparametric analyses were performed. Paired, two-tailed analysis was used to compare platelet and fibrin deposition on the grafts and in the propagated tails (InSTAT, GraphPAD software). The curves comparing platelet deposition were analyzed for significance by repeated measures ANOVA (SYSTAT, Inc).

	Results

Top Abstract Introduction Methods Results Discussion References

 
In Vitro Expression of Plasminogen Activators
Immunoreactive TPA and UPA were detected in conditioned media collected from the circuit in vitro (Fig 2) after overnight perfusion of collagen segments seeded with either untransduced ECs or ECs transduced with the TPA-containing or a-UPA–containing retroviral vectors. For both TPA-transduced and a-UPA–transduced ECs, vector-driven plasminogen activator expression was increased 9- to 10-fold over the levels of endogenous plasminogen activator present in perfusion media conditioned by untransduced ECs (Table 1). These results were quantitatively similar to the in vitro findings reported previously for these same cells grown in static culture.²² These data demonstrated that transduced ECs continue to secrete high levels of plasminogen activators after attachment to collagen segments and exposure to flow in vitro.

View this table: [in this window] [in a new window]   Table 1. Secretion of TPA and UPA by RetroviralTransduced Endothelial Cells

In Vivo Effects of Endothelial Cells on Thrombus Formation
Before initiating in vivo studies comparing the effects of untransduced and transduced ECs, the accumulation of ¹¹¹In-platelets and ¹²⁵I-fibrin on collagen segments alone without attached ECs was measured during 60 minutes of blood flow in exteriorized AV shunts. Platelets and fibrin accumulated rapidly on these unseeded collagen segments, reaching a plateau value by 60 minutes of 3.20±1.09x10⁹ platelets and 1.14±0.83 mg fibrin. Propagated thrombotic tails extending downstream from these unseeded collagen segments accumulated 10.8±5.26x10⁹ platelets and 1.94±1.40 mg fibrin.

To compare the antithrombotic effects of untransduced ECs to ECs transduced with TPA or a-UPA vectors, a series of 13 paired animal studies was carried out. For each of these studies, one collagen segment seeded with untransduced ECs (n=13) and a second collagen segment seeded with an equivalent number of TPA-transduced ECs (n=8), or a-UPA–transduced ECs (n=5), was placed in the exteriorized shunt of the same animal. Platelet deposition on collagen segments seeded with untransduced ECs was 1.98±0.69x10⁹ platelets at 60 minutes (compared with 3.20±1.09x10⁹ on unseeded segments; P=.003); fibrin accumulation on collagen segments was not affected by seeding with untransduced ECs (1.09±0.39 mg compared with 1.14±0.83 mg fibrin for unseeded segments; P=.5). Untransduced ECs significantly reduced platelet deposition but not fibrin accumulation in the propagated tails, that is, from 10.8±5.26x10⁹ to 6.09±3.31x10⁹ platelets (P=.014) and from 1.94±1.40 mg to 1.72±1.37 mg fibrin (P=.1).

Overexpression of TPA by transduced ECs exhibited local antithrombotic effects on platelet and fibrin accumulation (Fig 3 through 5 and Table 2) compared with the effects of untransduced ECs seeded onto collagen segments at equivalent density and interposed sequentially in the same AV shunts. TPA-transduced ECs reduced platelet deposition on the collagen segments from 2.05±0.47x10⁹ platelets to 1.13±0.82x10⁹ (P=.021; P=.026 by repeated measures ANOVA). Real-time images of platelet deposition on representative grafts seeded with either TPA-transduced or untransduced cells are shown in Fig 3. Fibrin accumulation on the collagen segments was 0.55±0.21 compared with 0.69±0.29 mg fibrin for untransduced ECs (P=.29). Platelet deposition was also significantly decreased in the propagated tail by TPA-transduced ECs (3.39±2.67x10⁹ versus 6.06±3.4x10⁹ platelets; P=.013; P=.10 by repeated measures ANOVA). Fibrin accumulation in the thrombus tails was 0.62±0.55 compared with 1.31±1.11 mg fibrin (P=.07). It is notable that although the magnitudes of the mean decrements in platelet and fibrin deposition by the TPA-transduced cells were not identical (approximately 50% for platelet deposition on both segments and tails as well as fibrin accumulation on tails; 25% for fibrin deposition on the collagen segment) in all cases, platelet and fibrin deposition decreased in concert. Assuming that the 95% confidence limits of the mean decrements extend 2 SEM, the magnitudes of the decrements were not significantly different.

View larger version (37K): [in this window] [in a new window]   Figure 3. Images of 111In-platelet deposition on vascular graft and in propagated thrombus tail. The deposition of 111In-labeled platelets is depicted in the upper image for a graft seeded with untransduced ECs exposed to flowing blood at 40 mL/min for 60 minutes from left to right. Lower image depicts the antithrombotic effects of attaching LtSN (a TPA-expressing vector)–transduced endothelial cells at equivalent density and exposing the graft to blood flow at 40 mL/min for 60 minutes. Both images were taken from grafts placed in the same baboon. Blood flow is from right to left. As levels of 111In emission increase, pixel color shifts from blue to green to yellow and finally to red.

View larger version (19K): [in this window] [in a new window]   Figure 4. Graphs show deposition of 111In platelets (A) and accumulation of 125I-fibrin (B) on segments of collagen-coated vascular graft seeded with ECs. Platelet deposition is shown in real time for untransduced ECs (open circles; n=13); LtSN (TPA)–transduced ECs (closed circles; n=8); and LUK+ASN (a-UPA)–transduced ECs (closed squares; n=5). Fibrin accumulation for the three graft types is shown at the 1-hour time point only. All data are presented as mean±SEM. In presenting the effects of TPA-transduced and a-UPA–transduced ECs versus untransduced ECs, values for platelet and fibrin accumulation on collagen segments bearing untransduced ECs represent overall means of data obtained from both of the paired studies. In contrast, the values of platelet and fibrin accumulation for untransduced ECs shown in Tables 2 and 3 represent means obtained in each of the two separate paired studies, comparing untransduced ECs with LtSN-transduced or LUK+ASN-transduced ECs, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 5. Graphs show deposition of 111In platelets (A) and accumulation of 125I-fibrin (B) in thrombus tails propagated downstream from grafts seeded at equivalent densities with either untransduced or transduced ECs. Platelet accumulation is shown in real time for untransduced ECs (open circles; n=13); LtSN (TPA)–transduced ECs (closed circles; n=8); and LUK+ASN (a-UPA)–transduced ECs (closed squares; n=5). Fibrin accumulation for the three graft types is shown at the 1-hour time point only. All data are presented as mean±SEM. In presenting the effects of TPA-transduced and a-UPA–transduced ECs versus untransduced ECs, values for platelet and fibrin accumulation on collagen segments bearing untransduced ECs represent overall means of data obtained from both of the paired studies. In contrast, the values of platelet and fibrin accumulation for untransduced ECs shown in Tables 2 and 3 represent means obtained in each of the two separate paired studies comparing untransduced ECs with LtSN-transduced or LUK+ASN-transduced ECs, respectively.

View this table: [in this window] [in a new window]   Table 2. Antithrombotic Effects of TPA-Transduced Endothelial Cells

Vector-driven expression of a-UPA by transduced ECs also produced local antithrombotic effects compared with the effects of untransduced ECs (Fig 4 and 5 and Table 3). a-UPA–transduced ECs reduced both platelet deposition and fibrin accumulation on collagen segments, that is, platelets decreased from 1.87±1.0x10⁹ to 1.14±0.53x10⁹ platelets (P=.16; P=.079 by repeated measures ANOVA), and fibrin accumulation on collagen segments fell from 0.92±0.50 to 0.32±0.11 mg (P=.039). In the propagated tail, platelet accumulation was reduced by a-UPA–transduced ECs to 3.86±3.06x10⁹ compared with 6.15±3.7x10⁹ platelets for untransduced ECs (P=.022; P=.021 by repeated measures ANOVA). Fibrin accumulation in the propagated tail was 1.32±0.57 mg compared with 2.37±1.61 mg fibrin for untransduced ECs (P=.12). Again, as detailed above for the TPA-transduced cells, decrements in fibrin and platelet accumulation on the segments and grafts were observed, although the magnitudes of individual reductions were not always of significant, uniform difference.

View this table: [in this window] [in a new window]   Table 3. Antithrombotic Effects of a-UPA–Transduced Endothelial Cells

The levels of TPA or UPA antigen failed to change in blood samples collected immediately distal to the EC-bearing thrombogenic segments compared with levels in blood sampled proximal to the segments (Tables 2 and 3; P>.46 for all comparisons). The absence of detectable increases in the levels of plasminogen activators across the devices bearing transduced ECs versus untransduced ECs indicates that the small numbers of attached transduced ECs enhanced only local thrombolysis. Moreover, because there was no detectable increase in systemic plasma levels of D-dimer, a specific biochemical marker of ongoing fibrinolysis (P>.48 for all comparisons), the local thrombolytic effects failed to produce detectable systemic evidence of increased fibrinolytic activity.

Measurements of Thrombus Formation In Vivo
Plasma biochemical tests of in vivo thrombosis were performed on peripheral blood collected before and 60 minutes after interposing collagen segments in AV shunts (Tables 2 and 3). For collagen segments seeded with untransduced ECs, the elevations in the 60-minute values of FPA and TAT were typical of those produced by thrombus forming locally (threefold to fivefold increases; P<.001 in both cases). Increases were also observed in plasma levels of ß-thromboglobulin and platelet factor 4, platelet-specific proteins secreted during thrombus formation, that is, from 6.6±2.2 to 21±17 and from 2.6±1.0 to 15±13, respectively (threefold- to sixfold increases; P<.05 in both cases). Similar results were obtained with collagen segments seeded with ECs transduced with the TPA and a-UPA vectors (Tables 2 and 3). No significant differences were detected between 60-minute plasma values measured for FPA, TAT, or D-dimer during experiments comparing untransduced ECs versus ECs transduced with either TPA or a-UPA vectors (Tables 2 and 3; P>.5 for all comparisons). Thus, unlike the results of platelet deposition and fibrin accumulation on seeded collagen segments, none of these systemic plasma measurements of ongoing thrombosis and fibrinolysis were sufficiently sensitive to distinguish the antithrombotic effects of transduced ECs from those of untransduced ECs. Similarly, none of these systemic plasma measurements could distinguish the relative importance of inhibiting thrombus formation (as determined by changes in FPA or TAT) from that of increasing fibrinolysis (as determined by changes in D-dimer) in the enhanced antithrombotic activity observed with ECs overexpressing plasminogen activators.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates that retroviral gene transfer of cDNAs encoding TPA or a-UPA amplifies plasminogen activator production 10-fold and confers antithrombotic efficacy in vivo for both platelet-rich and fibrin-rich thrombus formation. While others have postulated on the basis of in vitro studies that augmenting endothelial plasminogen activator production would reduce thrombotic events,^{34 35} this prediction has not been tested previously in vivo. Recent gene deletion studies involving plasminogen activator–deficient mice provide complementary evidence that thrombo-occlusive events are associated with decreased levels of plasminogen activator in vivo.³⁶ However, the elimination of plasminogen activator expression in those studies was not restricted to ECs. Thus, the experimental results in the present study constitute important direct evidence that endothelial overexpression of recombinant plasminogen activators produces antithrombotic benefits for both arterial-type and venous-type thrombosis.

The observed decreases in thrombus accumulation are most likely explained by direct local enhancement of thrombolysis. Increases in plasminogen activator–dependent fibrinolysis may reduce thrombus formation by four potential mechanisms: (1) direct reduction in fibrin content, (2) diminished retention of activated platelets to fibrin, (3) inhibition of fibrinogen-dependent platelet recruitment by early fibrinolytic cleavage products, and (4) decreased binding of thrombin to fibrin.^{37 38} While systemic markers of thrombosis and fibrinolysis were measured to help discriminate among these mechanisms, they failed to provide insight into the relative importance of these putative mechanisms.

It is likely that the success of this model in limiting thrombogenesis as well as the absence of detectable alterations in plasma markers of thrombosis and fibrinolysis reflect the exquisite localization of the enhanced thrombolytic effect. In the present experimental system, overexpressed plasminogen activators are either anchored to the cell surface or are secreted into the slow moving, poorly diffusible luminal boundary layer.³⁹ Within this boundary layer, the secreted plasminogen activators may further localize their effects by interacting with specific cell surface receptors⁴⁰ or forming fibrin. We conclude that fibrinolysis is enhanced within this thin boundary layer and that indirect blood markers of increased fibrinolysis are not sufficiently modified to produce changes that are detectable by traditional blood sampling techniques.

The two plasminogen activator constructs tested in this study, wild-type TPA and anchored urokinase, produced similar antithrombotic effects. This outcome is concordant with in vitro findings using cultured ECs transduced with these constructs to activate plasminogen in situ.²² Of note, concerns based on data contained in these and other in vitro studies of transduced ECs¹⁹ that wild-type TPA would be ineffective in vivo due to inactivation by EC-derived PAI-1 appear to be unfounded. The present data support the view that serpin-resistant TPA variants^{41 42} offer no advantage over wild-type TPA except when massive excesses of PAI-1 are present, such as in EC culture systems^{22 43} or in vivo after injecting very large amounts of PAI-1.⁴⁴ Specific testing of genetic constructs expressing serpin-resistant plasminogen activators in the present in vivo system would directly address this question.

It is important to emphasize both the strengths and the limitations of the experimental system used. An important strength is the use of a well-characterized nonhuman primate model of mural thrombosis. Baboons were chosen for this study because (1) baboons exhibit thrombotic and vascular response mechanisms closely simulating humans; (2) this particular thrombosis model is well characterized, reproducible, and quantitative, allowing detection of focal antithrombotic effects; and (3) as in humans, the formation of thrombus is abolished by either TPA or urokinase in this species.^{27 45 46} Accordingly, we believe that the results can reasonably be extrapolated to human thrombosis and fibrinolysis.

There are at least two significant limitations of the experimental system. First, while in the present study the period of observation was 60 minutes, it is theoretically possible that thrombo-occlusive events might develop many hours or days later. Realizing long-term persistence of these antithrombotic benefits will depend on both the ability of the transduced cells to survive in vivo and the capacity of these cells to continue secreting significant levels of recombinant gene products. In vitro, retrovirally transduced baboon endothelial cells express stable levels of recombinant gene products for several weeks (data not shown). However, data addressing the in vivo persistence of seeded EC and the continued ability of these seeded cells to express recombinant genes are more equivocal. Wilson et al¹⁸ demonstrated that transduced canine endothelial cells seeded onto Dacron grafts survived and continued to express a recombinant gene for at least 5 weeks, but the more recent results of Conte et al¹⁵ show that the level of recombinant gene expression from rabbit EC decays significantly within 1 week of reimplantation in vivo. Data from human trials of implantation of prosthetic devices seeded with autologous untransduced EC provide indirect evidence that seeded EC survive for at least 30 days and potentially as long as 3 years in vivo.^{47 48} It is apparent from these data that long-term trials testing the hypothesis that seeded, transduced EC can survive in vivo for prolonged periods of time are essential in defining the ultimate clinical promise of the short-term antithrombotic benefits demonstrated in the present study.

A second significant limitation of our data is that we compared the effects of transduced and untransduced ECs at subconfluent density, with contiguous exposure of thrombogenic collagen. This strategy was used because previous experiments showed that confluent coverage of collagen segments with untransduced ECs abolished thrombus accumulation,⁴⁹ rendering a confluent system unable to distinguish any additional antithrombotic effects mediated by elevated levels of plasminogen activators. However, mechanical interventional procedures, such as angioplasty and endarterectomy, produce variably complete denudation with adjacent largely intact endothelium and therefore comprise a clinical correlate with the experimental model. Because plasminogen activators are secreted directly into the slow-moving, poorly diffusible luminal boundary layer, the attachment of transduced ECs onto such denuded endovascular surfaces could be expected to produce significant thrombolytic benefits. Thus, genetic modification of ECs immediately upstream from these thrombogenic sites is predicted to reduce acute thrombo-occlusive events and improve late patency.

In summary, the present findings support the approach of establishing transduced ECs at sites of mechanical vascular injury or on luminal surfaces of implanted chronic cardiovascular grafts. Additionally, EC gene transfer may prove to be an effective targeting strategy for delivering interventional molecules locally at sites of denuding vascular injury without incurring possible adverse effects and without the commitment of resources required for systemic therapies using the same molecules.

	Selected Abbreviations and Acronyms

 

a-UPA = GPI-anchored UPA EC = endothelial cell FPA = fibrinopeptide A GPI = glycosylphosphatidylinositol PAI-1 = plasminogen activator inhibitor type-1 TAT = thrombin-antithrombin complex TPA = tissue-type plasminogen activator UPA = urokinase-type plasminogen activator

	Acknowledgments

 
This work was supported in part by grants HL-41619, HL-48667, RR-00165 and the Division of Intramural Research of the National Heart, Lung, and Blood Institute, National Institutes of Health. We acknowledge the valuable technical expertise of Deborah White in these studies.

Received September 20, 1994; revision received June 13, 1995; accepted August 29, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Harker LA, Mann KG. Thrombosis and fibrinolysis. In: Fuster V, Verstraete M, eds. Thrombosis in Cardiovascular Disorders. Philadelphia, Pa: WB Saunders Co; 1992:1-16.
   
2. Esmon CT. The regulation of natural anticoagulant pathways. Science. 1987;235:1348-1352. [Abstract/Free Full Text]
   
3. Hanson SR, Griffin JH, Harker LA, Kelly AB, Esmon CT, Gruber A. Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates. J Clin Invest. 1993;92:2003-2012.
   
4. Weksler BB, Marcus AJ, Jaffe EA. Synthesis of prostaglandin I-2 (prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci U S A. 1977;74:3922-3926. [Abstract/Free Full Text]
   
5. Moncada S. Prostacyclin and arterial wall biology. Arteriosclerosis. 1982;2:193-207. [Free Full Text]
   
6. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987;84:9265-9269. [Abstract/Free Full Text]
   
7. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526. [Medline] [Order article via Infotrieve]
   
8. De Graaf JC, Banga JD, Moncada S, Palmer RMJ, de Groot PG, Sixma JJ. Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions. Circulation. 1992;85:2284-2290. [Abstract/Free Full Text]
   
9. Levin EG. Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex. Proc Natl Acad Sci U S A. 1983;80:6804-6808. [Abstract/Free Full Text]
   
10. Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV. Cloning and expression of human tissue-type plasminogen activator cDNA in E coli. Nature. 1983;301:214-221. [Medline] [Order article via Infotrieve]
    
11. Dichek D, Quertermous T. Thrombin regulation of mRNA levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells. Blood. 1989;74:222-228. [Abstract/Free Full Text]
    
12. Zwiebel JA, Freeman SM, Kantoff PW, Cornetta K, Ryan US, Anderson WF. High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors. Science. 1989;243:220-222. [Abstract/Free Full Text]
    
13. Dichek DA, Neville RF, Zwiebel JA, Freeman SM, Leon MB, Anderson WF. Seeding of intravascular stents with genetically engineered endothelial cells. Circulation. 1989;80:1347-1353. [Abstract/Free Full Text]
    
14. Nabel EG, Plautz G, Boyce FM, Stanley JC, Nabel GJ. Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science. 1989;244:1342-1344. [Abstract/Free Full Text]
    
15. Conte MS, Birinyi LK, Miyata T, Fallon JT, Gold HK, Whittemore AD, Mulligan RC. Efficient repopulation of denuded rabbit arteries with autologous genetically modified endothelial cells. Circulation. 1994;89:2161-2169. [Abstract/Free Full Text]
    
16. Flugelman MY, Virmani R, Leon MB, Bowman RL, Dichek DA. Genetically engineered endothelial cells remain adherent and viable after stent deployment and exposure to flow in vitro. Circ Res. 1992;70:348-354. [Abstract/Free Full Text]
    
17. Shayani V, Newman KD, Dichek DA. Optimization of recombinant t-PA secretion from seeded vascular grafts. J Surg Res. 1994;57:495-504. [Medline] [Order article via Infotrieve]
    
18. Wilson JM, Birinyi LK, Salomon RN, Libby P, Callow AD, Mulligan RC. Implantation of vascular grafts lined with genetically modified endothelial cells. Science. 1989;244:1344-1346. [Abstract/Free Full Text]
    
19. Dichek DA, Nussbaum O, Degen SJF, Anderson WF. Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer. Blood. 1991;77:533-541. [Abstract/Free Full Text]
    
20. Lee SW, Kahn ML, Dichek DA. Expression of an anchored urokinase in the apical endothelial cell membrane. J Biol Chem. 1992;267:13020-13027. [Abstract/Free Full Text]
    
21. Lee SW, Ellis V, Dichek DA. Characterization of plasminogen activation by glycosylphosphatidylinositol-anchored urokinase. J Biol Chem. 1994;269:2411-2418. [Abstract/Free Full Text]
    
22. Dichek DA, Lee SW, Nguyen NH. Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors. Blood. 1994;84:504-516. [Abstract/Free Full Text]
    
23. Lisanti MP, Rodriguez-Boulan E. Polarized sorting of GPI-linked proteins in epithelia and membrane microdomains. Cell Biol Int. 1991;15:1023-1049.
    
24. Schneider PA, Hanson SR, Price TM, Harker LA. Confluent durable endothelialization of endarterectomized baboon aorta by early attachment of cultured endothelial cells. J Vasc Surg. 1990;11:365-372. [Medline] [Order article via Infotrieve]
    
25. Schneider PA, Hanson SR, Price TM, Harker LA. Durability of confluent endothelial cell monolayers on small-caliber vascular prostheses in vitro. Surgery. 1988;103:456-462. [Medline] [Order article via Infotrieve]
    
26. Hanson SR, Kotze HF, Savage B, Harker LA. Platelet interactions with Dacron vascular grafts: a model of acute thrombosis in baboons. Arteriosclerosis. 1985;5:595-603. [Abstract/Free Full Text]
    
27. Runge MS, Harker LA, Bode C, Ruef J, Kelly AB, Marzec UM, Caban R, Shaw S-Y, Haber E, Hanson SR. Enhanced thrombolytic and antithrombotic potency of a fibrin-targeted plasminogen activator in baboons. Circulation. In press.
    
28. Savage B, McFadden PR, Hanson SR, Harker LA. The relation of platelet density to platelet age: survival of low and high-density ¹¹¹Indium-labeled platelets in baboons. Blood. 1986;68:386-393. [Abstract/Free Full Text]
    
29. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
    
30. Harker LA, Hanson SR, Kirkman TR. Experimental arterial thromboembolism in baboons: mechanism, quantitation, and pharmacologic prevention. J Clin Invest. 1979;64:559-569.
    
31. Cadroy Y, Harker LA, Hanson SR. Inhibition of platelet-dependent thrombosis by low molecular weight heparin (CY222): comparison with standard heparin. J Lab Clin Med. 1989;114:349-357. [Medline] [Order article via Infotrieve]
    
32. Hanson SR, Harker LA. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone. Proc Natl Acad Sci U S A. 1988;85:3184-3188. [Abstract/Free Full Text]
    
33. Kelly AB, Marzec UM, Krupski W, Bass A, Cadroy Y, Hanson SR, Harker LA. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood. 1991;77:1006-1012. [Abstract/Free Full Text]
    
34. Sherry S. Fibrinolysis, Thrombolysis and Hemostasis: Concepts, Perspectives, and Clinical Applications. Malvern, Pa: Lea & Febiger; 1992:18-23.
    
35. Gimbrone MA Jr. Vascular endothelium: nature's blood container. In: Gimbrone MA Jr, ed. Vascular Endothelium in Hemostasis and Thrombosis. New York, NY: Churchill Livingstone; 1986:1-13.
    
36. Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, DeVos R, van den Oord JJ, Collen D, Mulligan RC. Physiological consequences of loss of plasminogen activator gene function in mice. Nature. 1994;368:419-424. [Medline] [Order article via Infotrieve]
    
37. Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest. 1990;86:385-391.
    
38. Lumsden AB, Kelly AB, Schneider PA, Krupski WC, Dodson T, Hanson SR, Harker LA. Lasting safe interruption of endarterectomy thrombosis by transiently infused antithrombin peptide D-Phe-Pro-ArgCH2Cl in baboons. Blood. 1993;81:1762-1770. [Abstract/Free Full Text]
    
39. Nemerson Y, Turitto VT. The effect of flow on hemostasis and thrombosis. Thromb Haemost. 1991;66:272-276. [Medline] [Order article via Infotrieve]
    
40. Bu G, Warshawsky I, Schwartz AL. Cellular receptors for the plasminogen activators. Blood. 1994;83:3427-3436. [Free Full Text]
    
41. Madison EL, Goldsmith EJ, Gerard RD, Gething MJ, Sambrook JF. Serpin-resistant mutants of human tissue-type plasminogen activator. Nature. 1989;339:721-724. [Medline] [Order article via Infotrieve]
    
42. Madison EL, Goldsmith EJ, Gerard RD, Gething MH, Sambrook JF, Bassel-Duby RS. Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1. Proc Natl Acad Sci U S A. 1990;87:3530-3533. [Abstract/Free Full Text]
    
43. van Mourik JA, Lawrence DA, Loskutoff DJ. Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J Biol Chem. 1984;259:14914-14921. [Abstract/Free Full Text]
    
44. Li X-K, Lijnen HR, Nelles L, Van Hoef B, Stassen JM, Collen D. Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1. Blood. 1992;79:417-429. [Abstract/Free Full Text]
    
45. Hanson SR, Harker LA. Baboon models of acute arterial thrombosis. Thromb Haemost. 1987;58:801-805. [Medline] [Order article via Infotrieve]
    
46. Del Zoppo GJ, Copeland BR, Harker LA, Waltz TA, Zyroff J, Hanson SR, Battenberg E. Experimental acute thrombotic stroke in baboons. Stroke. 1986;17:1254-1265. [Abstract/Free Full Text]
    
47. Fischlein T, Zilla P, Meinhart J, Puschmann R, Vesely M, Eberl T, Balon R, Deutsch M. In vitro endothelialization of a mesosystemic shunt: a clinical case report. J Vasc Surg. 1994;19:549-554. [Medline] [Order article via Infotrieve]
    
48. Zilla P, Deutsch M, Meinhart J, Puschmann R, Eberl T, Minar E, Dudezak R, Lugmaier H, Schmidt P, Noszian I, Fischlein T. Clinical in vitro endothelialization of femoropopliteal bypass grafts: an actuarial follow-up over three years. J Vasc Surg. 1994;19:540-548. [Medline] [Order article via Infotrieve]
    
49. Schneider P, Hanson S, Price T, Harker L. Preformed confluent endothelial cell monolayers prevent early platelet deposition on vascular prostheses in baboons. J Vasc Surg. 1988;8:2229-2235.
    

This article has been cited by other articles:

				S.K. Payeli, R. Latini, C. Gebhard, A. Patrignani, U. Wagner, T.F. Luscher, and F.C. Tanner Prothrombotic Gene Expression Profile in Vascular Smooth Muscle Cells of Human Saphenous Vein, but Not Internal Mammary Artery Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 705 - 710. [Abstract] [Full Text] [PDF]

				S. Zaitsev, K. Danielyan, J.-C. Murciano, K. Ganguly, T. Krasik, R. P. Taylor, S. Pincus, S. Jones, D. B. Cines, and V. R. Muzykantov Human complement receptor type 1-directed loading of tissue plasminogen activator on circulating erythrocytes for prophylactic fibrinolysis Blood, September 15, 2006; 108(6): 1895 - 1902. [Abstract] [Full Text] [PDF]

				F. Sharif, K. Daly, J. Crowley, and T. O'Brien Current status of catheter- and stent-based gene therapy Cardiovasc Res, November 1, 2004; 64(2): 208 - 216. [Abstract] [Full Text] [PDF]

				L. G. Melo, M. Gnecchi, A. S. Pachori, D. Kong, K. Wang, X. Liu, R. E. Pratt, and V. J. Dzau Endothelium-Targeted Gene and Cell-Based Therapies for Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1761 - 1774. [Abstract] [Full Text] [PDF]

				S. Wen, S. Graf, P. G. Massey, and D. A. Dichek Improved Vascular Gene Transfer With a Helper-Dependent Adenoviral Vector Circulation, September 14, 2004; 110(11): 1484 - 1491. [Abstract] [Full Text] [PDF]

				L.G Melo, M Gnecchi, A.S Pachori, K Wang, and V.J Dzau Gene- and cell-based therapies for cardiovascular diseases: current status and future directions Eur. Heart J. Suppl., September 1, 2004; 6(suppl_E): E24 - E35. [Abstract] [Full Text]

				J.-C. Murciano, S. Muro, L. Koniaris, M. Christofidou-Solomidou, D. W. Harshaw, S. M. Albelda, D. N. Granger, D. B. Cines, and V. R. Muzykantov ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface Blood, May 15, 2003; 101(10): 3977 - 3984. [Abstract] [Full Text] [PDF]

				M. Falkenberg, C. Tom, M. B. DeYoung, S. Wen, R. Linnemann, and D. A. Dichek Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth PNAS, August 6, 2002; 99(16): 10665 - 10670. [Abstract] [Full Text] [PDF]

				J. Ai Kim, C. C. Hedrick, Dangci Xie, and M. J. Fisher Adenoviral-Mediated Transfer of Tissue Plasminogen Activator Gene into Brain Capillary Endothelial Cells In Vitro Angiology, September 1, 2001; 52(9): 627 - 634. [Abstract] [PDF]

				P. Golino, P. Cirillo, P. Calabro', M. Ragni, D. D'Andrea, E. V. Avvedimento, F. Vigorito, N. Corcione, F. Loffredo, and M. Chiariello Expression of exogenous tissue factor pathway inhibitor in vivo suppresses thrombus formation in injured rabbit carotid arteries J. Am. Coll. Cardiol., August 1, 2001; 38(2): 569 - 576. [Abstract] [Full Text] [PDF]