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Cardiovascular Research 1997 35(3):459-469; doi:10.1016/S0008-6363(97)00153-3
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Gene therapy for arterial thrombosis

Giuseppe Vassalli and David A Dichek*

Gladstone Institute of Cardiovascular Disease, Department of Medicine, University of California, San Francisco, CA 94141-9100, USA

* Corresponding author. Gladstone Institute of Cardiovascular Disease, P.O. Box 419100, San Francisco, CA 94141-9100, USA. Tel.: +1 (415) 826-7500; fax: +1 (415) 285-5632; e-mail: david_dichek@quickmail.ucsf.edu

Received 10 April 1997; accepted 23 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Potential feasibility of...
 3 Candidate genes for...
 4 Summary and future...
 References
 
Conventional antithrombotic treatments with antiplatelet, anticoagulant, or fibrinolytic drugs are not uniformly successful and are associated with hemorrhagic side effects. Thus, new approaches to the prevention and treatment of arterial thrombosis are desirable. The gene transfer approach is particularly attractive because of its unique ability to express an antithrombotic gene at selected sites of the vessel wall (where thrombosis is threatened) while avoiding systemic anticoagulation. Clinical conditions potentially amenable to antithrombotic gene therapy include coronary artery bypass grafting, percutaneous transluminal coronary angioplasty, peripheral artery angioplasty or thrombectomy, intravascular stenting, and vascular graft prostheses. Gene therapy may prove effective in preventing subacute thrombosis in these settings and, eventually, may play an adjuvant role to systemic thrombolysis in the treatment of acute arterial occlusion. The introduction of an antithrombotic gene into the arterial wall can be achieved either by direct in vivo gene transfer (e.g., by luminal administration of a viral vector) or by in vitro genetic manipulation of cells before their seeding onto vascular grafts, stents, or denuded arteries. The direct gene transfer approach has been used to deliver antithrombotic genes to animal arteries in vivo. Antithrombotic genes used to date include those encoding enzymes of the prostacyclin synthetic pathway, nitric oxide synthase, the thrombin inhibitor hirudin, and thrombomodulin. The in vitro gene transfer approach has been used to enhance the fibrinolytic activity of vascular grafts by overexpressing plasminogen activators. If the initial successes of gene therapy for thrombotic disease in animal models are confirmed by longer-term experiments, and if new vectors are developed which permit prolonged transgene expression without inflammation, human studies can be initiated.

KEYWORDS Gene therapy; Thrombosis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Potential feasibility of...
 3 Candidate genes for...
 4 Summary and future...
 References
 
The limitations of current antithrombotic therapy have necessitated investigations of new approaches to the prevention and treatment of arterial thrombosis. Conventional antithrombotic treatments rely on the systemic administration of drugs designed to inhibit clot formation (e.g., coumarin anticoagulants) or to lyse existing clots (e.g., tissue-type plasminogen activator; t-PA). These antithrombotic agents are not uniformly successful and are associated with hemorrhagic complications.

Gene transfer approaches to the prevention of arterial thrombosis are based on widely accepted models of intravascular clot formation. These models predict that thrombosis will most likely occur in the presence of functional defects of the vessel wall. The potential of the arterial wall to participate actively in coagulation and fibrinolysis has long been recognized [1]. Endothelial cells produce molecules with antiplatelet (e.g., prostacyclin [2]), anticoagulant (e.g., thrombomodulin [3]), and fibrinolytic (e.g., t-PA [4]) activities. A failure of endothelial cells to fulfill their antithrombotic functions can result in clot formation [5]. In addition, increased amounts of prothrombotic and antifibrinolytic factors (e.g., plasminogen activator inhibitor-1 [6]) are produced in diseased arteries by endothelial cells and infiltrating mononuclear cells.

Gene therapy for arterial thrombosis is aimed at restoring the homeostatic balance between prothrombotic and antithrombotic factors, thereby enhancing the ability of the vessel wall to antagonize clot formation. The rationale of this approach is to express a protective gene at highest concentration at selected sites of the vessel wall, where thrombosis is imminent. For example, we have used retroviral vectors encoding either t-PA or a cell surface-anchored form of pro-urokinase to transduce baboon endothelial cells [7]. Gene transfer increased the expression of t-PA and cell surface-anchored pro-urokinase 10-fold in vitro and reduced thrombus formation in vascular grafts seeded with transduced cells in a baboon model of thrombosis in vivo. The focal antithrombotic effect of transduced endothelial cells appeared to be due to local enhancement of thrombolysis and had no systemic effects.

The challenges of antithrombotic gene therapy fall into three categories: identification of appropriate genes as targets for manipulation, construction of efficient and nontoxic gene transfer vectors, and technical development of clinical gene transfer protocols.

Several candidate genes for antithrombotic gene therapy have been identified. These candidate genes encode important anticoagulant and fibrinolytic proteins (e.g., thrombomodulin [8], t-PA [9], and urokinase [10]), as well as key cellular enzymes and proteins involved in the synthesis of nonprotein anticoagulant molecules such as prostacyclin [11]and endothelial cell heparan sulfates [12].

Recent advances have been made in the development of biological vectors that are suitable for introducing genes into the vessel wall [13]. Adenoviral vectors are currently the vectors-of-choice for vascular gene delivery. They are highly efficient [14–17], but their use is limited by a short duration of transgene expression due to a T cell-mediated immune response to viral proteins expressed from the vector backbone [18–20]. This problem has not yet been resolved, but recent studies suggest that the immune response to adenovirus may be minimized or eliminated either by engineering of the vectors to decrease expression of viral genes or by suppression of the immune system. Indeed, an adenoviral vector lacking all viral genes expressed a recombinant gene in mouse skeletal muscle for up to 3 months, with no detectable inflammation [21]. Future studies will determine whether these exciting findings can be extended to other tissues and other species.

The development of clinically applicable vascular gene transfer protocols is also a significant challenge. In studies performed in animal models, various clinically relevant procedures have been used to introduce genes of interest into the vessel wall, either surgically or percutaneously. Percutaneous gene delivery systems include double-balloon catheters [22], perforated perfusion [23]or infusion [24]catheters, and hydrogel catheters [25]. Topical injection of vectors into the periarterial sheath has also been described [26]. This latter protocol is suitable for surgical approaches. An alternative approach to direct gene delivery to the vessel wall in vivo involves the harvest and genetic manipulation of vascular cells in vitro before their reimplantation to the donor organism in vivo [27]. Examples of such an ex vivo gene transfer approach include seeding of intravascular stent devices [28]and vascular graft prostheses [7]with genetically modified endothelial cells that overexpress fibrinolytic genes.

As a consequence of the complexity of these challenges, only one human trial in vascular gene therapy has been initiated. This trial involves a single intraarterial injection of naked DNA encoding vascular endothelial growth factor (VEGF) to induce therapeutic angiogenesis in patients with peripheral vascular disease [29, 30]. Thus, virtually all of the available data on gene therapy for arterial thrombosis have been obtained in in vitro experiments or in vivo animal models. Based on the results of these studies, the feasibility of gene therapy for specific thrombotic diseases is discussed in the first part of the present review. In the following sections, we discuss potential candidate genes for antithrombotic gene therapy and we outline future research directions.


   2 Potential feasibility of gene therapy in thrombotic diseases
Top
 Abstract
 1 Introduction
 2 Potential feasibility of...
3 Candidate genes for...
 4 Summary and future...
 References
 
The design of gene transfer strategies for preventing arterial thrombosis must take many factors into account. These factors include the biological effect of the transgene, the time course of expression, the efficiency and safety of the gene transfer vector, the anatomy of the diseased artery, the tolerance of distal tissues to transient ischemia, and the availability of a gene delivery device and protocol that are suitable for a specific clinical condition.

2.1 Coronary artery disease
Intravascular clot formation is a major cause of acute myocardial infarction and contributes to the majority of sudden deaths in patients with coronary artery disease [31]. Thrombolysis for acute coronary artery occlusion must be started early, ideally within 1 h after the onset of symptoms [32]. Since achievement of maximal transgene expression after adenovirus-mediated gene transfer into the vessel wall takes at least 1 day [33], gene therapy is not suitable as primary thrombolytic therapy for acute coronary occlusion. Nevertheless, it is tempting to speculate that gene therapy will eventually play an adjuvant role to systemic fibrinolysis in the treatment of acute thrombosis. The local overexpression of a fibrinolytic gene might be useful in preventing subacute reocclusion, rebound thrombogenesis after heparin treatment, and chronic arterial narrowing. This last potential effect is suggested by continuous blood flow monitoring studies showing that recurrent thrombus deposition contributes to the development of chronic stenosis in injured arteries [34]. The therapeutic effect of antithrombotic gene therapy in the coronary circulation might be enhanced by the delivery of a gene that has both antithrombotic and antiproliferative functions (e.g., hirudin [35]).

Gene transfer into coronary arteries poses special technical problems. Prolonged incubation of a gene transfer vector solution in the arterial lumen is desirable because in vitro expression of recombinant protein correlates directly with the duration of exposure of cells to vector DNA [36]. A modified perfusion balloon catheter that permits perfusion of the distal vasculature while the transgene is injected into the vessel wall through laser-generated pores may provide a solution to this problem. Perfusion balloon catheters have been used to deliver genes into canine [23]and porcine coronary arteries [33], but these devices require further refinement before they can be used for human gene therapy. Improvements of these catheters must be directed at increasing the efficiency of gene transfer, providing adequate perfusion of the distal vascular bed, and avoiding systemic dissemination of the gene transfer vector during or after the infusion.

2.2 Coronary artery bypass graft (CABG) and percutaneous transluminal coronary angioplasty (PTCA)
Antiplatelet and anticoagulant agents are effective in decreasing thrombotic occlusion after CABG [37]and PTCA [38]. However, thrombotic occlusion after these procedures has not yet been eliminated. Gene transfer approaches may be particularly attractive for CABG and elective PTCA because they might improve the outcomes without substantially increasing the risk. Delivery of a therapeutic gene to a bypass conduit can be easily performed during CABG, as the conduit is manipulated ex vivo before implantation. Catheter-mediated gene transfer could be carried out with a gene delivery perfusion catheter (exchanged over a guidewire) immediately after the deflation of the PTCA balloon catheter. Alternatively, it is possible that new three-compartment PTCA balloon catheters will be developed that permit simultaneous distension of the stenotic arterial segment at therapeutic pressures and introduction of a gene of interest into the vessel wall, while ensuring distal perfusion during the whole procedure.

2.3 Intracoronary stents
The recent development of ultrasound-guided techniques for stent deployment in coronary arteries has allowed stent placement to be carried out with antiplatelet therapy alone (i.e., without anticoagulation) [39]. However, subacute coronary artery events still occur in 1.9% of patients receiving only antiplatelet therapy [40]. Additionally, in-stent restenosis rates of 13% have been reported even in the most optimistic studies [41]. A theoretical solution to the problem of stent thrombosis and restenosis is to promote rapid vascular healing and restoration of a nonthrombogenic luminal surface. To accomplish this, endothelial cells have been ‘seeded’ onto the stent surface to create an endothelial monolayer [42]. The hypothesis that seeding will prevent stent thrombosis is based on the clinical observation that clot formation occurs rarely after complete reendothelialization of the stent surface.

Seeded endothelial cells could potentially be engineered to express antithrombotic recombinant genes. In an initial study we used retrovirus-mediated gene transfer to insert the gene for human t-PA into cultured endothelial cells from sheep saphenous veins. These cells were then seeded onto Palmaz-Schatz stents [28]. High level secretion of t-PA was demonstrated after the transduced cells were seeded onto the stents. After expansion of the stent in vitro, much of the layer of genetically modified cells remained adherent to the stent. Some of the seeded cells survived on the stent surface for at least 10 days after stent deployment in sheep femoral arteries and exposure to flow, as determined by polymerase chain reaction [43]. Before this approach is useful in humans, it will be necessary to overcome several limitations. Stent seeding is impractical because endothelial cells must be harvested from the patient and genetically modified in vitro. Moreover, cell adherence to stent surfaces under flow conditions needs to be improved substantially. For these reasons, alternative methods such as ultrasound-guided stent deployment [39]and heparin-coated stents [41]are currently more attractive than is the use of seeded stents.

2.4 Peripheral arterial disease
Gene therapy for acute peripheral artery thrombosis faces obstacles similar to those outlined above for coronary arteries. However, gene transfer into arteries of the lower extremities is technically less demanding because the vessels are more uniformly accessible to catheters and because skeletal muscle has a higher ischemic tolerance than cardiac muscle. Several studies have documented efficient percutaneous gene transfer into iliofemoral arteries in various animal models [16, 22]. Overexpression of a gene with antithrombotic functions in peripheral arteries will be most suitable for preventing subacute thrombosis after angioplasty or thrombectomy. In this setting gene therapy will likely play an adjuvant role to anticoagulant and antiplatelet drugs, which will still be required in the acute phase of the intervention [44].

2.5 Cerebrovascular disease
The extracranial carotid arteries are foci for thrombosis and embolism and therefore are also potential targets for antithrombotic gene therapy. Gene delivery to the extracranial carotid arteries by surgical approaches has been successfully performed in several animal models [15, 44, 45]. In a clinical setting, luminal or perivascular delivery of an antithrombotic gene performed during carotid endarterectomy might improve the long-term success of the procedure. Use of catheter-mediated antithrombotic gene therapy as an alternative (rather than adjuvant) to surgical endarterectomy is not currently attractive. Catheter-mediated gene delivery to the carotid arteries is technically challenging because of the risk of cerebral ischemia and embolization of plaque debris. Delivery of a marker gene to rat intracranial arteries has been achieved with an adenoviral vector injected into the cerebrospinal fluid [46]. This modality has not yet been used to deliver a therapeutic gene, and the intracranial arteries are not usually a site of primary thrombotic disease.

2.6 Pulmonary thromboembolic disease
We previously reported that adenovirus-mediated gene transfer into rat pulmonary arteries is feasible, albeit at low levels, with a surgical (but not a percutaneous) approach [47]. The great majority of the thrombi found in the pulmonary vasculature originate in peripheral veins, and a potent antithrombotic effect in the venous system is therefore required to prevent pulmonary thromboembolism. Gene delivery is probably not a preferred therapy for a first episode of venous thromboembolism, as this is effectively treated with a brief course of anticoagulant therapy. However, a recent study indicates that after a second episode of pulmonary thromboembolism anticoagulation should be continued for at least 6 months and, potentially, indefinitely [48]. In this setting, antithrombotic gene therapy might be considered, as it would represent an attractive alternative to prolonged courses of oral medication. From a theoretical point of view, the delivery of an antithrombotic gene to lower-extremity arteries during transient blockade of the venous return by tourniquets might result in effective delivery of the gene to the venous system. If the secreted gene product were cleared significantly before recirculation, a locally enhanced anticoagulant activity with relatively less systemic anticoagulation might be achieved. A significant concern in this context is the difficulty of reversing antithrombotic gene therapy should a patient with genetically enhanced anticoagulant or thrombolytic activity start to bleed.

2.7 Prosthetic vascular grafts
Small-diameter (≤4 mm) prosthetic vascular grafts are inferior to autologous arteries and veins in both coronary arterial and infrainguinal revascularization procedures [49]. One way to improve the performance of small-diameter vascular prostheses would be to seed them with an autologous endothelial monolayer, which might survive at the graft surface for prolonged periods of time in vivo after graft implantation [50]. Firm evidence that endothelial seeding of vascular grafts is beneficial in humans is somewhat scant [51]. In one study the 32-month patency rate of human lower-extremity 6-mm bypass grafts was 85% for seeded grafts compared with 55% for unseeded grafts (p<0.05 and p = 0.07 by Kaplan-Meier life-table analysis using the generalized Wilcoxon and Savage tests, respectively) [52]. Although endothelial seeding seems to decrease platelet accumulation [53], no improvement of graft patency was found in another study [54].

In an attempt to enhance vascular graft patency, we have used gene transfer techniques to increase the fibrinolytic activity of endothelial cells that were subsequently seeded on vascular prostheses. Transduction of baboon endothelial cells with retroviral vectors encoding either t-PA or a cell surface-anchored form of urokinase plasminogen activator (see below) increased plasminogen activator expression 10-fold in vitro. Seeding of collagen-coated segments of vascular grafts with transduced cells reduced thrombus formation in a baboon model of graft thrombosis (Fig. 1) [7]. These results are encouraging, but they were obtained over a time course of only 1 h. To improve graft patency, gene therapy must continue to be effective for many years.


Figure 1
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  		Fig. 1 Deposition of 111In-labeled platelets as a quantitative measure of the antithrombotic effect of endothelial cells overexpressing plasminogen activators seeded onto collagen-coated segments of vascular grafts. Grafts were seeded at equivalent densities with either untransduced or transduced endothelial cells and were interposed in exteriorized femoral arteriovenous shunts in baboons. Platelet accumulation is shown in real time for untransduced endothelial cells (open circles; n = 13), endothelial cells transduced with a retroviral vector containing a t-PA expression cassette (closed circles; n = 8), and endothelial cells transduced with a retroviral vector containing an expression cassette for cell surface-anchored pro-urokinase (a-uPA; open squares; n = 5). All data are presented as mean±S.E.M. There was a significant decrease in platelet deposition on collagen graft segments seeded with endothelial cells overexpressing t-PA (p = 0.026 by repeated measures ANOVA) vs. grafts seeded with untransduced cells. From: Dichek et al., 1996 [7]; reproduced with the permission of the American Heart Association.

 

   3 Candidate genes for antithrombotic gene therapy
Top
 Abstract
 1 Introduction
 2 Potential feasibility of...
 3 Candidate genes for...
4 Summary and future...
 References
 
Potential candidate genes for gene therapy of arterial thrombosis include those encoding proteins with antiplatelet, anticoagulant, or fibrinolytic activities. Subdivision into these three categories is imperfect, as many gene products have antithrombotic activity based on more than one effect (e.g., hirudin likely has both antiplatelet and anticoagulant effects). Some of these genes are listed in Table 1. Neither this table nor the following paragraphs are intended as an exhaustive list of genes with antithrombotic activity, but rather serve as an overview of those genes either used to date or that appear most likely to be useful in gene transfer studies.


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  		Table 1 Candidate genes for antithrombotic gene therapy

 
3.1 Genes with antiplatelet activity
Biological inhibitors of platelet aggregation include prostacyclin, nitric oxide (NO), and thrombin inhibitors. Prostacyclin also inhibits vasoconstriction, vascular smooth muscle cell proliferation, and the interaction of monocytes with the endothelium [55]. Initial attempts to treat thrombosis with systemic prostacyclin infusion failed because of unacceptable systemic complications [56, 57]. In vivo adenovirus-mediated gene transfer of cyclooxygenase-1 (the rate-limiting step of prostacyclin synthesis) into porcine carotid arteries increased prostacyclin synthesis 4-fold at 10 days after infection and completely eliminated arterial thrombosis after balloon injury (Fig. 2) [44]. Notably, however, initial administration of high-dose heparin was required because gene transfer did not protect against thrombus deposition immediately after injury.


Figure 2
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  		Fig. 2 Occurrence of zero-flow and frequency of cyclic flow variations as a measure of thrombus formation in balloon-injured porcine carotid arteries. Arteries were transduced with an adenoviral vector that contains the cyclooxygenase gene (Ad-COX-1), a control vector with no foreign gene (Ad-null), or with buffer alone. The dose of Ad-COX-1 was 3x1010 and 5x109 plaque-forming units. Cyclic flow variations, as defined as changes in cyclic flow >25%, are indicated as mean±S.D. during a 10-day monitoring period. High dose Ad-COX-1 abolished zero-flow episodes and cyclic flow variations. Comparisons of all the other values were statistically insignificant. Modified from: Zoldhelyi et al., 1996 [44]; reproduced with the permission of the American Heart Association.

 
Endothelium-derived NO inhibits platelet adhesion and aggregation, vascular smooth muscle cell proliferation, and vasoconstriction [58]. The antiplatelet effect of NO is illustrated by a recent study showing that a novel NO-releasing agent, proline NO adduct, inhibits platelet-dependent thrombus formation in the baboon arteriovenous shunt [59]. Inducible NO synthase (NOS) expressed by medial and neointimal smooth muscle cells antagonized platelet adhesion to injured rat carotid arteries [60]. Gene transfer of endothelial NOS (eNOS) into rat carotid arteries with a Sendai virus/liposome vector restored eNOS expression in balloon-injured arteries, resulting in a decreased neointima formation [61], potentially mediated, at least in part, by an antithrombotic effect. Adenovirus-mediated gene transfer of human constitutive eNOS into injured rat carotid arteries also inhibited neointima formation [62]. Single aerosol delivery of this adenoviral vector to the lung decreased collagen-induced platelet aggregation in explanted arteries in vitro and decreased neointima formation in injured rat carotid arteries in vivo [63]. These studies illustrate not only that NOS may be an effective gene therapy agent but also that aerosol delivery to the lung and subsequent dissemination via the circulation may represent an alternative approach to antithrombotic gene therapy.

3.2 Genes with anticoagulant activity
Anticoagulant gene products include hirudin, thrombomodulin, tissue factor pathway inhibitor (TFPI), and specific factor Xa inhibitors. The leech anticoagulant hirudin is the most potent and specific inhibitor of thrombin known [64]. Thrombin effects fibrinogen cleavage [65], platelet activation [66], and smooth muscle cell proliferation [67]. Intravenously administered hirudin has been successful in preventing both thrombosis [68]and neointima formation [69]after arterial injury in animal models. Clinical studies have evaluated the efficacy of hirudin both as an adjunct to thrombolytic therapy [70]and as an inhibitor of restenosis after angioplasty [71]. Although this drug has shown some promise as a therapeutic agent [72], the GUSTO IIa trial showed that hirudin delivered systemically can also cause a systemic coagulopathy [73]. Recent data from the GUSTO IIb substudy in patients with acute coronary syndromes show that hirudin is associated with greater thrombin inhibition and less rebound thrombin formation than heparin [74]. This is important because rebound generation of thrombin after coronary interventions may contribute to subacute thrombosis [75].

We have used an adenoviral vector to express hirudin in vascular cells in vitro and in rat carotid arteries in vivo [35]. A significant decrease in neointima formation was observed in arteries transduced with the hirudin cDNA (Fig. 3). Both antithrombotic activity and the inhibition of smooth muscle cell proliferation may have contributed to the decrease in neointima formation after hirudin gene transfer. Systemic partial thromboplastin times were not affected by local hirudin expression, suggesting that this local delivery approach can avoid the systemic side effects of conventional antithrombotic treatments.


Figure 3
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  		Fig. 3 Adenovirus-mediated gene transfer of hirudin (AdHV-1.2) reduced neointima formation in balloon-injured rat carotid arteries. Intima/media area ratios were measured in histologic sections of rat carotid arteries treated with no virus (n = 14), with an adenoviral vector expressing bacterial β-galactosidase (n = 8) as a control, or AdHV-1.2 (n = 8). ANOVA showed significant differences between the three groups (p<0.02). Results of unpaired two-tailed t-tests between the experimental and the control groups are shown. Both antithrombotic activity and the inhibition of smooth muscle cell proliferation may have contributed to the decrease in neointima formation after hirudin gene transfer. From: Rade et al., 1996 [35]; reproduced with the permission of Nature America Inc.

 
Thrombomodulin has also been used in antithrombotic gene transfer experiments. This glycoprotein is predominantly expressed on the surface of endothelial cells and plays a role in the feedback inhibition of clotting by stimulating thrombin-mediated activation of protein C, which degrades factors Va and VIIIa [76, 77]. Adenovirus-mediated gene transfer of thrombomodulin into cultured human endothelial cells increased thrombomodulin expression on the cell surface, enhanced protein C activation, and decreased clot formation in an in vitro model [78].

The tissue factor pathway inhibitor (TFPI) is synthesized by serum-stimulated fibroblasts and plays an important role in regulating blood coagulation induced by tissue factor [79]. Local infusion of a TFPI solution to injured rabbit carotid arteries suppressed platelet aggregation and thrombosis, as assessed by transmission electron microscopy, and inhibited neointima formation [80]. A single local TFPI infusion at the site of angioplasty resulted in a persistent suppression of mural platelet thrombosis for 12 h in injured porcine carotid arteries [81]. Based on these data, TFPI is an attractive candidate for antithrombotic gene therapy.

Among coagulation factor inhibitors, the specific factor Xa inhibitor antistasin (ATS) has attracted particular attention. ATS has anticoagulant and antithrombotic actions [82]and may block the mitogenic effect of factor Xa on vascular smooth muscle cells [83]. These activities may explain the inhibitory effect of recombinant ATS on neointima formation after balloon injury in atherosclerotic femoral arteries in rabbits [84]. ATS also prevented vascular graft thrombosis in baboons [85]. Finally, ATS accelerated the t-PA-induced reperfusion of thrombosed femoral arteries and prevented their reocclusion in dogs [86]. This ability of ATS to potentiate the fibrinolytic activity of plasminogen activators makes it a particularly attractive candidate for antithrombotic gene therapy.

3.3 Genes with fibrinolytic activity
As a first step toward the development of plasminogen activators as gene therapy agents, we constructed a retroviral vector expressing human t-PA [28]. In this vector, the endogenous human t-PA promoter sequences, which appear to be active only at low levels in endothelial cells [87, 88], were replaced with sequences from the simian virus-40 (SV-40) early promoter. In vitro, secreted endothelial cell plasminogen activator activity was increased by one to two orders of magnitude over the low level normally present in the conditioned medium of cultured endothelium. However, a substantial proportion of the secreted recombinant human t-PA was inactive due to complex formation with plasminogen activator inhibitor-1 (PAI-1). Similar results were reported by other groups [89]. Since injured endothelial cells may secrete large amounts of PAI-1 in vivo [90], and since the plasminogen activator pro-urokinase is not inhibited by PAI-1, we constructed a pro-urokinase molecule fused to a carboxyl-terminal glycolipid anchoring signal [91]. This anchoring signal targets protein expression specifically to the apical (i.e., luminal) endothelial cell surface. Specific localization of this anchored pro-urokinase molecule to the surface of transduced cells prevents clearance by blood flow, minimizes the potential for PAI-1 inhibition of the expressed plasminogen activator, and permits optimal interactions with cell-bound plasminogen [92, 93]. Localization of plasminogen activators along the luminal surface optimizes endovascular fibrinolysis. When this anchored pro-urokinase molecule was expressed in endothelial cells by means of retroviral vectors, the recombinant pro-urokinase was detected specifically in the apical endothelial cell membrane. Recombinant pro-urokinase was easily activated to 2-chain urokinase by plasmin [91]. Thus, after initiation of fibrinolysis, the cell surface pro-urokinase would be converted to enzymatically active urokinase, enhancing cell surface fibrinolysis.

We subsequently used retroviral vectors expressing either t-PA or this cell surface-anchored pro-urokinase to transduce baboon endothelial cells, which were then seeded onto collagen-coated segments of vascular grafts [7]. The antigenic levels of t-PA and anchored pro-urokinase each increased 10-fold in the medium perfusing the corresponding transduced endothelial cells in an in vitro perfusion circuit. Seeded vascular grafts were then interposed in exteriorized arteriovenous femoral shunts in baboons. The antithrombotic effect of the transduced cells was quantitated by measuring the deposition of 111In-labeled platelets and the accumulation of 125I-labeled fibrin on graft segments bearing sparsely attached, transduced endothelial cells. The presence of t-PA- or anchored u-PA-transduced cells on collagen segments resulted in a decreased 111In-platelet deposition and 125I-fibrin accumulation on collagen surfaces compared with untransduced cells (Fig. 1). The systemic levels of t-PA, urokinase, fibrinopeptide A (FPA), and thrombin-antithrombin complex (TAT) were not increased by transduced endothelial cells, indicating the absence of systemic effects of locally enhanced thrombolysis.

In a study designed to evaluate the retention of these genetically modified cells in a more physiologic setting, we seeded autologous sheep endothelial cells expressing human t-PA onto 4-mm-diameter Dacron grafts. The seeded grafts were placed both in vitro in a nonpulsatile flow system and in vivo as femoral and carotid interposition grafts in the donor sheep [94]. Two hours after exposure to flow in vitro, 68% of endothelial cells transduced with t-PA were retained on the grafts as compared with 81% of untransduced cells (p<0.05). On implantation in vivo, however, cells transduced with t-PA were retained at a very low rate (median, 0%; range, 0–11%). Addition of the protease inhibitor aprotinin to the in vitro graft perfusion media significantly increased the retention of cells transduced with t-PA, suggesting that the proteolytic activity of t-PA was responsible for the loss of the seeded cells. Thus, increased thrombolytic activity induced by plasminogen activator gene transfer may actually decrease the adherence of endothelial cells, owing to proteolytic digestion of cell–matrix contacts. Effective antithrombotic gene therapy may require the use of genes without proteolytic activity, such as hirudin.


   4 Summary and future directions
Top
 Abstract
 1 Introduction
 2 Potential feasibility of...
 3 Candidate genes for...
 4 Summary and future...
References
 
The potential of gene transfer approaches for therapy of arterial thrombosis is suggested by in vitro data from studies in which endothelial cells were transduced with genes with antithrombotic activities and by experimental in vivo data showing both transgene expression in the vessel wall and successful inhibition of thrombosis. Local expression of a gene with antithrombotic activity provides a unique opportunity to decrease thrombus deposition or promote thrombolysis at specific arterial sites, while avoiding the systemic side effects of conventional anticoagulant therapy. As a result of the time course of transgene expression after arterial gene delivery, gene therapy strategies are more suitable for the subacute prevention of clot formation in states of increased thrombogenicity than for acute thrombolysis in the setting of arterial occlusion. Potential clinical indications for antithrombotic gene therapy include the early and subacute phases after therapeutic manipulations such as PTCA, peripheral artery angioplasty, intravascular stenting, and arterial bypass surgery. Achievement of prolonged overexpression of antithrombotic genes in the vasculature is not currently feasible, due to the short duration of transgene expression mediated by available vector systems. If new vectors are developed, and if the initial successes of gene therapy for thrombotic disease in animal models are confirmed by longer-term experiments, human studies can be initiated. These studies would most logically be directed at investigating the safety and efficacy of antithrombotic gene therapy, as compared with conventional antithrombotic drug treatments.

Thrombosis is in many ways an excellent candidate for gene therapy. It is a focal process, for which the pathophysiology is well defined, and for which potentially therapeutic genes with antithrombotic activity have been cloned. Although gene therapy for arterial thrombosis is still in its infancy, important concepts have been proven in the last few years. Vascular cells, including those of nonhuman primates, have been transduced with biologically active antithrombotic genes. Vectors and techniques for high efficiency in vivo gene delivery to blood vessels have been described. In vivo expression of at least four of these genes (t-PA, urokinase, hirudin, and cyclooxygenase) has resulted in significant local therapeutic effects with no systemic toxicity [7, 35, 44]. The potential of gene therapy for treatment of thrombotic disease is evident from the results of these animal studies. Although significant problems involving vector development remain to be solved, we speculate that the potential of antithrombotic gene therapy will be further realized in the context of clinical trials within the coming years.

Time for primary review 12 days.


   Acknowledgements
 
G. Vassalli was supported by grants of the Swiss National Science Foundation and the Swiss Association Against Hypertension.


   References
Top
 Abstract
 1 Introduction
 2 Potential feasibility of...
 3 Candidate genes for...
 4 Summary and future...
 References
 

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