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(Circulation. 1996;93:1319-1320.)
© 1996 American Heart Association, Inc.

Articles

Improving the Interface Between Biomaterials and the Blood

The Gene Therapy Approach

Alexander W. Clowes, MD

From the Department of Surgery, University of Washington School of Medicine, Seattle.

Correspondence to Alexander W. Clowes, MD, Department of Surgery, University of Washington School of Medicine, BB442 HSB, Box 356410, Seattle, WA 98195-6410.

Key Words: Editorials • plasminogen activators • vessels • genes • endothelium

	Introduction

Top Introduction References

 
Synthetic grafts can substitute for diseased arteries and function well as long as there is high blood flow. In low-flow situations, they are prone to sudden thrombosis. They also provoke a wound-healing response from the adjacent vessels and the surrounding tissue that under some circumstances narrows the lumen and reduces blood flow.¹ Despite 50 years of investigation and development, we still lack "smart biomaterials" that possess the properties characteristic of normal vessels needed for the regulation of coagulation and luminal diameter. What can we do to improve this situation?

A great deal of effort has been put into making materials with nonthrombotic and nonanticoagulant surfaces. Although these materials are relatively inert in the short term, they are soon modified by the deposition of proteins from the blood, and they do not necessarily form smooth junctions with the adjacent arteries. One way to improve their performance is to encourage the formation of a surface on the biomaterial that mimics the surface of a normal vessel. Most investigators would consider a monolayer of endothelial cells as the right surface, even though other cell types (eg, vascular smooth muscle cells, mesothelial cells) and certain antithrombotic proteins might be able to form a suitable covering.^{2 3 4} If the endothelium is the right surface, then certain assumptions must be made. First, endothelium can be seeded onto the graft or induced to grow from local sources to form a confluent layer at the surface. Second, the endothelium over a graft behaves like endothelium over a normal artery and expresses the appropriate antithrombotic and anticoagulant molecular program to prevent thrombosis and lyse fibrin. Third, the endothelium is renewable and the surface repairable. Fourth, the endothelium regulates vascular diameter by changing vessel tone, mass, or both.

Only some of these assumptions have been tested. Endothelial cells can be harvested from autologous sources, propagated, and seeded onto a graft and can survive at the luminal surface for periods of time in vivo after graft implantation.² The seeded cells may be replaced in time by cells derived from local sources. It is also clear that microvascular endothelial cells can be harvested and applied directly to the graft without a cell culture step (endothelial "sodding"). The graft structure and material can be altered in such a way as to encourage endothelial ingrowth not only from the adjacent artery but also from the granulation tissue surrounding the graft.¹

Does any of this make a difference to the long-term performance of the graft? The evidence is sketchy. In humans, endothelial seeding seems to decrease platelet accumulation and may improve lower-extremity bypass patency, although the studies are limited and not always in agreement.^{2 5 6 7} The benefits of seeding seem modest at best. Can anything be done to improve the situation?

Dunn et al⁸ have proposed that grafts seeded with endothelial cells overexpressing tissue-type plasminogen activator (TPA) should exhibit increased local fibrinolytic activity and a reduced tendency to generate occlusive thrombi. Endothelial cells normally express TPA but might not be able to secrete sufficient amounts to combat the significant fibrin deposits on a synthetic surface exposed to blood. A TPA overexpression strategy in endothelium exploits the natural tendency of the endothelium in vivo to express fibrinolytic and anticoagulant proteins.

Human TPA was introduced into sheep venous endothelial cells by use of a retroviral vector. These transduced cells were then seeded into synthetic grafts and tested either in an in vitro flow system or in vivo. Because the transduced gene was stably incorporated into the genome, the investigators expected to observe continuous, increased expression of TPA. They found, somewhat to their surprise, that the level of TPA expression declined as the grafts were exposed to flow. Further investigation of this somewhat disappointing result revealed that the likely mechanism was not inactivation of the transduced gene but instead a decrease in endothelial cell retention on the graft surface. The endothelial cells appeared to weaken or sever their attachments to the underlying substrate, probably by generating excessive amounts of plasmin.

These observations raise a number of questions about gene therapy in general and, in particular, about the effects of gene transfer on local homeostatic mechanisms. Can gene expression really be targeted specifically to endothelial cells on a graft? In this case, can TPA be overexpressed on the luminal surface but not on the abluminal surface so as to increase fibrinolysis without affecting cell attachment? What effect does overexpression of TPA have on overall proteolytic balance? Is overexpression of TPA the best way to improve the function of the synthetic conduit? Should the approach instead be to overexpress components of the anticoagulant system? The investigators have proposed to overexpress factor X_a or thrombin inhibitors such as antistasin or hirudin locally; membrane components of the protein C/S system or heparin-like molecules might also be considered. Will these molecules have the desired effect, and in the future, will we be able to avoid trading one complication (fibrin accumulation) for another (endothelial loss because of increased plasmin)? We have no answers as yet, but it does seem clear that molecular strategies that take into account all aspects of the biology of the system are more likely to work. Many of the molecular targets of antithrombotic or fibrinolytic pharmacology have critical functions in several systems. For example, integrins are required for platelet adhesion and cell attachment, and interference with their function might reduce both thrombosis and cell migration and proliferation.^{9 10}

It could be argued that the gene transfer approach to improving the function of biomaterials is overly complex and fundamentally misdirected. Perhaps the most direct and elegant way to address the complications of thrombosis and scarring is to modify the material itself and thereby elicit a more appropriate response from the blood and the surrounding tissue. We know that anticoagulant and antithrombotic functions, wall mass, and luminal diameter are tightly controlled in normal arteries, but we understand little about their control in healed synthetic conduits. We think that the endothelium is the controlling element. It expresses genes that maintain an anticoagulant and fibrinolytic state in the blood but can shift to a procoagulant and antifibrinolytic pattern in response to stimuli. What is missing is a clear understanding of what regulates the balance. For example, what regulates TPA and plasminogen activator inhibitor-1 (PAI-1) expression? Blood flow does. Do the underlying matrix and the physical properties of the wall also affect TPA and PAI-1? Until we understand how to regulate endothelial "happiness" by subtly adjusting the local environment, we are stuck with either systemic or local pharmacology to control the function of the vessel substitutes. The reason for pursuing the local pharmacological approach is very simple: local pharmacology targets the drug of interest to the site of pathology and thereby avoids the problems of systemic toxicity. Systemic toxicity is a very real issue with anticoagulant, antithrombotic, and antineoplastic drugs. In an ideal world, we would have true biological substitutes for diseased vessels. The saphenous vein or the internal mammary artery certainly come close to the ideal, but even these conduits have problems at late times after implantation, and they are in very short supply. They, too, might benefit from local pharmacology, perhaps local gene therapy, that would increase their resistance to scarring, atherosclerotic change, and thrombosis.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

Top Introduction References

 

1. Clowes AW. Intimal hyperplasia and graft failure. Cardiovasc Pathol. 1993;2:179S-186S.
   
2. Williams SK. Endothelial cell transplantation. Cell Transplant. 1995;4:401-410. [Medline] [Order article via Infotrieve]
   
3. Yue X, van der Lei B, Schakenraad JM, van Oene GH, Kuit JH, Feijen J, Wildevuur CRH. Smooth muscle cell seeding in biodegradable grafts in rats: a new method to enhance the process of arterial wall regeneration. Surgery. 1988;103:206-212. [Medline] [Order article via Infotrieve]
   
4. Visser MJT, Van Bockel JH, Van Muijen GNP, Van Hinsbergh VWM. Cells derived from omental fat tissue and used for seeding vascular prostheses are not endothelial in origin: a study on the origin of epithelioid cells derived from omentum. J Vasc Surg. 1991;13:373-381. [Medline] [Order article via Infotrieve]
   
5. Örtenwall P, Wadenvik H, Kutti J, Risberg B. Endothelial cell seeding reduces thrombogenicity of Dacron grafts in humans. J Vasc Surg. 1990;11:403-410. [Medline] [Order article via Infotrieve]
   
6. Herring M, Smith J, Dalsing M, Glover J, Compton R, Etchberger K, Zollinger T. Endothelial seeding of polytetrafluoroethylene femoral popliteal bypasses: the failure of low-density seeding to improve patency. J Vasc Surg. 1994;20:650-655. [Medline] [Order article via Infotrieve]
   
7. Zilla P, Deutsch M, Meinhart J, Puschmann R, Eberl T, Minar E, Dudczak 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]
   
8. Dunn PF, Newman KD, Jones M, Yamada I, Shayani V, Virmani R, Dichek DA. Seeding of vascular grafts with genetically modified endothelial cells: secretion of recombinant TPA results in decreased seeded cell retention in vitro and in vivo. Circulation. 1996;93:1439-1446. [Abstract/Free Full Text]
   
9. EPIC Investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. N Engl J Med. 1994;330:956-961. [Abstract/Free Full Text]
   
10. Schwartz SM, DeBlois D, O'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ Res. 1995;77:445-465.[Free Full Text]
    

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