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cummunication

natively, the iridium-labeled platelet scan may not be an adequate method for determining if the graft is covered by endothelium. Since grafts wrapped with a porous PTFE film were used for these clinical studies, and unwrapped grafts were used in earlier baboon studies, similarly wrapped grafts have been placed in the baboon and studied both by morphology and indium-labeled platelet scanning. Although endothelialization did occur, it appeared to be retarded. Differences in platelet accumulation could be seen when large thrombi formed on nonendothelialized areas of PTFE. These findings suggest that the wrap may retard angiogenesis.

9. Golden MA, Au YPT, Kirkman TR, et al. Platelet derived growth factor activity and mRNA expression in healing vascular grafts in baboons. The association in vivo of PDGF mRNA and protein with cellular proliferation. J Clin Invest 1991;87:406-14. 10. Golden MA, Au YPT, Kenagy RD, Clowes AW. Growth factor gene expression by intimal cells in healing polytetratluoroethylene grafts. J VASC SURG 1990;11:580-85. 11. Kohler TR, Kirkman TR, Clowes AW. Intimal hyperplasia in polytetratluoroethylene (PTFE) grafts is reduced by increase

flow. [Abstract] FASEB J 1990;4:1153. 12. Ortenwall P, Wadenvik H, Risberg B. Reduced platelet deposition on seeded versus unseeded segments of polytetrafluoroethylene grafts: clinical observations after a six-month follow-up. J VASC SURG 1989;10:374-80.

Conclusions The factors regulating microvessel ingrowth have not been defined although it is quite clear that a number of factors carried in the blood and lodged in the thrombus within the interstices of the graft could stimulate microvesse1 ingrowth. Healing of porous grafts by angiogenic mechanisms has been demonstrated in several species, but not in humans. Although Dacron grafts are sufficiently porous to allow capillary ingrowth, the Dacron material itself appears to inhibit angiogenesis. Highly porous PTFE grafts heal by an angiogenic mechanism in juvenile baboons, but do not appear to do so in adult humans. Failure of human grafts to heal may be due to species differences, differences as a result of aging, or to retardation of capillary ingrowth by the wrap surrounding the graft. Alexander W. Cluwes, MD Ted fibh, MD univtity of wmbin~on Seattle, Wish.

REFERENCES 1. Clowes AW, Gown AM, Hanson SR, Reidy MA. Mechanisms of arterial graft failure. I. Role of cellular proliferation in early healing of PTFE prostheses. Am J Path01 1985;118:43-54. 2. Clowes AW, Kirkman TR, Clowes MM. Mechanisms of arterial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses. J VASC SURG 1986;3:877-84. 3. Clowes AW, Kirkman TR, Reidy MA. Mechanisms of arterial graft healing. III. Rapid transmural capillary ingrowth provides a source of intimal porous PTFE prostheses. 4. Golden MA, Hanson

endothelium and smooth muscle Am J Path01 1986;123:220-30.

in

SR, Kirkman TR, Schneider PA, Clowes AW. Healing of polytetrafluoroethylene arterial grafts is influenced by graft porosity. J VASC SURG 1990;11:838-45.

5. Zacharias RK, Kirkman TR, Clowes AW. Mechanisms of healing in synthetic grafts. J VASC SURG 1987;6:429-36. 6. Greisler HP, Lam TM, Henderson S, Cabusao E, Tattersall CW, Kim DU. Vascular graft healing - kinetics of cell proliferation. [Abstract] FASEB J 1990;4: 1149. 7. Fingerle J, Johnson R, Clowes AW, Majesky MW, Reidy MA. Role of platelets in smooth muscle cell proliferation and

migration after vascular injury in rat carotid artery. Proc Nat1 Acad Sci USA 1989;86:8412-6. 8. Zacharias RK, Kirkman TR, Kenagy RD, Bowen-Pope DF, Clowes AW. Growth factor production by polytetrafluoroethylene vascular grafts. J VASC SURG 1988;7:606-10.

GENETIC MANIPULATION ENDOTHELIAL CELLS IN VASCULAR PROSTHESES

OF SEEDED

Endothelial cells that have been seeded in vascular prostheses may be genetically modified in such a manner so as to influence both luminal and abluminal events that contribute to graft failure. Endothelial cell seeding of synthetic conduits has been demonstrated to improve graft patency in experimental models, but application of genetic engineering to this technology has only recently been possible. The relevance of endothelium in graft function relates to the profound effects of these cells in controlling surface coagulation and subjacent cellular proliferation. Certain unique characteristics of endothelium make it an attractive target organ for gene transfer. The most obvious is its immediate interface with the bloodstream, a factor that allows luminal release of various proteins with both local paracrine effects on thrombotic activities, or more distant endocrine effects, such as clot lysis, and inhibition of platelet aggregation at the blood-surface interface. Similarly, endothelium, because of its close apposition to smooth muscle cells, allows one to consider genetic modification of a number of interactions between these two tissues regarding both vasomotion and cellular proliferation. The former may be quite important in regard to basic regulatory mechanisms involved in hypertension or in control of blood pressure, whereas the latter has been implicated in a number of disorders including migration of cells from the media to the intimal region where they may participate in the evolution of arteriosclerotic plaque. Earlier experiments with endothelial cell seeding have suggested that the endothelium that lines a prosthetic graft functions in a relatively normal manner with release of prostacyclin, lessening of platelet adherence and aggregation along the surface of these hybrid conduits, and improved patency of small caliber grafts under conditions of low flow. However, these cells, unlike undisturbed endothelium in the normal arterial circulation, may release various cytokines and growth factors that contribute to neutrophil-related thrombotic events on the surface and proliferation of subjacent cells. Certainly, in the case of

Volume 13 Number 5 May 1991

porous grafts, transinterstial ingrowth of mesenchymal cellscauses a gradual thickening of the inner capsule over a period of time, despite the fact that an intact endothelial surface is present.’ The technology of seeding endothelial cells onto prosthetic materials of various constructs, may offer particular advantages if the cell’s function is genetically modified. Applied molecular genetics has allowed rather consistent modification of cell function by gene transfer. Genetic material (DNA) in the form of specificgene constructs may be introduced into host cell genome using a variety of vectors. Recently, both retroviruses aswell asencapsidition of DNA into liposomes have gained appeal when considering in vivo studies of gene transfer.2’3Direct transfer into skeletal muscle without vector carriers also has been described.4These means of transfection provide integration of multiple copies of a specificgene into the genome, with its subsequent expression usually being unregulated. This random integration and constitutive expression represents the current state of genetic engineering, but site specific insertions and regulated expression are theoretically possible. The relative simplicity of molecular genetics as a means of altering a cell’sprotein production, makes this technology attractive for application in a variety of experimental and clinical settings. Recombinant gene expression in endothelial cells has been successful,with production of many proteins including tissue-type plasminogen activator (t-PA), B-galactosidase,adenosine deaminase, and growth hormone. A number of specific studies have been directed at endothelial cell function both in vitro aswell as in vivo, and the results of these experiments are relevant in the application of genetic engineering to improve the function of vascular prostheses.

In vitro studies with genetically motied endothelium Zwiebel et aL5 documented that endothelial cellscould serve as recipients of functioning recombinant genes in vitro. In their experiments, genes encoding for neomycin resistance, human adenosine deaminase, and rat growth hormone were inserted in rabbit aortic endothelial cells by use of retroviral transfer. Expression of all three genes was confirmed in these studies. In certain of their experiments, the endothelial cells were grown in culture on siliconecoated polyurethane vascular prostheses. In another in vitro study, Dichek et al6 transferred genes encoding for the production of p-galactosidase and human t-PA into cultivated sheep endothelial cells. These transduced cells were subsequently seeded onto stainless steel stems. The latter were of a type applicable for intraarterial use during balloon angioplasty procedures. In their studies, p-galactosidase was evident with X-gal staining of the lac-Z transduced cellsthat covered the stems. Similarly, measurementi of t-PA within the culture media confirmed the fact that cells transduced with this gene were producing this protein at levels much higher than those transduced with the 1acZ gene for p-galactosidase.The implication of these latter studies was

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that excess constitutive expression of t-PA may occur without conferring disadvantages to other cell functions or the growth of endothelial cells,and that this might enhance a protective function that seemsto be absent with the usual cellular incorporation of most prostheses used in the clinical setting. Brothers et al.’ assessedthe affect of genetic transduction on canine endothelial cell prostocyclin (6-ketoPGF,) production and cell growth. In their studies, adult canine venous endothelium was transfected with the lac-Z gene in combination with the Tn5 selectablemarker gene. Transduced cellsconsistently revealed a slower proliferation rate than uninfected cells. Similarly basal production of 6-ketoPGF, during log phase growth was less in transduced cells compared to noninfected cells. However, prostenoid production differences were not noted among the two cell lines once cells had reached confluence. Alterations such as these may be relevant in gene transfer clinically, and may differ for the progeny of each transduction event in relation to the particular gene or vector utilized.

In vivo studies with genetically modified endothelium Nabel et al.’ undertook a series of studies that documented recombinant gene expression in vivo within endothelial cells placed in the iliofemoral arteries of Yucatan minipigs. The endothelium in their experiments was derived from jugular veins and transduced with both the lac-Z and Tn5 genes using a retroviral vector. Cells were selectedin vitro, and a nearly pure population of genetically modified cells were then transplanted into denuded segments of the iliofemoral arteries. This was accomplished by use of a double balloon catheter specifically designed for isolation of a vascular segment during such instillation procedures. Arterial segments that were explanted 2 to 4 weeks after transfer of endothelial cells documented the presence of p-galactosidaseproduction, particularly along the surface cells where the transplanted cellshad spread to form a lining layer. l3-galactosidaseactivity was not apparent in vascular segments seeded with nontransduced cells. In additional experiments Nabel et al9 documented the presence of site specific gene expression with in vivo studies involving direct gene transfer. These latter experiments used a similar animal model and catheter delivery system, except that the retroviral vector containing the fi-galactosidasegene was directly instilled within the vessel after the introduction of polybrene to increasethe efficiency of infection. The recombinant lac-Z reporter gene was expressedfor at least 5 months. This activity was observed within all three layers of the vesselwall. These authors also reported DNA transfection using liposomes as vectors, with similar site specific gene expression noted up to 6 weeks. In these experiments DNA transfection appeared limited to the arterial wall where the genetic material was instilled, being absent from the liver, lung, kidney and spleen.

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Wilson et al.” implanted porous Dacron vascular prostheses,as interposition carotid artery grafts in a canine model, that had been seeded with genetically modified endotheliurn that had been transduced with the lac-Z gene. Grafts examined 5 weeks after implantation revealed expressionof p-galactosidaseactivity among the progeny of seeded cellswhich in areashad grown to confluence along the surface of the graft. These studies have been duplicated by others, including the author, using relatively impervious expanded polytetratluoroethylene conduits, and suggest the relative applicability of this form of hybrid organ for studies using genetically modified endothelial cells.

Continuing

research efforts

The future of genetic engineering in modifying synthetic graft function will depend on overcoming a number of obstacles.Currently, the use of retrovitus vectors limits the size of the gene that may be inserted to approximately 8 kb. Similarly, the transfection rate with retroviral vectors is relatively low, and in vitro methods of increasing the proportion of transduced cells, such as the use of the Tn5 gene with subsequent exposure to G418, may alter other functions of surviving cells.In this regard the constitutive, unregulated, expression of protein production by endothelial cellsthat have had random insertions of genetic material into their genome, may alter other important cellfunctions. One must first identify the molecular basisfor events related to increased surface thrombosis, as well as those factors contributing to the progressive increase in inner capsule thickness accompanying proliferation of myointimal cells.A major advance will occur if such events can be specifically defined and down regulators for these events determined, or the molecular basisidentified for competing events that negate these thrombotic and proliferative cellular complications accompanying graft implantation. Growth promoting factors associated with vessel wall modeling and angiogenesis include platelet-derived growth factor, basic fibroblast growth factor (FGF), interleukin’ (11-l),and insulin-lie growth factor I (IGF-1). Identification of the genetic regulation of these growth promoting factors might provide insight as to how to modify their expression in the in vivo setting, especially within the inner

capsule and anastomotic regions of grafts where proliferative cellular events appear to be the major causeof graft failure. If such factors can be related to simple protein production, then the nucleotide sequence of the responsible gene may be derived, and by recombinant means the effect of such gene constructions on cell function may be

tested

in vitro

and then in in vivo settings.

Early

candidate genes for endothelial cell modification include t-PA, thrombomodulin, and transforming growth factor (TGF-P). In regard to celhrlar proliferative events, agents that might down regulate t-PA production may prove important,

in that t-PA

appears

involved

with

matrix

changesthat facilitate cell growth. Obviously other agents such as the heparins and prostenoids are important in vascular remodeling,

but their genetic control

is complex

enough that specificmodification of a single gene structure to effect these substances is not likely to occur in the immediate future. Nevertheless genetic modification of endothelial cellsand even vascular smooth muscle cells may lead to improved vascular prosthesis function. James C. Stanley, MD Untie&~ of Michigan Ann Arbor, Mid.

REFERENCES 1. Stanley JC, Burke1 WE, Ford JW, et al. Enhanced patency in endothelial cell seeded small diameter externally supported Dacron iliofemoral interposition grafts. Surgery 1982;92: 994-1005. 2. Cepko CL, Roberts BE, Mulligan RC. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 1984;37:1053. 3. Felger PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci 1987;84:7423. 4. Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science 1990;247:1465-8. 5. 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;243:220-2. 6. Dichek DA, Neville RF, Zwiebel JA, Freeman SM, Leon MB, Anderson WF. Seeding of intravascular stems with genetically engineered endothelial cells. Circulation 1989;80:1347-53. 7. Brothers TE, Judge LM, Wilson JM, Burke1 WE, Stanley JC. Effect of genetic transduction on in vitro canine endothelial cell prostanoid production and growth. Surg Forum 1990; 41:337-9. 8. Nabel EG, Plautz G, Boyce FM, Stanley JC, Nabel GJ. Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 1989;244:1342-4. 9. Nabel EG, Plautz G, Nabel GJ. Site-specific gene expression in vivo by direct gene transfer into the arterial wall. Science 1990;249:1285-8. 10. 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: 1344-6.

IWLATIONSHIP BE’IWEEN ANASTOMOTIC HEMODYNAMICS AND INTIMAL THICKENING Intimal

thickening

is a characteristic

of the normal

adaptive and healing response of arteries and usually is self-limiting. Under certain circumstances, however, intimal proliferation is progressive and results in lumen stenosis.Such intimal hyperplasia is particularly prominent at prosthetic vascular graft anastomoses in lower extremity bypassesand frequently leads to graft failure. The precise factors that regulate

and control

intimal

thickness

are

incompletely understood but include hemodynamic forces. Wall shear stresshas been shown to be inversely related to intimal thickness in both human and experimental arteries. In the human carotid artery intimal thickness and plaque localization is related to low wall shear stress.’ In experi-