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Journal of VASCULAR SURGERY

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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-

Volume 13 Number 5 May 1991

mental aortic coarctations in monkeys, intimal thickening is inhibited in areas of high shear stressand promoted in areas of low wall shear stress.’ Oscillation of wall shear stressdirection and increased particle residence time have also been implicated in intimal tbickening.3v4These hemodynamic factors are strongly influenced by changes in blood flow and heart rate, which are dependent on activity levels and exercise.Thus an improved understanding of anastomotic hemodynamic conditions under varying flow conditions may lead to new insights into the mechanism and control of intimal hyperplasia. The purpose of this investigation is to (1) characterize the near-wall flow field in model end-to-side anastomoses and to relate the findings to experimental anastomotic intimal thickening, and (2) to determine the influence of exerciseflow conditions on the anastomotic flow field. Method Iliofemoral bypass grafts were constructed in mongrel dogs with saphenous vein grafts and polytetrafluoroethylene prostheses. In vivo flow conditions were measured with an electromagnetic flowmeter and pulsed Doppler ultrasonography, and the vessels were then pressureperfusion fixed, excised, and cast to preserve in vivo geometry. Intimal thickening was assessedin chronic experiments in which animals were fed an atherogenic diet for 8 weeks. After pressure-perfusion fixation, each anastomosis was serially sectioned, and the distribution of lesions was mapped by use of quantitative morphometry and three dimensional reconstruction. Data from casts of animal anastomoses were used to construct two large-scale (7.5 times in vivo dimensions) transparent silicone rubber models of the distal end-to-side anastomosis. The graft-to-host vessel diameter ratio was 1: 1, and the hood length-to-vessel diameter ratio was 4 : 1 and 8:l. The models were placed in a pulsatile flow system that allowed physiologic flow waveforms to be used. Flow patterns were visualized by the injection of small (500 urn) almost neutrally buoyant particles illuminated with either flood lights or helium-neon laser light. Standard VHS video and 35 mm photography were used to record particle motion. Pulsatile flow conditions were modeled after measured canine femoral artery flow waveforms. The anastomotic flow field was assessedunder simulated resting and exerciseflow conditions. Resting flow conditions were defined as a heart rate of 80 beats/minute and a peak Reynolds number of 750. Exercise flow conditions were defined as a heart rate of 140 beats/minute and a peak Reynolds number of 1500. During exercisethere was a two and one-half-fold increase in mean flow compared to resting. Results Flow patterns in the area of end-to-side anastomoses are remarkably complex. Under resting conditions a large area of flow separation develops in the anastomotic sinus

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with flow stasisand prominent, particle trapping. This was most prominent in the proximal portion of the anastomosis. Particles also accumulated at the lateral walls. In the distal portion of the anastomosis, where flow exited into the distal outaow artery, there was rapid flow and short particle residence times. Particles tended to reside in the anastomosis for extended periods, even to the point of permanent accumulation along the side wall. Along the floor of the anastomosis in the host vesseloscillation of shear stress was prominent whereas wall shear stresswas unidirectional distally. Under exercisecon&tins, the areaof Ilow separation was significantly reduced and anastomotic stasiswas virtually eliminated. Trapped particles along the lateral walls began to clear immediately asa result of more vigorous secondary flow patterns. All particleswere gone within 5 to 6 pulsatile cyclesof exerciseflow. The wall shear was notably larger in magnitude, and relatively strong vortexes were created which removed stagnant particles and cleared out the anastomotic region. Along the floor of the anastomosis, the area subjected to oscillating shear stresswas reduced. Histologic sections revealed intimal thickening at the suture line along the lateral wall of the anastomotic sinus in a region of prolonged particle residence time as well as along the floor of the anastomosis in the region of flow separation and shear stressoscillation. Exerciseconditions decreasedparticle residencetime significantly and increased the levelsof wall shear stress,both at the lateral walls of the sinus as well as along the floor where the maximum flow velocities entering the anastomosis were directed. Discussion These results indicate that hemodynamic characteristics known to promote intimal thickening, namely low wall shear stress, flow separation, increased particle residence time, and oscillation in shear stress are present within vasculargraft anastomoses.These factors may play a role in intimal thickening. However, the precise role these factors play, if any, in anastomotic intimal hyperplasia are unknown. It is known, however, that the normal artery wall requires an optimum wall shear for homeostasis, and that the artery wall remodels its structure if wall shear departs from this value over a sustained period of time? The focal nature of intimal thickening, intimal hyperplasia, and atherosclerosismay well be related artery wall responsesto local conditions of exposure to low wall shear.14 Furthermore, the eventual evolution of a developing plaque, at least until it reaches certain limits,5 may be influenced by the local shear stressand the residence time of atherogenic substances.Although excessiveintimal thickening appears to be prevented in arteries where the mean wall shearstress exceedsapproximately 15 dynes/cm*, recent studies in our laboratory suggest that the determining factor may be the maximum wall shear experienced during the cycle regardlessof direction and not simply the mean shear value.* The conditions of low wall shear stressand flow stasis that exist at rest in our model anastomoses were reversed

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Journal of VASCULAR SURGERY

Special communication

during exercise. Similar elimination of adverse hemodynamic conditions have been demonstrated in an aortic model under exercise flow conditions.6 Since stasis has been related not only to intimal thickening but also to graft thrombosis, a structured program of exercise that would periodically “clear” the anastomosis may be beneficial in extending graft patency and minimizing intimal thickening. The amount, frequency and duration of exercise periods necessary for clinical benefit requires further investigation. Christoj~her

K. Zarins,

MD

Don P. Giddens, PhD University of Chicago Chicago,

Ill.

REFERENCES:

1. Zarins CK, Giddens DP, Bharadavaj BK, Sottiurai VS, Mabon RF, Glagov S. Carotid bifurcation atherosclerosis: quantitation of plaque localization with flow velocity profiles and wall shear stress. Circ Res 1983;53:502-14. 2. Bassiouny HS, Lieber BB, Giddens DP, Xu Cl’, Glagov S, Zarins CK. Quantitative inverse correlation of wall shear stress with experimental intimal thickening. Surg Forum 1988;39: 328-30. 3. Ku DN, Giddens DP, Zarins CK, Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation: positive correlation between plaque location and low and oscillating shear stress. Arteriosclerosis 1985;5:293-302. 4. Giddens DP, Zarins CK, Glagov S. Response of arteries to near-wall fluid dynamic behavior. Appl Mech Rev 1990;43: S98-102. 5. Glagov S, Weisenberg E, Zarins CK, Stankunavicius R, Kolettis G. Compensatory enlargement of human atherosclerotic coronary arteries. N Engl J Med 1987;316: 1371-j. 6. Ku DN, Glagov S, Moore JE, Zarins CK. Flow parterns in the abdominal aorta under simulated postprandial and exercise conditions: an experimental study. J VASC SURG 1989;9:30916.

THE BLOOD-MATERIALS INTERACTION DOWNSTREAM EFFECTS IN PROSTHETIC CONDUITS

AND

The most common cause of delayed failure of prosthetic arterial grafts is the development of neointimal hyperplasia at the anastomoses as originally described by DeWeese.’ Because these lesions are restricted to the anastomotic site, we became interested in the possibility that mechanical factors at the anastomosis played a role. After developing plastic models of various anastomotic configurations, we were able to demonstrate ringlike areas of separated flow at the end-to-side anastomosis.’ These areas of flow separation occur at both inflow and outflow anastomoses and are highly dependent upon the angle of anastomosis and the flow split between the runoffvessels.“* Based on what we had learned from the plastic models, we devised an animal model in which we could control the presence or absence of flow separation at the anastomoses.3 Using Dacron grafts, we were unable to demonstrate a relationship between the presence or absence of flow separation and the development of anastomotic hyperpla-

sia. However, we did note a consistently greater amount of hyperplasia at the distal anastomosis as compared with the proximal anastomosis. These observations led us to our current hypothesis that interaction of blood with the surface of the conduit produces substances that aggravate or augment the hyperplastic response at the outflow or downstream anastomosis. For that reason, we referred to the lesion as downstream anastomotic hyperplasia and identified it as the primary mechanism of failure of Dacron grafts in our modeL3 We then considered the role of the underlying prosthetic material and designed a study to determine whether there was a significant difference between Dacron and polytetrafluoroethylene (PTFE) in the development of downstream anastomotic hyperplasia. In this study we used end-to-end anastomoses in the carotid position to minimize the possibility that any flow disturbances were playing a role. That study confirmed an increase in anastomotic hyperplasia at the downstream anastomosis for both Dacron and PTFE, and we concluded that this is probably the final common pathway for failure of all prosthetic arterial grafts.’ From our work and that of others it is clear that hyperplasia occurs at all anastomoses between a prosthetic graft and a host artery. Many events are occurring at these anastomoses, and consequently it has been difficult to determine precisely any cause and effect relationships. At the anastomosis, there is, in essence, an ongoing wound healing response. In this case, the wound healing is complicated by the presence of a foreign body and a variety of stresses, i.e. hoop stress, normal stress, and shear stress. The foreign body response is in itself poorly understood but is probably a function of the material structure and warrants continued investigation. Sumpio et aL5 and others have demonstrated the effects of pulsatile stress on cell growth.6 The hyperplasia itself consists of modified smooth muscle cells or myofibroblasts whose migration, growth and secretory patterns are influenced by a myriad of growth factors and cytokines in a resilient biologic response system.’ Our approach has been to focus on one small part of this and that is the mechanism leading to augmentation of hyperplasia at the outSlow anastomosis. It is our hypothesis that the blood-materials interaction produces substances that modify the cellular response. Shortly after implantation of a prosthetic device, regardless of the substrate material, the surface rapidly becomes coated with proteins and ultimately develops a pseudointima consisting of fibrin with enmeshed red cells. The surface itself activates Hagemann factor, initiating a cascade of sequences the end result of which is the activation of three important components: (1) the coagulation cascade with the production of fibrin by thrombin, (2) the activation of platelets directly by the surface or indirectly through thrombin, and (3) production of plasmin, which in addition to its lytic activity, also initiates the complement cascade with activation of white blood cells.’ Thus the blood-materials interaction is extremely complex with the production of