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Transgraft Infusion of Heparin to Prevent Early Thrombosis of Expanded PTFE Grafts in Canine Femoral Veins
Transgraft Infusion of Heparin to Prevent Early Thrombosis of Expanded PTFE Grafts in Canine Femoral Veins Changyi Chen, MD, John D. Hughes, MD, Samer G. Mattar, MB, ChB, Stephen R. Hanson, PhD, and Alan B. Lumsden, MD, Atlanta, Georgia
Recently we designed an expanded polytetrafluoroethylene (ePTFE)-based local infusion device that delivers therapeutic agents directly through the graft wall in the region adjacent to the upstream anastomosis, thereby achieving a high drug concentration downstream along the graft-blood interface. In this study we evaluated the effects of infusing heparin by this method on graft patency and neointimal hyperplasia in a canine model of femoral vein replacement. Five dogs underwent bilateral femoral vein replacement with the device. In each case one graft was infused with continuous heparin (48 U/kg/day) while the contralateral control graft received phosphate-buffered saline solution for 14 days. All heparin-treated grafts were patent and all control grafts were thrombosed at 14 days. There was no significant difference in systemic activated partial thromboplastin time among samples taken preoperatively, at 48 hours, and at 14 days of implantation (p > 0.5). There was no significant difference in neointimat hyperplasia between the upstream and downstream anastomoses in heparintreated grafts. These data demonstrate that the transgraft infusion of heparin preserved venous ePTFE graft patency without measurable systemic anticoagulation. Thus this approach may represent an attractive strategy for maintaining patency of synthetic venous grafts. (Ann Vasc Surg 1996;10:147-155.)
Venous disease is a common problem that often causes significant morbidity and sometimes death. Superior vena caval syndrome, inferior vena caval syndrome, postphlebitic disease, traumatic loss of veins, and congenital absence or displacement of veins all represent serious lesions that are potentially correctable. Although collected reports indi-
From the Division of Vascular Surgery, Department of Surgery, and the Division of Hematology/Oncology (S.R.H.), Department of Medicine, Emory University School of Medicine, Atlanta, Ga. Presented at the Twentieth Annual Meeting of the Peripheral Vascular Surgery Society, New Orleans, La., June 10, 1995. Supported in part by research grants HL 31469 and HL 48667 from the National Institutes of Health and by a grant from the American Heart Association. Reprint requests: Alan B. Lumsden, MD, Surgery Research, 1639 Pierce Dr., 5105 WMB, Atlanta, GA 30322.
cate that a considerable number of grafts have been placed in various portions of the h u m a n venous system, success rates for replacement prostheses have consistently been low in small-caliber vein replacement or reconstruction because of early graft thrombosis and anastomotic stenosis.l5 Expanded polytetrafluoroethylene (ePTFE) has been used for segmental venous replacement of vena cava, portal, and external iliac veins, 69 Patency rates in large-caliber veins appear promising but those in femoral vein replacement with ePTFE are very low, with thrombosis usually occurring within 24 to 48 hours, l° For satisfactory results in venous reconstruction, therefore, prevention of thrombus formation is mandatory. Patency may be maintained by overcoming those factors known to cause thrombosis including operative trauma, anastomotic strictures, low-pressure systems, slow intraluminal blood flow, host reaction to grafts, and relative high thrombogenicity of the substi147
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tutes. 11'12 Adjunctive techniques to improve patency of venous grafts include the use of arteriovenous fistulas, systemic anticoagulants, and antithrombotic materials bonded to the prosthetic wall. ~318However, all of these techniques are associated with side effects that limit their use. Recently we designed and characterized an ePTFE-based local drug delivery device that selectively concentrates infused pharmacologic agents specifically within those blood layers (boundary layers) adjacent to the graft wall and at downstream anastomotic sitesJ 9 In our previous study we administered heparin locally by means of this approach and evaluated its effects on graft patency and neointimal hyperplasia formation in a rabbit model of inferior vena cava replacement. 2° Data from this study demonstrate that the transgraft infusion of heparin significantly increases inferior vena cava graft patency in the rabbit and markedly reduces downstream anastomotic neointimal hyperplasia and cell proliferation without measurable systemic anticoagulation. However, the design of the infusion device used in the rabbit experiment delivered heparin at the midportion of the graft, thus highlighting the significant difference in findings between the upstream and downstream anastomoses as a result of the minimal delivery of heparin to upstream sites. The unchecked development of neointimal hyperplasia at the upstream anastomosis may lead to graft failure. To overcome this problem we modified the delivery device to infuse drug immediately adjacent to the upstream anastomosis, thereby preventing graft thrombosis and perhaps reducing neointimal thickening at both anastomoses. The purpose of the present study was to evaluate the effect of this modified system for improving patency and reducing anastomotic neointimal hyperplasia of femoral vein replacements in a canine model, which is more clinically relevant. MATERIAL AND METHODS Local I n f u s i o n D e v i c e The local infusion device was constructed from ringed ePTFE clinical vascular graft material (Gore-Tex, W.L. Gore & Associates, Inc., Flagstaff, Ariz.) as previously describedJ 9'2° For modification of the device, a cuff-reservoir was made adjacent to one end of a segment of ePTFE graft (6 m m internal diameter and 4.5 cm in length), which delivers heparin directly through the graft wall in the region adjacent to the upstream anastomosis, thereby achieving a high drug concentration downstream along the graft-blood inter-
face (Fig. 1). In view of the proximity of the infusion site to the upstream anastomosis, it is likely that heparin diffuses to this area and causes biologic effects. An implantable osmotic pump (model 2ML2, Alza Corp., Palo Alto, Calif.) was connected to the tubing of the device. These osmotic pumps delivered at a predictable rate of 5 b~l/hr over a period of 14 days. Pumps for treated grafts were loaded with heparin (20,000 U/2 ml; Elkins-Sinn, Inc., Cherry Hill, N.J.), whereas control pumps were loaded with phosphate-buffered saline solution (PBS). Treated grafts received local infusion of heparin for 14 days at the rate of 60 U/hr, which was equivalent to an average rate of 2 U/kg/hr. Satisfactory drug delivery was confirmed at the time of harvest by observations of pump residual volume and reservoir tubing patency. Animal Procedures Five adult male mongrel dogs weighing 30.56 _+ 3.96 kg were used in this study. All animal procedures and care were performed in accordance with the "Principles of Laboratory Animal Care" and the "Guidelines for the Care and Use of Laboratory Animals" (NIH publication No. 80-23, revised 1985). Anesthesia was induced with thiopental sodium (10 to 20 mg/kg intravenously) administered via endotracheal intubation and maintained with i% to 2.5% isoflurane. Under sterile conditions, an approximate 8 cm segment of femoral vein was dissected bilaterally and clamped proximally and distally. Systemic heparin (100 U/kg) was given prior to clamping. A 2 cm segment of femoral vein was excised. An ePTFE-based infusion device was implanted bilaterally in the femoral vein in an end-to-end anastomosis using running 6-0 polypropylene sutures. On completion of the anastomosis, an osmotic pump preloaded with heparin was connected to the infusion device on one side, and another osmotic pump preloaded with PBS was connected to the infusion device on the contralateral side as a control. These pumps were implanted in subcutaneous pockets. The incision was closed with 3-0 polyglactin sutures. Bromodeoxyuridine (BrdU, Sigma Chemical Co., St. Louis, Mo.), in a dose of 50 mg/kg dissolved in 50 ml normal saline solution, was administered intraperitoneally 24 hours prior to euthanasia at i4 days. The animals were anesthetized as previously described, and the femoral veins and grafts were exposed. Patency of grafts was determined by direct inspection, by blood
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Fig. 1. ePTFE-based local infusion device. A, Device consists of a silicone rubber cuff-reservoir glued in position between the two rings of a 6 rnm internal diameter ePTFE graft (30 p,m internodal distance). The hub is attached to a 1 m m internal diameter silicone rubber catheter tube, which is then attached to an osmotic pump. 13, A soluble agent from an osmotic pump will pass through the graft wall, enter the bloodstream in highest concentration at the graft wall (boundary layer), and be transported downstream (axially) by blood flow (convection), with slower mixing in the radial direction (i.e., toward the center stream blood flow), occurring primarily through diffusive mechanisms. Long arrow indicates direction of blood flow.
flow measurement using an ultrasonic flowmeter (model T201, two-channel, Transonic Systems, Inc., Ithica, N.Y.), and later by histologic analysis. A sternotomy was performed, and Ringer's solution was infused at 120 m m Hg pressure through a wide-bore needle into the left ventricle while the animal was synchronously exsanguinated via a cannula placed in the right atrium. Once the blood was cleared from the circulatory system, the whole animal was perfusion fixed in situ for 20 minutes at 120 m m Hg pressure using 2.5% glutaraldehyde. Grafts with 3 cm segments of attached femoral vein were harvested and fixed in 10% buffered formalin for 4 hours and then in 70% alcohol. Blood was drawn for activated partial thromboplastin time (aPTF) assay preoperatively, at 48 hours, and at 14 days of implantation.
Histology and Morphometry Following fixation, the grafts were cut longitudinally at both the upstream and downstream anastomoses to observe the anastomotic neointimal tissues, cut transversely at the remaining femoral vein and middle portions of the grafts at 3 m m
intervals, processed through graded ethanol, infiltrated with xylene and paraffin, and then embedded in paraffin blocks. Five-micrometer sections were cut and stained with hematoxylin and eosin and Verhoeff-Masson's stain. Pannus tissue ingrowth overlying the luminal surface of the graft adjacent to the anastomoses was considered to be neointima. Morphometric measurements of the thickness and area of neointima were performed by computer image analysis software (Optimas, Bioscan, Inc., Edmonds, Wash.) on a magnified image relayed from a microscope-mounted video camera to a digitizing pad and video monitor (Thomas Optical, Columbus, Ga. ) as previously described. 2°
Immunocytochemistry The avidin-biotin complex immunoperoxidase procedure (LSAB Kit, Dako Corp., Carpenteria, Calif.) was used to identify determinants characterizing vascular smooth muscle cells (SMCs) and proliferating cells as previously described. 2° Briefly, SMCs were identified by specific ~-actin immunostaining with HHF35 monoclonal antibody (Dako Corp.). Proliferating cells were iden-
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tiffed with anti-BrdU monoclonal antibody (Dako Corp.). BrdU-positive cells were quantified manually using a cell-counting technique per highpower field ( x 400) on a micrometer grid. In each field all cells were counted and the number of positively stained cells was expressed as a percentage of total cells to arrive at the BrdU index. A m i n i m u m of 10 fields were quantified per section.
Statistical Analysis Statistical analysis was performed on a Macintosh Quadra 650 computer using Excel 4.0 statistical software. Chi-square analysis was used to determine significant differences in patency rates between the heparin-treated grafts and the control grafts. The paired Student's t test was used to compare aPTf values among samples taken from different time points. The paired Student's t test was also used to determine the significance of neointimal thickness, neointimal area, and cell proliferation rates between the upstream and downstream anastomoses of the treated grafts. Results were considered significant at p < 0.05.
RESULTS Graft Patency At harvest all osmotic pumps were functioning. All heparin-treated grafts (5 of 5) were patent and all PBS-treated grafts (5 of 5) were throm-
bosed. The volume blood flow rate through the patent grafts at 14 days averaged 8 0 _ I8 ml/min. Thus the transgraft infusion of heparin resulted in a significant increase in graft patency as compared with the PBS-treated grafts (p-0.025).
M e a s u r e m e n t s of Clotting Time Systemic aPTF measurements were 10.I _+ 0.2 seconds, i0.9 +_ 0.6 seconds, and 10.1 _+ 0.9 seconds for samples taken before local heparin infusion, at 48 hours, and at 14 days of continuous local heparin infusion, respectively. The results of the systemic aPTF determinations revealed no significant difference before and during local heparin therapy (p > 0.05). N e o i n t i m a l Hyperplasia Histologic examination showed the presence of thrombus in all of the control grafts. Organized thrombus was seen at regions of the grafts close to both the upstream and downstream anastomoses (Fig. 2, A and C), and thrombus in the central region remained unorganized (Fig. 2, B). Thus the control grafts were excluded from morphometric analysis because of the confounding effects of graft thrombosis on the development of neointimal hyperplasia and because of the difficulty in distinguishing anastomotic neointimal hyperplasia from organized thrombus.
Fig. 2. Control grafts were thrombosed. A, Upstream anastomosis. Organized thrombus was seen around the suture line (longitudinal section).
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Fig. 2, c o n t ' d . 13, Midportion of the graft. Unorganized thrombus was present (transverse section). C, Downstream anastomosis. Organized thrombus was seen around the suture line (longitudinal section). (Verhoeff-Masson's stain; x 20.)
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Table I. Anastomotic neointimal hyperplasia and cell proliferation (BrdU index) of heparin-treated grafts Neointima
Upstream anastomosis
Downstream anastomosis
Difference (%)
p Value
A r e a ( m m 2) Thickness (mm) B r d U i n d e x (%)
1.19 - 0.67 0.53 --_ 0.19 14.41 _+ 2.51
1.08 --+ 0.97 0.43 ~ 0.19 10.48 ± 2.49
8 19 27
0.6295 0.1319 0.0073
In the heparin-treated grafts neointimal hyperplasia occurred at both the upstream and downstream anastomoses (Fig. 3, A and C). Morphometric determinations of neointimal area and neointimal thickness were slightly higher at the upstream anastomoses but were not significant as compared with the downstream anastomoses (Table I). The major cellular component in the neointima was SMC, as identified by a-actin immunostaining. Pseudointima was seen in the middle portions of patent grafts (0.40 _+ 0.09 m m thickness, Fig. 3, C). Cell P r o l i f e r a t i o n Vascular SMC proliferation was determined by BrdU immunostaining. The BrdU labeling index was significantly higher (p = 0.0073) in the neointimal tissue at the upstream anastomoses (14.41% _+ 2.51%) as compared with the downstream anastomoses (10.48% m 2.49%). Thus the local transgraft infusion of heparin through the modified device reduces neointimal SMC proliferation by 27% (Table I). DISCUSSION Standard heparin for systemic effect needs to be administered by continuous intravenous infusion or by regular subcutaneous injections two or three times a day. It is associated with acute complications such as hemorrhage, anaphylaxis, thrombocytopenia, and delayed embolus and with chronic complications such as alopecia, osteoporosis, pathologic fracture, and ascorbic acid deficiency. 16 For these reasons standard heparin is unlikely to be accepted as long-term therapy in vascular surgery. However, local infusion of heparin in small doses may be useful for certain long-term therapy without major complications. A major advantage of local drug administration is the potential to greatly reduce systemic side effects, since the absolute amount of compound
administered is relatively small. An ePTFE-based local infusion device delivers therapeutic agents directly through the graft wall in the region adjacent to the upstream anastomosis, thereby achieving a high drug concentration downstream along the graft-blood interface (boundary layer). This concept has been supported by previous computational and experimental studies in vitro (unpublished data). Computational fluid dynamics was used to calculate the infusate concentration on the graft wall. Experimental techniques such as flow visualization and local sampiing were also used to document high infusate concentrations along the wall downstream from the graft. The theoretical and laboratory measurements were in agreement and were used to estimate local heparin concentrations in the present experiments. In the present model (80 ml/min blood flow through a 6.0 m m internal diameter graft) the infusate is predicted to be diluted approximately 400-fold along the graft wall over a distance of 4 to 5 cm downstream according to theoretical modeling of mass transport under these flow conditions. Because the infusate concentration of heparin was i0,000 U/ml, the concentration of heparin along the graft wall and downstream anastomosis is calculated to have been approximately 25 U/ml, a level expected to produce profound inhibition of both coagulation and vascular cell proliferation. 2~x3 The average infusion rate of heparin in a treated graft was 1 U/min. If this amount were infused so as to mix uniformly with all of the incoming blood (80 ml/min), the mean heparin concentration at all points within the graft would have been 0.0125 U/ml (1 U/min - 80 ml/min), that is, heparin levels along the graft wall would have been approximately 2000-fold lower than those achieved by the boundary layer infusion approach. The consequences of heparin recirculation can also be readily evaluated. Based on estimates of the half-life of heparin in vivo (tl/2 = 1 hour) and dog plasma volume ( - 4 0 ml/kg), the steady-
Fig. 3. Heparin-treated grafts were patent. A, Upstream anastomosis. Pannus tissue ingrowth to form anastomotic neointimal hyperplasia (longitudinal section). B, Midportion of graft. Pseudointima was present (longitudinal section). C, Downstream anastomosis. Anastomotic neointimal hyperplasia was seen (longitudinal section). (Verhoeff-Masson's stain; ×20.)
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state plasma heparin level is directly calculated according to first-order clearance kinetics as 0.072 U/ml, a minimal anticoagulation dose whose predicted level is consistent with the absence of detectable effects observed on the aPTF clotting time m e a s u r e m e n t s (see Results). The ratio of local boundary layer heparin level (25 U/ml) to systemic circulating level is thus estimated as 350:1 in this large animal model, indicating that ~dth this m e t h o d very high drug levels can be localized to a target vessel surface while at the same time minimizing systemic bleeding or other u n w a n t e d side effects. Although we currently do not have an effective means of local sampling and direct m e a s u r e m e n t of the boundary layer drug concentration in vivo, the results of these and other animal experiments are consistent with predictions based on in vitro studies and computational modeling. ~9"2° The approach of transgraft infusion of heparin in vein replacement has been evaluated in our previous study of rabbit inferior vena cava replacement 2° and in the present study of dog femoral vein replacement (Table II). Striking effects on outcome events were achieved in both animal models, preventing early thrombosis, preserving graft patency, and reducing anastomotic neointimal hyperplasia. In this study vce have modified the delivery device to infuse drug immediately adjacent to the upstream anastomosis in an a t t e m p t to reduce neointimal hyperplasia at both anastomoses. In heparin-treated grafts the neointimal thickness at the upstream anasto-
moses was slightly higher than that at the downstream anastomoses, and the difference between t h e m was 19% (not significant), whereas the difference was 88% (highly significant) in the rabbit model. 2° Although the cell proliferation rate at the upstream anastomosis was still significantly higher than that at the downstream anastomosis, the difference between t h e m was 27%, which is considerably lower t h a n the 72% in the rabbit model. 2° This observation demonstrates that transgraft infusion of heparin through the redesigned infusion device treats both the downstream anastomosis and, at least in part, the upstream anastomosis. Heparin infusion adjacent to the upstream anastomosis m a y prevent pannus tissue ingrowth from the edge of native vessel. Heparin diffusion to the upstream anastomosis m a y play a role in inhibiting cell proliferation and reducing neointimal growth. The dog model is relevant in terms of clinical applicability of the infusion device. Compared to rabbits, the hemodynamics and blood vessels of dogs more closely resemble those of humans. Furthermore, peripheral veins, longer grafts, and internal controls have been used in the dog model, providing more reliable and clinically relevant studies (Table II). There appears to be a critical period of approximately 1 to 2 weeks after venous implantation w h e n the conduit is at particular risk of thrombosis. 2'5'm In this study the 2-week time period was selected specifically to permit us to evaluate the use of this approach to modulate graft thrombosis, although we are unable to c o m m e n t on the pros-
T a b l e II. Comparison of rabbit and dog models of transgraft infusion of heparin in vein replacement Rabbit model Infusion device Replaced vein Heparin dose Graft patency Systemic aPTT Neointimal thickness
4 mm i.d., 2.5 cm in length, infusion from middle portion of device Inferior vena cava 30 U/hr, equivalent to 7 U/kg/hr
Dog model 6 mm i.d., 4.5 cm in length, infusion from region of device adjacent to the upstream anastomosis Femoral vein 60 U/I~, equivalent to 2 U/kg/hr
100%
100%
Blood flow Control setting
No significant change 88% reduction at downstream anastomosis* 72% reduction at downstream anastomosis* 27 ml/min Saline infusion in separate animals
Clinically relevant
Less relevant
No significant change 19% reduction at downstream anastomosis 27% reduction at downstream anastomosis* 80 ml/min PBS infusion in contralateral site of same animals More relevant
BrdU index
*Student's t test analysis showed significant difference.
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pects for long-term patency of ePTFE grafts in the venous system. This approach could be extended for long-term local infusion of heparin if this were found necessary to maintain patency. Further studies with different time courses are needed to evaluate long-term patency of vein replacement by means of this approach. Another potential study using this approach is the transplantation of prosthetic and autogenous valves into ePTFE grafts to further optimize patency results and reduce venous hypertension. Although this approach is presently being used exclusively as a research tool, it may represent an attractive strategy for antithrombotic therapy in venous replacement with synthetic graft materials.
CONCLUSION Prosthetic grafts in the venous systems are of limited usefulness because of early thrombotic occlusion, especially in small-caliber veins. Adjunctive therapy to improve patency of venous grafts includes systemic anticoagulation, which is associated with serious side effects. A major advantage of local drug administration is the potential to greatly reduce systemic side effects, since the absolute amounts of compound administered is relatively small. A new ePTFE-based local infusion device has been used to deliver therapeutic agents such as heparin, in this case directly through the wall of a synthetic graft, thereby achieving high drug concentrations in the blood fluid boundary layer along the graft wall and at the downstream anastomotic site. This approach has been demonstrated to have striking effects on preserving ePTFE graft patency and reducing anastomotic neointimal hyperplasia and cell proliferation without systemic anticoagulation in the animal models. Reduced neointimal hyperplasia at both upstream and downstream anastomoses was achieved by delivering heparin immediately adjacent to the upstream anastomosis. This approach may have potential clinical application for antithrombotic therapy in venous replacement with synthetic graft materials. We thank Ms. Carolyn Suwyn for her expert technical assistance in tissue processing and histology procedures and Ms. Beverly Noe for laboratory assistance. REFERENCES 1. Gloviczki P, Pairolero PC, Toomey BJ, et al. Reconstruction of large veins for nonmalignant venous occlusive disease. J Vasc Surg 1992;16:750-761,
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2. Dale WA, Scott HW Jr. Grafts of the venous system. Surgery 1963;53:52-74. 3. Norton L, Eiseman B. Replacement of portal vein during pancreatectomy for carcinoma. Surgery 1975;77:280-284. 4. Florian A, Cohn LH, Dammin GJ, et al. Small vessel replacement with Gore-Tex (expanded polytetrafluoroethylene). Arch Surg 1976;111:267-270. 5. Dale WA. Reconstructive venous surgery. Arch Surg 1979; 114:1312-1318, 6. Scherck JP, Kerstein MD, Stansel HC Jr. The current status of vena cava replacement. Surgery 1974;76:209-233. 7. Scheinin TM, Jude JR. Experimental replacement of the SVC: Effect of temporary increase in blood flow. J Thorac Cardiovasc Surg 1964;48:781-790. 8. Symbas PW, Foster JH, Scott HW. Experimental vein grafting in the portal venous system. Surgery 1961;50:97-101. 9. Plate G, Einarsson E, Eklof B, et al. Iliac vein obstruction associated with acute lliofemoral venous thrombosis: Results of early reconstruction using polytetrafluoroethylene grafts. Acta Chir Scand 1985; 151:607-611. 10. Faulkner RT, Cowan GS Jr, Rothouse L. Early failure of expanded polytetrafluoroethylene in femoral vein replacement. Arch Surg 1979;114:939-943. l 1. Haimovici H, Hoffert PW, Zinicola N, et al. An experimental and clinical evaluation of grafts in the venous system. Surg Gynecol Obstet 1970;131:1173-1186. 12. Inokuchi K, Ono K. Experimental venous replacement by transplanted synthetic grafts. Surgery 1966;60:862-869. 13. J o h n s o n V, Eseman B. Evaluation of arteriovenous shunt to maintain patency of venous autograft. Am J Surg 1969;118: 915-920. 14. Wilson SE, Jabour A, Stone R, et al. Patency of biologic and prosthetic inferior vena cava grafts with distal limb fistula. Arch Surg 1978;113:1174-1179. 15. Gruss JD, Bartels D, Kawai S, et al. Application of plastic material in the PALMA operation. Angiology 1979;1:51-57. 16. Gervin AS. Complications of heparin therapy. In Swan L, ed. Venous Surgery in the Lower Extremities, 1st ed. St. Louis: WH Green, Inc, 1975, pp 307-325. 17. Gott VL, Leininger RI, Falb RD, et al. In vivo and in vitro evaluation of newer non-graphited heparinized surfaces. Surgery 1968;63:60-68. 18. Najjar FB, Gott VL. The use of small-diameter Dacron grafts with wall-bonded heparin for venous and arterial replacement: Canine studies and preliminary clinical experience. Surgery 1970;68:1053-1063. 19. Scott NA, Nunes GL, King SB, et al. Local delivery of an antithrombin inhibits placelet-dependent thrombosis. Circulation 1994;90:1951-1955. 20. Chen C, Hanson SR, Lumsden AB. Boundary layer infusion of heparin prevents thrombosis and reduces neointimal hyperptasia in venous PTFE grafts without systemic anticoagulation. J Vasc Surg 1995;22:237-247. 21. Guyton JR, Rosenberg RD, Clowes AW, et al. Inl~bition of rat arterial smooth muscle cell proliferation by heparin. I. In vitro studies with anticoagulant and non-anticoagulant heparin. Circ Res 1980;46:625-634. 22. Caste[lot J J, Wright TC, Karnovsky MJ. Regulation of vascular smooth muscle cell growth by heparin and heparin sulfates. Semin Thromb Hemost 1987;13:489-503. 23. Majesky MW, Schwartz SM, Clowes /VIM, et al. Heparin regulates smooth muscle S phase entry in the injured rat carotid artery. Circ Res 1987;61:296-300.
Recently we designed an expanded polytetrafluoroethylene (ePTFE)-based local infusion device that delivers therapeutic agents directly through the graft wall in the region adjacent to the upstream anastomosis, thereby achieving a high drug concentration downstream along the graft-blood interface. In this study we evaluated the effects of infusing heparin by this method on graft patency and neointimal hyperplasia in a canine model of femoral vein replacement. Five dogs underwent bilateral femoral vein replacement with the device. In each case one graft was infused with continuous heparin (48 U/kg/day) while the contralateral control graft received phosphate-buffered saline solution for 14 days. All heparin-treated grafts were patent and all control grafts were thrombosed at 14 days. There was no significant difference in systemic activated partial thromboplastin time among samples taken preoperatively, at 48 hours, and at 14 days of implantation (p > 0.5). There was no significant difference in neointimat hyperplasia between the upstream and downstream anastomoses in heparintreated grafts. These data demonstrate that the transgraft infusion of heparin preserved venous ePTFE graft patency without measurable systemic anticoagulation. Thus this approach may represent an attractive strategy for maintaining patency of synthetic venous grafts. (Ann Vasc Surg 1996;10:147-155.)
Venous disease is a common problem that often causes significant morbidity and sometimes death. Superior vena caval syndrome, inferior vena caval syndrome, postphlebitic disease, traumatic loss of veins, and congenital absence or displacement of veins all represent serious lesions that are potentially correctable. Although collected reports indi-
From the Division of Vascular Surgery, Department of Surgery, and the Division of Hematology/Oncology (S.R.H.), Department of Medicine, Emory University School of Medicine, Atlanta, Ga. Presented at the Twentieth Annual Meeting of the Peripheral Vascular Surgery Society, New Orleans, La., June 10, 1995. Supported in part by research grants HL 31469 and HL 48667 from the National Institutes of Health and by a grant from the American Heart Association. Reprint requests: Alan B. Lumsden, MD, Surgery Research, 1639 Pierce Dr., 5105 WMB, Atlanta, GA 30322.
cate that a considerable number of grafts have been placed in various portions of the h u m a n venous system, success rates for replacement prostheses have consistently been low in small-caliber vein replacement or reconstruction because of early graft thrombosis and anastomotic stenosis.l5 Expanded polytetrafluoroethylene (ePTFE) has been used for segmental venous replacement of vena cava, portal, and external iliac veins, 69 Patency rates in large-caliber veins appear promising but those in femoral vein replacement with ePTFE are very low, with thrombosis usually occurring within 24 to 48 hours, l° For satisfactory results in venous reconstruction, therefore, prevention of thrombus formation is mandatory. Patency may be maintained by overcoming those factors known to cause thrombosis including operative trauma, anastomotic strictures, low-pressure systems, slow intraluminal blood flow, host reaction to grafts, and relative high thrombogenicity of the substi147
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tutes. 11'12 Adjunctive techniques to improve patency of venous grafts include the use of arteriovenous fistulas, systemic anticoagulants, and antithrombotic materials bonded to the prosthetic wall. ~318However, all of these techniques are associated with side effects that limit their use. Recently we designed and characterized an ePTFE-based local drug delivery device that selectively concentrates infused pharmacologic agents specifically within those blood layers (boundary layers) adjacent to the graft wall and at downstream anastomotic sitesJ 9 In our previous study we administered heparin locally by means of this approach and evaluated its effects on graft patency and neointimal hyperplasia formation in a rabbit model of inferior vena cava replacement. 2° Data from this study demonstrate that the transgraft infusion of heparin significantly increases inferior vena cava graft patency in the rabbit and markedly reduces downstream anastomotic neointimal hyperplasia and cell proliferation without measurable systemic anticoagulation. However, the design of the infusion device used in the rabbit experiment delivered heparin at the midportion of the graft, thus highlighting the significant difference in findings between the upstream and downstream anastomoses as a result of the minimal delivery of heparin to upstream sites. The unchecked development of neointimal hyperplasia at the upstream anastomosis may lead to graft failure. To overcome this problem we modified the delivery device to infuse drug immediately adjacent to the upstream anastomosis, thereby preventing graft thrombosis and perhaps reducing neointimal thickening at both anastomoses. The purpose of the present study was to evaluate the effect of this modified system for improving patency and reducing anastomotic neointimal hyperplasia of femoral vein replacements in a canine model, which is more clinically relevant. MATERIAL AND METHODS Local I n f u s i o n D e v i c e The local infusion device was constructed from ringed ePTFE clinical vascular graft material (Gore-Tex, W.L. Gore & Associates, Inc., Flagstaff, Ariz.) as previously describedJ 9'2° For modification of the device, a cuff-reservoir was made adjacent to one end of a segment of ePTFE graft (6 m m internal diameter and 4.5 cm in length), which delivers heparin directly through the graft wall in the region adjacent to the upstream anastomosis, thereby achieving a high drug concentration downstream along the graft-blood inter-
face (Fig. 1). In view of the proximity of the infusion site to the upstream anastomosis, it is likely that heparin diffuses to this area and causes biologic effects. An implantable osmotic pump (model 2ML2, Alza Corp., Palo Alto, Calif.) was connected to the tubing of the device. These osmotic pumps delivered at a predictable rate of 5 b~l/hr over a period of 14 days. Pumps for treated grafts were loaded with heparin (20,000 U/2 ml; Elkins-Sinn, Inc., Cherry Hill, N.J.), whereas control pumps were loaded with phosphate-buffered saline solution (PBS). Treated grafts received local infusion of heparin for 14 days at the rate of 60 U/hr, which was equivalent to an average rate of 2 U/kg/hr. Satisfactory drug delivery was confirmed at the time of harvest by observations of pump residual volume and reservoir tubing patency. Animal Procedures Five adult male mongrel dogs weighing 30.56 _+ 3.96 kg were used in this study. All animal procedures and care were performed in accordance with the "Principles of Laboratory Animal Care" and the "Guidelines for the Care and Use of Laboratory Animals" (NIH publication No. 80-23, revised 1985). Anesthesia was induced with thiopental sodium (10 to 20 mg/kg intravenously) administered via endotracheal intubation and maintained with i% to 2.5% isoflurane. Under sterile conditions, an approximate 8 cm segment of femoral vein was dissected bilaterally and clamped proximally and distally. Systemic heparin (100 U/kg) was given prior to clamping. A 2 cm segment of femoral vein was excised. An ePTFE-based infusion device was implanted bilaterally in the femoral vein in an end-to-end anastomosis using running 6-0 polypropylene sutures. On completion of the anastomosis, an osmotic pump preloaded with heparin was connected to the infusion device on one side, and another osmotic pump preloaded with PBS was connected to the infusion device on the contralateral side as a control. These pumps were implanted in subcutaneous pockets. The incision was closed with 3-0 polyglactin sutures. Bromodeoxyuridine (BrdU, Sigma Chemical Co., St. Louis, Mo.), in a dose of 50 mg/kg dissolved in 50 ml normal saline solution, was administered intraperitoneally 24 hours prior to euthanasia at i4 days. The animals were anesthetized as previously described, and the femoral veins and grafts were exposed. Patency of grafts was determined by direct inspection, by blood
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Fig. 1. ePTFE-based local infusion device. A, Device consists of a silicone rubber cuff-reservoir glued in position between the two rings of a 6 rnm internal diameter ePTFE graft (30 p,m internodal distance). The hub is attached to a 1 m m internal diameter silicone rubber catheter tube, which is then attached to an osmotic pump. 13, A soluble agent from an osmotic pump will pass through the graft wall, enter the bloodstream in highest concentration at the graft wall (boundary layer), and be transported downstream (axially) by blood flow (convection), with slower mixing in the radial direction (i.e., toward the center stream blood flow), occurring primarily through diffusive mechanisms. Long arrow indicates direction of blood flow.
flow measurement using an ultrasonic flowmeter (model T201, two-channel, Transonic Systems, Inc., Ithica, N.Y.), and later by histologic analysis. A sternotomy was performed, and Ringer's solution was infused at 120 m m Hg pressure through a wide-bore needle into the left ventricle while the animal was synchronously exsanguinated via a cannula placed in the right atrium. Once the blood was cleared from the circulatory system, the whole animal was perfusion fixed in situ for 20 minutes at 120 m m Hg pressure using 2.5% glutaraldehyde. Grafts with 3 cm segments of attached femoral vein were harvested and fixed in 10% buffered formalin for 4 hours and then in 70% alcohol. Blood was drawn for activated partial thromboplastin time (aPTF) assay preoperatively, at 48 hours, and at 14 days of implantation.
Histology and Morphometry Following fixation, the grafts were cut longitudinally at both the upstream and downstream anastomoses to observe the anastomotic neointimal tissues, cut transversely at the remaining femoral vein and middle portions of the grafts at 3 m m
intervals, processed through graded ethanol, infiltrated with xylene and paraffin, and then embedded in paraffin blocks. Five-micrometer sections were cut and stained with hematoxylin and eosin and Verhoeff-Masson's stain. Pannus tissue ingrowth overlying the luminal surface of the graft adjacent to the anastomoses was considered to be neointima. Morphometric measurements of the thickness and area of neointima were performed by computer image analysis software (Optimas, Bioscan, Inc., Edmonds, Wash.) on a magnified image relayed from a microscope-mounted video camera to a digitizing pad and video monitor (Thomas Optical, Columbus, Ga. ) as previously described. 2°
Immunocytochemistry The avidin-biotin complex immunoperoxidase procedure (LSAB Kit, Dako Corp., Carpenteria, Calif.) was used to identify determinants characterizing vascular smooth muscle cells (SMCs) and proliferating cells as previously described. 2° Briefly, SMCs were identified by specific ~-actin immunostaining with HHF35 monoclonal antibody (Dako Corp.). Proliferating cells were iden-
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tiffed with anti-BrdU monoclonal antibody (Dako Corp.). BrdU-positive cells were quantified manually using a cell-counting technique per highpower field ( x 400) on a micrometer grid. In each field all cells were counted and the number of positively stained cells was expressed as a percentage of total cells to arrive at the BrdU index. A m i n i m u m of 10 fields were quantified per section.
Statistical Analysis Statistical analysis was performed on a Macintosh Quadra 650 computer using Excel 4.0 statistical software. Chi-square analysis was used to determine significant differences in patency rates between the heparin-treated grafts and the control grafts. The paired Student's t test was used to compare aPTf values among samples taken from different time points. The paired Student's t test was also used to determine the significance of neointimal thickness, neointimal area, and cell proliferation rates between the upstream and downstream anastomoses of the treated grafts. Results were considered significant at p < 0.05.
RESULTS Graft Patency At harvest all osmotic pumps were functioning. All heparin-treated grafts (5 of 5) were patent and all PBS-treated grafts (5 of 5) were throm-
bosed. The volume blood flow rate through the patent grafts at 14 days averaged 8 0 _ I8 ml/min. Thus the transgraft infusion of heparin resulted in a significant increase in graft patency as compared with the PBS-treated grafts (p-0.025).
M e a s u r e m e n t s of Clotting Time Systemic aPTF measurements were 10.I _+ 0.2 seconds, i0.9 +_ 0.6 seconds, and 10.1 _+ 0.9 seconds for samples taken before local heparin infusion, at 48 hours, and at 14 days of continuous local heparin infusion, respectively. The results of the systemic aPTF determinations revealed no significant difference before and during local heparin therapy (p > 0.05). N e o i n t i m a l Hyperplasia Histologic examination showed the presence of thrombus in all of the control grafts. Organized thrombus was seen at regions of the grafts close to both the upstream and downstream anastomoses (Fig. 2, A and C), and thrombus in the central region remained unorganized (Fig. 2, B). Thus the control grafts were excluded from morphometric analysis because of the confounding effects of graft thrombosis on the development of neointimal hyperplasia and because of the difficulty in distinguishing anastomotic neointimal hyperplasia from organized thrombus.
Fig. 2. Control grafts were thrombosed. A, Upstream anastomosis. Organized thrombus was seen around the suture line (longitudinal section).
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Fig. 2, c o n t ' d . 13, Midportion of the graft. Unorganized thrombus was present (transverse section). C, Downstream anastomosis. Organized thrombus was seen around the suture line (longitudinal section). (Verhoeff-Masson's stain; x 20.)
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Table I. Anastomotic neointimal hyperplasia and cell proliferation (BrdU index) of heparin-treated grafts Neointima
Upstream anastomosis
Downstream anastomosis
Difference (%)
p Value
A r e a ( m m 2) Thickness (mm) B r d U i n d e x (%)
1.19 - 0.67 0.53 --_ 0.19 14.41 _+ 2.51
1.08 --+ 0.97 0.43 ~ 0.19 10.48 ± 2.49
8 19 27
0.6295 0.1319 0.0073
In the heparin-treated grafts neointimal hyperplasia occurred at both the upstream and downstream anastomoses (Fig. 3, A and C). Morphometric determinations of neointimal area and neointimal thickness were slightly higher at the upstream anastomoses but were not significant as compared with the downstream anastomoses (Table I). The major cellular component in the neointima was SMC, as identified by a-actin immunostaining. Pseudointima was seen in the middle portions of patent grafts (0.40 _+ 0.09 m m thickness, Fig. 3, C). Cell P r o l i f e r a t i o n Vascular SMC proliferation was determined by BrdU immunostaining. The BrdU labeling index was significantly higher (p = 0.0073) in the neointimal tissue at the upstream anastomoses (14.41% _+ 2.51%) as compared with the downstream anastomoses (10.48% m 2.49%). Thus the local transgraft infusion of heparin through the modified device reduces neointimal SMC proliferation by 27% (Table I). DISCUSSION Standard heparin for systemic effect needs to be administered by continuous intravenous infusion or by regular subcutaneous injections two or three times a day. It is associated with acute complications such as hemorrhage, anaphylaxis, thrombocytopenia, and delayed embolus and with chronic complications such as alopecia, osteoporosis, pathologic fracture, and ascorbic acid deficiency. 16 For these reasons standard heparin is unlikely to be accepted as long-term therapy in vascular surgery. However, local infusion of heparin in small doses may be useful for certain long-term therapy without major complications. A major advantage of local drug administration is the potential to greatly reduce systemic side effects, since the absolute amount of compound
administered is relatively small. An ePTFE-based local infusion device delivers therapeutic agents directly through the graft wall in the region adjacent to the upstream anastomosis, thereby achieving a high drug concentration downstream along the graft-blood interface (boundary layer). This concept has been supported by previous computational and experimental studies in vitro (unpublished data). Computational fluid dynamics was used to calculate the infusate concentration on the graft wall. Experimental techniques such as flow visualization and local sampiing were also used to document high infusate concentrations along the wall downstream from the graft. The theoretical and laboratory measurements were in agreement and were used to estimate local heparin concentrations in the present experiments. In the present model (80 ml/min blood flow through a 6.0 m m internal diameter graft) the infusate is predicted to be diluted approximately 400-fold along the graft wall over a distance of 4 to 5 cm downstream according to theoretical modeling of mass transport under these flow conditions. Because the infusate concentration of heparin was i0,000 U/ml, the concentration of heparin along the graft wall and downstream anastomosis is calculated to have been approximately 25 U/ml, a level expected to produce profound inhibition of both coagulation and vascular cell proliferation. 2~x3 The average infusion rate of heparin in a treated graft was 1 U/min. If this amount were infused so as to mix uniformly with all of the incoming blood (80 ml/min), the mean heparin concentration at all points within the graft would have been 0.0125 U/ml (1 U/min - 80 ml/min), that is, heparin levels along the graft wall would have been approximately 2000-fold lower than those achieved by the boundary layer infusion approach. The consequences of heparin recirculation can also be readily evaluated. Based on estimates of the half-life of heparin in vivo (tl/2 = 1 hour) and dog plasma volume ( - 4 0 ml/kg), the steady-
Fig. 3. Heparin-treated grafts were patent. A, Upstream anastomosis. Pannus tissue ingrowth to form anastomotic neointimal hyperplasia (longitudinal section). B, Midportion of graft. Pseudointima was present (longitudinal section). C, Downstream anastomosis. Anastomotic neointimal hyperplasia was seen (longitudinal section). (Verhoeff-Masson's stain; ×20.)
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state plasma heparin level is directly calculated according to first-order clearance kinetics as 0.072 U/ml, a minimal anticoagulation dose whose predicted level is consistent with the absence of detectable effects observed on the aPTF clotting time m e a s u r e m e n t s (see Results). The ratio of local boundary layer heparin level (25 U/ml) to systemic circulating level is thus estimated as 350:1 in this large animal model, indicating that ~dth this m e t h o d very high drug levels can be localized to a target vessel surface while at the same time minimizing systemic bleeding or other u n w a n t e d side effects. Although we currently do not have an effective means of local sampling and direct m e a s u r e m e n t of the boundary layer drug concentration in vivo, the results of these and other animal experiments are consistent with predictions based on in vitro studies and computational modeling. ~9"2° The approach of transgraft infusion of heparin in vein replacement has been evaluated in our previous study of rabbit inferior vena cava replacement 2° and in the present study of dog femoral vein replacement (Table II). Striking effects on outcome events were achieved in both animal models, preventing early thrombosis, preserving graft patency, and reducing anastomotic neointimal hyperplasia. In this study vce have modified the delivery device to infuse drug immediately adjacent to the upstream anastomosis in an a t t e m p t to reduce neointimal hyperplasia at both anastomoses. In heparin-treated grafts the neointimal thickness at the upstream anasto-
moses was slightly higher than that at the downstream anastomoses, and the difference between t h e m was 19% (not significant), whereas the difference was 88% (highly significant) in the rabbit model. 2° Although the cell proliferation rate at the upstream anastomosis was still significantly higher than that at the downstream anastomosis, the difference between t h e m was 27%, which is considerably lower t h a n the 72% in the rabbit model. 2° This observation demonstrates that transgraft infusion of heparin through the redesigned infusion device treats both the downstream anastomosis and, at least in part, the upstream anastomosis. Heparin infusion adjacent to the upstream anastomosis m a y prevent pannus tissue ingrowth from the edge of native vessel. Heparin diffusion to the upstream anastomosis m a y play a role in inhibiting cell proliferation and reducing neointimal growth. The dog model is relevant in terms of clinical applicability of the infusion device. Compared to rabbits, the hemodynamics and blood vessels of dogs more closely resemble those of humans. Furthermore, peripheral veins, longer grafts, and internal controls have been used in the dog model, providing more reliable and clinically relevant studies (Table II). There appears to be a critical period of approximately 1 to 2 weeks after venous implantation w h e n the conduit is at particular risk of thrombosis. 2'5'm In this study the 2-week time period was selected specifically to permit us to evaluate the use of this approach to modulate graft thrombosis, although we are unable to c o m m e n t on the pros-
T a b l e II. Comparison of rabbit and dog models of transgraft infusion of heparin in vein replacement Rabbit model Infusion device Replaced vein Heparin dose Graft patency Systemic aPTT Neointimal thickness
4 mm i.d., 2.5 cm in length, infusion from middle portion of device Inferior vena cava 30 U/hr, equivalent to 7 U/kg/hr
Dog model 6 mm i.d., 4.5 cm in length, infusion from region of device adjacent to the upstream anastomosis Femoral vein 60 U/I~, equivalent to 2 U/kg/hr
100%
100%
Blood flow Control setting
No significant change 88% reduction at downstream anastomosis* 72% reduction at downstream anastomosis* 27 ml/min Saline infusion in separate animals
Clinically relevant
Less relevant
No significant change 19% reduction at downstream anastomosis 27% reduction at downstream anastomosis* 80 ml/min PBS infusion in contralateral site of same animals More relevant
BrdU index
*Student's t test analysis showed significant difference.
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pects for long-term patency of ePTFE grafts in the venous system. This approach could be extended for long-term local infusion of heparin if this were found necessary to maintain patency. Further studies with different time courses are needed to evaluate long-term patency of vein replacement by means of this approach. Another potential study using this approach is the transplantation of prosthetic and autogenous valves into ePTFE grafts to further optimize patency results and reduce venous hypertension. Although this approach is presently being used exclusively as a research tool, it may represent an attractive strategy for antithrombotic therapy in venous replacement with synthetic graft materials.
CONCLUSION Prosthetic grafts in the venous systems are of limited usefulness because of early thrombotic occlusion, especially in small-caliber veins. Adjunctive therapy to improve patency of venous grafts includes systemic anticoagulation, which is associated with serious side effects. A major advantage of local drug administration is the potential to greatly reduce systemic side effects, since the absolute amounts of compound administered is relatively small. A new ePTFE-based local infusion device has been used to deliver therapeutic agents such as heparin, in this case directly through the wall of a synthetic graft, thereby achieving high drug concentrations in the blood fluid boundary layer along the graft wall and at the downstream anastomotic site. This approach has been demonstrated to have striking effects on preserving ePTFE graft patency and reducing anastomotic neointimal hyperplasia and cell proliferation without systemic anticoagulation in the animal models. Reduced neointimal hyperplasia at both upstream and downstream anastomoses was achieved by delivering heparin immediately adjacent to the upstream anastomosis. This approach may have potential clinical application for antithrombotic therapy in venous replacement with synthetic graft materials. We thank Ms. Carolyn Suwyn for her expert technical assistance in tissue processing and histology procedures and Ms. Beverly Noe for laboratory assistance. REFERENCES 1. Gloviczki P, Pairolero PC, Toomey BJ, et al. Reconstruction of large veins for nonmalignant venous occlusive disease. J Vasc Surg 1992;16:750-761,
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2. Dale WA, Scott HW Jr. Grafts of the venous system. Surgery 1963;53:52-74. 3. Norton L, Eiseman B. Replacement of portal vein during pancreatectomy for carcinoma. Surgery 1975;77:280-284. 4. Florian A, Cohn LH, Dammin GJ, et al. Small vessel replacement with Gore-Tex (expanded polytetrafluoroethylene). Arch Surg 1976;111:267-270. 5. Dale WA. Reconstructive venous surgery. Arch Surg 1979; 114:1312-1318, 6. Scherck JP, Kerstein MD, Stansel HC Jr. The current status of vena cava replacement. Surgery 1974;76:209-233. 7. Scheinin TM, Jude JR. Experimental replacement of the SVC: Effect of temporary increase in blood flow. J Thorac Cardiovasc Surg 1964;48:781-790. 8. Symbas PW, Foster JH, Scott HW. Experimental vein grafting in the portal venous system. Surgery 1961;50:97-101. 9. Plate G, Einarsson E, Eklof B, et al. Iliac vein obstruction associated with acute lliofemoral venous thrombosis: Results of early reconstruction using polytetrafluoroethylene grafts. Acta Chir Scand 1985; 151:607-611. 10. Faulkner RT, Cowan GS Jr, Rothouse L. Early failure of expanded polytetrafluoroethylene in femoral vein replacement. Arch Surg 1979;114:939-943. l 1. Haimovici H, Hoffert PW, Zinicola N, et al. An experimental and clinical evaluation of grafts in the venous system. Surg Gynecol Obstet 1970;131:1173-1186. 12. Inokuchi K, Ono K. Experimental venous replacement by transplanted synthetic grafts. Surgery 1966;60:862-869. 13. J o h n s o n V, Eseman B. Evaluation of arteriovenous shunt to maintain patency of venous autograft. Am J Surg 1969;118: 915-920. 14. Wilson SE, Jabour A, Stone R, et al. Patency of biologic and prosthetic inferior vena cava grafts with distal limb fistula. Arch Surg 1978;113:1174-1179. 15. Gruss JD, Bartels D, Kawai S, et al. Application of plastic material in the PALMA operation. Angiology 1979;1:51-57. 16. Gervin AS. Complications of heparin therapy. In Swan L, ed. Venous Surgery in the Lower Extremities, 1st ed. St. Louis: WH Green, Inc, 1975, pp 307-325. 17. Gott VL, Leininger RI, Falb RD, et al. In vivo and in vitro evaluation of newer non-graphited heparinized surfaces. Surgery 1968;63:60-68. 18. Najjar FB, Gott VL. The use of small-diameter Dacron grafts with wall-bonded heparin for venous and arterial replacement: Canine studies and preliminary clinical experience. Surgery 1970;68:1053-1063. 19. Scott NA, Nunes GL, King SB, et al. Local delivery of an antithrombin inhibits placelet-dependent thrombosis. Circulation 1994;90:1951-1955. 20. Chen C, Hanson SR, Lumsden AB. Boundary layer infusion of heparin prevents thrombosis and reduces neointimal hyperptasia in venous PTFE grafts without systemic anticoagulation. J Vasc Surg 1995;22:237-247. 21. Guyton JR, Rosenberg RD, Clowes AW, et al. Inl~bition of rat arterial smooth muscle cell proliferation by heparin. I. In vitro studies with anticoagulant and non-anticoagulant heparin. Circ Res 1980;46:625-634. 22. Caste[lot J J, Wright TC, Karnovsky MJ. Regulation of vascular smooth muscle cell growth by heparin and heparin sulfates. Semin Thromb Hemost 1987;13:489-503. 23. Majesky MW, Schwartz SM, Clowes /VIM, et al. Heparin regulates smooth muscle S phase entry in the injured rat carotid artery. Circ Res 1987;61:296-300.