The role of an endothelial cell lining in limiting distal anastomotic intimal hyperplasia of 4-mm-I.D. Dacron grafts in a canine model Linda M. Graham,*,+Thomas E. Brothers; Christopher K. Vincent,* William E. Burkel: and James C. Stanleyf *Department of Surgery, Case Western Reserve University, #Department of Surgery, University of Michigan, and the §Department of Anatomy and Cell Biology, University of Michigan The effect of an endothelial cell (EC) lining on intimal hyperplasia at the distal anastomosis of . Dacron grafts was assessed in a canine model. Enzymatically derived autologous EC were used to seed 14 to 17 cm long, 4 mm I.D., knitted Dacron aortoiliac grafts implanted in an end-to-side manner in six dogs (Group I). Unseeded grafts were similarly implanted in six control dogs (Group 11). All animals received acetylsalicylic acid (325 mg PO qd) 24 h prior to graft placement and for 2 weeks postoperatively. Distal anastomotic intimal hyperplasia (AIH) and luminal surface EC coverage were quantitated at the conclusion of a

16-week study period. Patency for Group I and Group I1 grafts were 90% and 55%, respectively ( p = 0.07). Maximum AIH, defined as the maximum reduction of luminal cross-sectional area at the distal anastomosis, was not significantly different between Group I (13.1 2 8.0%) and Group I1 (15.1 f 7.3%) conduits. However, AIH was inversely related to the extent of luminal EC coverage (r = -0.6, p c 0.05), thus greater endothelialization was associated with decreased AIH. These data support the idea that EC coverage of the luminal surface of prosthetic vascular grafts may limit the development of AIH.

INTRODUCTION

Anastomotic intimal hyperplasia (AIH) is a recognized cause of infrainguinal prosthetic vascular graft failure in the clinical setting. AIH, especially evident at distal anastomoses in small-caliber conduits, must be prevented if optimal long-term patency is to be achieved. Seeding of prosthetic conduits with autologous endothelial cells (EC) promotes the early development of a luminal endothelial lining and improves the patency of small caliber grafts in experimental animals.’-4 These EC linings are functional as evidenced by prostacyclin production and diminished graft-platelet interaction^.^^ Furthermore, the inner capsule in the midgraft region of EC seeded grafts is thinner than in unseeded grafts?” However, the effect of an EC lining on the develSupported by a grant from the Department of Veterans Affairs. ‘To whom correspondence should be addressed at Surgery Service 112w Veterans Administration Medical Center, 10701 East Boulevard, Cleveland, OH 44106. Journal of Biomedical Materials Research, Vol. 25, 525-533 (1991) CCC 0021-9304/91/040525-09$04.00 0 1991 John Wiley & Sons, Inc.

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opment of AIH has not been assessed in these studies. The potential for EC seeding and antiplatelet agents to promote patency and reduce anastomotic intimal hyperplasia in ePTFE grafts has been the subject of recent studies in our laboratory.*-" The relation between EC luminal coverage and AIH in Dacron grafts is the subject of the present investigation. MATERIALS A N D METHODS

Adult mongrel dogs weighing 20 to 36 kg underwent bilateral aortoiliac graft placement, using 4 mm I.D., 14- to 17-cm-long conduits of noncrimped knitted Dacron with an external polypropylene spiral support coil (USCI Surgical Products, C. R. Bard, Inc., Billerica, MA). Each animal received either two grafts seeded with autologous EC (Group I, n = 6) or two unseeded grafts (Group 11, n = 6). Use of separate animals for EC seeded and unseeded grafts prevented potential cross-inoculation of unseeded conduits with EC or systemic effects which might alter graft healing. Hematologic and coagulation studies, including platelet count and platelet aggregation in response to ADP, thrombin, and collagen were performed on all animals. On th.e basis of these studies, dogs were paired and entered into opposite treatment groups. Acetylsalicylic acid, 325 mg PO qd, was administered to all animals beginning 24 h prior to graft implantation and continuing for 14 days postoperatively. Dogs were cared for in compliance with NIH guidelines "for the care and use of laboratory animals" [NIH Publication 8523, revised 19851. Animals undergoing graft placement had anesthesia induced with intravenous thiamylal sodium and maintained with halothane/N20/02. Hydration was provided by infusion of lactated Ringer's solution, 15 mL/kg/hr IV, during the operative procedure. Penicillin G benzathine and procaine hydrochloride, 450,000 IU each, were administered intramuscularly immediately prior to surgery. A 10-cm segment of external jugular vein was removed for endothelial cell harvest using sequential incubations in 0.1% trypsin (Difco, Detroit, MI) and collagenase CLS type I, 630 U/ml (Worthington, Freehold, NJ) as previously reported.I2 EC pellets, containing 8 x lo5 cells (range 4 to 15 x lo5 cells), obtained by centrifugation at 1508, were resuspended in two 0.5mL aliquots of M199 culture medium. One aliquot of M199 containing autologous EC (Group I) or M199 alone (Group 11) was added to 10 mL of blood and used to preclot each Dacron graft. Prior to implantation all prostheses were flushed five times with 5 mL of blood containing; heparin sodium, 200 IU/mL. EC seeding density ranged from 6 to 24 x lo3 cells/cm2 of graft surface in Group I animals. The infrarenal aorta and distal iliac arteries were exposed through a midline abdominal incision. Following systemic anticoagulation with1 heparin sodium, 100 IU/kg IV, the aorta and iliac arteries were cross-clamped. The grafts were anastomosed in an end-to-side manner to the infrarenal aorta proximally and to the external iliac arteries distally with continuous 6-0 polypropylene suture. Care was taken to assure that proximal anastomoses were aligned and equal in size, and that the arteriotomy for each dis-

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tal anastomosis was precisely 2 cm in length to minimize differences in hemodynamics between anastomoses in various animals. Blood flow was established through both grafts simultaneously.The anticoagulant effect of the heparin sodium was reversed with protamine sulfate, 1.0 mg/kg IV Ligation of the median sacral and proximal external iliac arteries excluded major lower extremity collateral blood flow. Completion arteriograms were performed in all animals to verify the technical adequacy of graft placement. Graft patency was determined by assessment of femoral artery pulses three times per week for the first 2 weeks and weekly thereafter for a total of 16 weeks, unless bilateral graft occlusion was documented. In such animals prostheses were removed immediately. At the time of sacrifice, animals were anesthetized as they had been for graft implantation and heparin sodium, 150 IU/kg, was administered intravenously. The arteries proximal and distal to the grafts were isolated and cannulated, and the grafts were flushed with phosphate-buffered saline until the effluent was clear. The infrarenal aorta, aortoiliac grafts, and proximal femoral arteries were then removed in toto. The midportion of each excised graft was divided longitudinally, with one half processed for light microscopy and the other for scanning electron microscopy (SEM). The endothelial coverage of each specimen was evaluated by an observer blinded to the treatment group. Coverage was considered 100%if the specimen was completely covered with endothelium and 0% if no endothelium was observed. Partial coverage was quantitated using a grid and determining the presence or absence of endothelium at ten intersections per field. At least 10 random fields were evaluated per section. Anastomotic regions were fixed in glutaraldehyde, dehydrated in ethanol, embedded in glycol methacrylate, sectioned at 2-mm intervals perpendicular to the longitudinal axis of the anastomoses, and stained with methylene bluebasic fuchsin. The first section studied was at the heel of the anastomosis (Fig. l),and sequential sections were evaluated throughout the anastomosis. The arterial and graft circumference at the intima-media and inner capsulegraft junctions were measured using a computer-linked digitizer, and the area of the original lumen was calculated. The area of arterial intimal hyperplasia and inner capsule thickening was measured from an average of nine sections per anastomosis. AIH was defined as the sum of the areas of arterial intimal hyperplasia and graft inner capsule, and was expressed as a percentage of the area of the original lumen. This determination eliminated measurement errors due to magnification. Statistical analysis of morphometric data utilized the unpaired t-test, and graft patencies were compared using the Chi-square test and Fisher’s test for 2 x 2 contingency tables. RESULTS

At 16 weeks, 9 of 10 (90%) EC seeded grafts in Group I dogs were patent compared to 6 of 11 (55%) unseeded grafts in Group I1 animals. This difference in patency approached, but did not reach, statistical significance ( p = 0.07). In Group I, one dog died as a result of an anesthetic complication

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Inner Capsule,

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Area of Inner Capsule + Area of lntimal Hyperplasia Area of Original Lumen

Figure 1. Serial sections through the distal anastomosis were taken at 2-mm intervals. The area of the inner capsule of the graft and the area of the intimal hyperplasia of the artery were measured using a computerlinked digitizer. Anastomotic intimal hyperplasia (AIH) was defined as the sum of these areas and expressed as a percentage of the area of the original lumen.

and was not included in any calculation of results, and one graft in another dog thrombosed within 2 weeks after implantation. In Group I1 animals, one graft thrombosed at 1 week, one at 3 weeks, and three at more than 4 weeks after implantation. On macroscopic evaluation graft occlusions which occurred more than 4 weeks after implantation appeared to be due to accumulation of tissue at the distal anastomosis. One animal in Group I1 which exhibited an occluded graft at 1 week was sacrificed after developing paralysis following interval angiography at 4 weeks. The occluded graft in this dog was included in patency calculations, but the contralateral patent graft was excluded from study since the 16-week endpoint was not reached. The variability in thrombotic tendency between dogs may skew patency results when analyzed on a per graft basis even when dogs are paired on the basis of platelet and coagulation parameters. Therefore, graft patency in Group I and I1 was also analyzed by animal. In Group I, 4 of 5 dogs had bilateral patent grafts at 16 weeks compared to 2 of 6 dogs in Group 11. Again, this difference in patency approached, but did not reach, statistical significance (p = 0.1) due to the small number of observations. Similarly, patency differences approached, but did not reach, statistical significance (p = 0.1) when data were analyzed in a paired fashion on the basis of the original pairing of dogs with respect to their thrombotic potential. Histologic studies revealed a cellular lining of most grafts that was characteristic of endothelium on SEM. Luminal surfaces of all but two EC seeded grafts were essentially completely covered by such cells ranging from 92% to 100% coverage. The remaining two seeded grafts exhibited 10% and 35% endothelial cell coverage of the surface. Unseeded grafts exhibited variable (35 to 100%)luminal surface coverage by EC cells.

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No significant differences in AIH were observed at distal anastomoses of Group I and Group I1 grafts that were patent at least 4 weeks postoperatively. Anastomotic luminal compromise, due to thickened inner capsule of the graft and intimal hyperplasia of the adjacent artery, was similar in the two groups, being 7.8 f 5.8% ( x k SD) in Group I EC seeded grafts compared to 7.6 k 6.5% in Group I1 unseeded grafts. Since the maximum area of intimal hyperplasia may be more important than the mean value in effecting graft patency, this measurement was also compared. Maximum AIH was similar for Group I and for Group I1 grafts, being 13.1 +- 8.0% and 15.1 f 7.376, respectively. However, if data were combined from both groups, comparison of maximum area of AIH to extent of endothelialization (Fig. 2) revealed a significant negative correlation [r = -0.6, p < 0.05, SE(b) = 6.161. DISCUSSION

The effect of an EC lining on the development of AIH in synthetic grafts is a topic of considerable speculation. Previous studies in our laboratory using canine thoracoabdominal and iliofemoral Dacron graft models demonstrated the efficacy of EC seeding in reducing inner capsule thickness? but these investigations did not assess the effect of an EC surface on anastomotic hyperplasia. Therefore, this study was undertaken to investigate the effect of 30

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an EC lining on AIH. EC seeding was used to promote the early development of a graft lining composed of endothelium. However, due to lack of uniformity in the efficiency of seeding as well as the early healing of some control grafts, presumably due to growth of capillaries through the interstices of the porous Dacron grafts, no difference was noted in AIH between seeded and control grafts. However, when AIH was related to the EC surface coverage, a significant negative correlation was seen suggesting that endothelial coverage of the luminal surface of a graft may reduce development of AIH. Although an EC surface will not alter all factors potentially contributing to AIH such as compliance mismatch between artery and graft or flow alterations with boundary layer separation, it might lessen activation of circulating blood elements including platelets, leukocytes, and componeiits of the coagulation and complement systems. An EC lining in a prosthetic graft could decrease platelet deposition and thrombus formation by generation of prostacyclin, antithrombotic factors, and plasminogen activators. Decreased platelet activation would lessen the release of platelet-derived growth factor (PDGF), a mitogen for SMC. We have previously demonstrated that development of an EC surface on Dacron and ePTFE grafts is accompanied by decreased deposition of indium labeled platelet^:'^ as well as by increased platelet survival time and increased platelet serotonin ~ o n t e n tSimilarly, .~ the rapid development of an EC lining may minimize deposition of leukocytes which also release growth factors for SMC.14 The contribution of platelets and other blood elements to the development of intimal hyperplasia in grafts is unclear. Several studies have demonstrated the effectiveness of ASA and dipyridamole in decreasing intimal thickening in vein graft^,'"^ and anastomotic hyperplasia in Dacron and ePTFE grafts,l”’ but others have been unable to confirm such an effect.” Recent studies from our laboratory suggest that ASA is more effective in limiting AIH than other antiplatelet agents, including specific inhibitors of cyclooxygenase and thromboxane ~ynthetase.”’~ Similarly, comparison of ASA-dipyridamole to a pure platelet antiaggregatory agent, ticlopidine, revealed reduction in platelet deposition on ePTFE grafts by both regimens.” However, only ASA-dipyridamole reduced anastomotic intimal hyperplasia and improved 3-month patency. Furthermore, clinical trials of antiplatelet agents suggest that platelet inhibition alone does not prevent AIH.2’ An EC surface may also limit AIH by direct inhibition of SMC proliferation. Conditioned medium from cultures of confluent EC, but not those in exponential growth phase, contains a heparinlike substance that inhibits SMC proliferation.” It is postulated that EC in vivo secrete a heparinlike substance that regulates the growth of underlying SMC. This suggestion receives support from the finding that in injured arteries intimal and medial SlLlC proliferation stops after EC coverage is ree~tablished.’~ The role of heparinlike substances in vivo remains to be defined. Exogenous heparin has been shown to suppress SMC proliferation in injured arteries apparently by inhibiting SMC in the G I growth phase preventing entry into the growth ~ycle.’~,’~ Heparin also inhibits SMC migration from the media to the inth~ia.’~ How-

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ever, EC coverage does not necessarily inhibit SMC proliferation. Clowes and colleagues have demonstrated EC and SMC proliferation at anastomoses of ePTFE grafts despite complete endothelial coverage, perhaps a consequence of chronic EC inj~ry.2"'~ Although EC produce SMC growth inhibitors, EC also produce SMC growth factors which could adversely affect graft healing and long-term patency. These mitogens include interleukin-1, PDGF, and other less welldefined SMC growth f a ~ t o r s .Although ~ ~ , ~ ~ these mitogens are secreted at high levels in vitro, the level of growth factor production in vivo is unclear. Cultured human umbilical vein and bovine aortic EC express the c-sis gene for PDGF at moderate levels, but freshly harvested EC from these same sources express very low levels of this gene.3oThis suggests that mitogen production by EC in vivo may be much lower than that in vitro. Production of SMC mitogens by EC is a likely explanation for SMC proliferation in the inner capsule of ePTFE grafts in regions underlying the endothelial cells at the leading edge of pannus and at the anastomo~es.2~ In fact, Zacharias and colleagues31have documented markedly increased mitogenic activity in perfusates of endothelial lined ePTFE grafts implanted in baboons for 2 weeks compared to perfusates of native artery. However, in grafts implanted for longer than 3 months, SMC mass does not increase despite continued SMC division; thus, SMC proliferation balances SMC l0ss.2~Studies have demonstrated that EC mitogen production can be regulated in vitro, suggesting that such regulatory processes may also occur in viv0.3233 Understanding the conditions which regulate mitogen production by EC in vivo may be very important in defining the role of EC in AIH. Although in the present study AIH was similar in EC seeded and unseeded grafts, it was inversely correlated with the actual extent of EC coverage of the graft. This observation supports the idea that the presence of intact endothelium on a graft's luminal surface may contribute to the control of anastomotic hyperplasia. The basis for this control awaits elucidation, but may include such diverse factors as a reduction in activated circulating blood elements that are stimulatory to SMC proliferation as well as production of antithrombotic or antiproliferative agents by the EC. References 1. L. M. Graham, W. E. Burkel, J.W. Ford, D.W. Vinter, R. H. Kahn, and J.C.

Stanley, "Immediate seeding of enzymatically derived endothelium in Dacron vascular grafts. Early experimental studies with autologous canine cells," Arch. Surg., 115, 1289-1294 (1980). 2. J.C. Stanley, W. E. Burkel, J.W. Ford, D.W. Vinter, R. H. Kahn, W.M. Whitehouse Jr., and L.M. Graham, "Enhanced patency of smalldiameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells," Surgery, 92, 994-1005 (1982). 3. B.T. Allen, J. A. Long, R. E. Clark, G. A. Sicard, K.T. Hopkins, and M. J. Welch, "Influence of endothelial cell seeding on platelet deposition and patency in small-diameter Dacron arterial grafts," J. Vusc. Surg., 1, 224-233 (1984).

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18.

E.C. Douville, R. E Kempczinski, L. K. Birinyi, and G. R. Ramalanjaona, ”Impact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetraf luoroethylene graft,” J. Vasc. Surg., 5, 544-550 (1987). G. P. Clagett, W.E. Burkel, J. B. Sharefkin, J.W. Ford, H. Hufnagel, D.W. Vinter, R. H. Kahn, L. M. Graham, J.C. Stanley, and P.W. Ramwell, ”Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells,” Circulation, 69, 632-639 (1984). W.M. Whitehouse, Jr., T.W. Wakefield, D.W. Vinter, J.W. Ford, D. P. Swanson, J.H. Thrall, J.W. Froelich, L.E. Brown, W.E. Burkel, L.M. Graham, and J. C. Stanley, ”Indium-111-oxine labeled platelet imaging of endothelial seeded Dacron thoracoabdominal vascular prostheses in a canine model,“ Trans. Am. SOC. Artif. Intern. Organs, 29, 183-187 (1983). W. E. Burkel, J.W. Ford, D.W. Vinter, R. H. Kahn, L. M. Graham, and J.C. Stanley, “Fate of knitted Dacron velour vascular grafts seeded with enzymatically derived autologous canine endothelium,”Trarns.Am. SOC. Artif. Intern. Organs, 28, 178-182 (1982). L. M. Graham, J.C. Stanley, and W. E. Burkel, ”Influence of endothelial cell seeding and antiplatelet drugs on patency of prosthetic vascular grafts, in Endothelialization of Vascular Grafts,” P. P. Zilla, R. D. Fasol, and M. Deutsch (eds.), Karger, Basel, 1987, pp. 57-63. L.M. Graham, C.K. Vincent, T.E. Brothers, K.A. Harrell, D. Darvishian, R. Sell, W. E. Burkel, and J.C. Stanley, ”Efficacy of antiplatelet agents in promoting patency and reducing anastomotic hyperplasia of endothelial cell seeded and unseeded e-PTFE grafts,” Surg. Forum, 39, 348-350 (1988). L.M. Graham, T.E. Brothers, D. Darvishian, K.A. Harrell, C.K. Vincent, W. E. Burkel, and J.C. Stanley, ”Effects of thromboxane synthetase inhibition on patency and anastomotic hyperplasia of vascular grafts,” J. Surg. Res., 46, 611-615 (1989). T. E. Brothers, C. K. Vincent, D. Darvishian, K. A. Harrell, W. E. Burkel, J.C. Stanley, and L. M. Graham, ”Effects of duration of acetylsalicylic acid administration on patency and anastomotic hyperplasia of ePTFE grafts,” Trans. Am. SOC.Artif. Intern. Organs, 35, 558-560 (1989). J.W. Ford, W.E. Burkel, and R.H. Kahn, ”Isolation of adult canine venous endothelium for tissue culture,” In Vifro, 17, 44-50 (1981). T.W. Wakefield, B. Lindblad, L.M. Graham, W.M. Whitehouse Jr., S. D. Ripley, N. A. Petry, S. A. Spaulding, W. E. Burkel, and J.C. Stanley, ”Nuclide imaging of vascular graft-platelet interactions: Comparison of indium excess and technetium subtraction techniques,” J. Surg. Res., 40, 388-394 (1986). K. Shimokado, E.W. Raines, D.K. Madtes, T.B. Barrett, E. P. Benditt, and R. Ross, ”A significant part of macrophage-derived growth factor consists of at least two forms of PDGF,” Cell, 43, 277-286 (1985). M. P. Metke, J.T. Lie, V. Fuster, M. Josa, and M.P. Kaye, “Reduction of intimal thickening in canine coronary bypass vein grafts with dipyridamole and aspirin,” Am. J. Cardiol., 43, 1144-1148 (1979). R.L. McCann, P-0. Hagen, and J.C.A. Fuchs, “Aspirin and dipyridamole decrease intimal hyperplasia in experimental vein grafts,” Ann. Surg., 191, 238-243 (1980). P-0. Hagen, Z-G. Wang, E. M. Mikat, and D. 8. Hackel, ’Xntiplatelet therapy reduces aortic intimal hyperplasia distal to small diameter vascular prostheses (PTFE) in nonhuman primates,” Ann. Surg., 195, 328-339 (1982). R.W. Oblath, EO. Buckley Jr., R.M. Green, S.I. Schwartz, and J.A. DeWeese, ”Prevention of platelet aggregation and adherence to prosthetic vascular grafts by aspirin and dipyridamole,” Surgery, 84/37-44 (1978).

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19. L.M. Rainwater, G. Plate, P. Gloviczki, R.C. Bahn, L. H. Hollier, and M. P. Kaye, ”Morphologic quantitation of pseudointima and effects of antiplatelet drugs on vascular prostheses in goats,” Am. J. Surg., 148, 195-202 (1984). 20. K. J. Hansen, H.R. Howe, T.A. Edgerton, K.B. Faust, N.D. Kon, K.R. Geisinger, and J. H. Meredith, ”Ticlopidine versus aspirin and dipyridamole: Influence on platelet deposition and three-month patency of polytetraf luoroethylene grafts,” J. Vasc. Surg., 4, 174-178 (1986). 21. A.W. Clowes, “The role of aspirin in enhancing arterial graft patency,” J. Vasc. Surg., 3, 381-385 (1986). 22. J. J. Castellot Jr., M. L. Addonizio, R. Rosenberg, and M. J. Karnovsky, “Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth,” J. Cell. Biol., 90, 372-379 (1981). 23. A.W. Clowes, M. A. Reidy, and M. M. Clowes, ”Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium,” Lab. Invest., 49, 327-333 (1983). 24. A.W. Clowes and M. J. Karnowsky, “Suppression by heparin of smooth muscle cell proliferation in injured arteries,” Nature, 265, 625-626 (1977). 25. A.W. Clowes and M. M. Clowes, “Kinetics of cellular proliferation after arterial injury. IV. Heparin inhibits rat smooth muscle mitogenesis and migration,” Circ. Res., 58, 839-845 (1986). 26. A.W. Clowes, A.M. Gown, S.R. Hanson, and M.A. Reidy, “Mechanisms of arterial graft failure. 1. Role of cellular proliferation in early healing of PTFE prostheses,” Am. J. Pathol., 118, 43-58 (1985). 27. A.W. Clowes, T. R. Kirkman, and M. M. Clowes, ”Mechanisms of arterial graft failure. 11. Chronic endothelial and smooth muscle cell proliferation in healing polytetraf luoroethylene prostheses,” 1. Vasc. Surg., 3, 877-884 (1986). 28. P. Miossec, D. Cavender, and M. Ziff, ”Production of interleukin 1 by human endothelial cells,” J. Immunol., 136, 2486-2491 (1986). 29. P. E. DiCorleto and D. F. Bowen-Pope, “Cultured endothelial cells produce a platelet-derived growth factor-like protein,” Proc. Natl. Acad. S C ~LISA., . 80, 1919-1923 (1983). 30. T. B. Barrett, C. M. Gajdusek, S. M. Schwartz, J. K. McDougall, and E. P. Benditt, “Expression of the sis gene by endothelial cells in culture and in vivo,“ Proc. Natl. Acad. Sci. U.S.A., 81, 6772-6774 (1984). 31. R. K. Zacharias, T.R. Kirkman, R.D. Kenagy, D. F. Bowen-Pope, and A.W. Clowes, “Growth factor production by polytetrafluoroethylene vascular grafts,” J. Vasc. Surg., 7, 606-610 (1989). 32. P. L. Fox and P. E. DiCorleto, “Regulation of production of a plateletderived growth factor-like protein by cultured bovine aortic endothelial cells,” J. Cell. Physiol., 121, 298-308 (1984). 33. P. L. Fox and P. E. DiCorleto, ”Modified low density lipoproteins suppress production of a platelet-derived growth factor-like protein by cultured endothelial cells,“ Proc. Natl. Acad. Sci. U.S.A., 83, 4774-4778 (1986).

Received March 22,1990 Accepted October 1,1990