seeding of intracoronary stents with immortalized human microvascular endothelial cells

Seeding of intracoronary stents with immortalized human microvascular endothelial cells

Intracoronary stents are effective in decreasing the complications associated with acute closure during coronary angioplasty. A major complication associated with the use of coronary stents is acute thrombotic occlusion. It has been postulated that the stent loses its thrombogenic potential after it becomes covered with a layer of endothelial cells. Human dermal microvascular endothelial cells were transfected with a plasmid containing the simian virus 40 large T-antigen gene. Stents were placed in culture media with cells for 2 weeks. Seeding efficiency of the stent with the endothelial cells was assessed by scanning electron microscopy. Balloon-expandable coronary stents placed in cell culture with immortalized human microvascular endothelial cells showed near-complete coverage after 2 weeks. After balloon inflation, persistence of cells on the stent was noted only on the lateral aspect of the balloon-expanded stents. If these stents were placed in culture, complete recovery of the monolayer was noted after 3 days. Stents were then covered with endothelial cells and frozen for 4 days. After thawing, the cells adhered to the devices and divided to form a monolayer in tissue culture. Seeded balloon-expandable stents were frozen for 4 months, thawed, and then implanted in a pig coronary artery. Human endothelial cells were identified on the stent 4 hours after deployment. These studies demonstrate the feasibility of using a human microvascular endothelial cell line to seed an uncoated metal stent. The cells remain adherent to the stent, are functional after freezing, and remain on the stent at least 3 hours after intracoronary implantation. (AM HEART J 1995;129: 860-6.)

Neal A. Scott, MD, PhD, a Francisco J. Candal, b Keith A. Robinson, PhD, a

and Edwin W. Ades, PhD b Atlanta, Ga.

Intracoronary stenting is an important adjunct in the treatment of the acute complications associated with percutaneous transluminal coronary angioplasty (PTCA). Patients who have acute closure of the tar- get vessel during the PTCA procedure have a high incidence of myocardial infarction and emergent coronary artery bypass graft surgery. 1 When coronary stents are used to treat acute closure, the incidence of myocardial infarction and emergent surgery is significantly decreased. 2' 3 Since the clinical intro- duction of coronary stenting in 1987, 4 the enthusiasm for this procedure has been tempered by the compli-

From the aAndreas Gruentzig Cardiovascular Center, Emory University Hospital; and the bBiological Products Branch, Center for Disease Control and Prevention. Dr. Scott was supported in part by a grant from the Robert Wood Johr~son Foundation for Minority Faculty Development. Received for publication Aug. 3, 1994; accepted Sept. 20, 1994.

Reprint requests: Neal A. Scott, MD, PhD, Andreas Gruentzig Cardiovas- cular Center, Emory University Hospital, Suite F-606, 1364 Clifton Rd., Atlanta, GA 30322.

4 /1 /61630

cations associated with the sudden thrombotic occlusion of the device. Despite concomitant treatment with aspirin, Dipyridamole, heparin, and warfarin, the incidence of thrombotic occlusion of coronary stents is between 2% and 25 %.5-9 In addition, the incidence of hemorrhagic complications associated with this aggressive anticoagulant regimen is also significant.2, 3

The blood-metal interface causes platelet deposition and is responsible for the significant thrombotic potential of coronary stents. It has been assumed that once the metal is covered by a layer of endothelial cells, the stents are no longer thrombogenic. 1° Prior studies have demonstrated that fibronectin-coated coronary stents can be seeded in vitro with vascular endothelial cells n' 12 and that a number of the cells remain adherent to the stent after balloon expansion of the stent and placement in a flow chamber for 2 hours. 13 Although these studies clearly demonstrated the feasibility of the concept of seeding stents with endothelial cells, many questions remained concern- ing the potential clinical utility of these devices.

860

Volume 129, Number 5 American Heart Journal Scott et al. 861

Repor t s of endothel ia l cell seeding have consis- t en t ly descr ibed the necessi ty of coat ing the pros the- sis with a ma t r ix to faci l i tate the a t t a c h m e n t of endothel ial cells. TM 12,14 However , these mat r ixes (fi- b ronec t in and collagen) are th rombogenic z5' 16 and, if some of the cells are lost dur ing s ten t dep loyment , could increase s ten t thrombogenic i ty . In addit ion, the logistics of using autologous endothel ia l cells for seeding s tents are impractical . Mos t P T C A proce- dures are pe r fo rmed in c o m m u n i t y hospi ta ls where the relat ively sophis t ica ted equ ipmen t and personnel necessary to isolate and ma in ta in viable endothel ial cell cul tures are not available. Even if these resources were available, the mos t i m p o r t a n t use of coronary s tents is for the t r e a t m e n t of acute closure. Al though specific coronary lesions have an increased likelihood for complicat ions, 17 acute closure dur ing P T C A is largely unpred ic tab le . Therefore , to be effectively used, autologous endothel ia l cells would have to be harves ted and seeded onto s tents for all pa t ien ts un- dergoing PTCA. This concept is clearly prohibi t ive in t e rms of costs and personnel.

Recent ly Ades et al is descr ibed the successful im- mor ta l iza t ion of a line of h u m a n de rmal microvascular endothel ia l cells. These cells re ta in mos t of the pheno typ ic character is t ics of endothel ia l cells af ter over 50 passages in culture. The deve lopmen t of this cell line allows the seeding of coronary s tents with homologous endothel ia l cells. The use of homologous cells could potent ia l ly decrease the logistical prob- lems associated with autologous endothel ial cells.

Th is s tudy was pe r fo rmed to de te rmine whether an uncoa ted s ten t could be seeded with a novel h u m a n microvascular endothel ia l cell line, whether the cells r emained on the s ten t a f ter s ten t expansion in vi tro and in vivo, and whether the h u m a n endothel ia l cells covering the s ten t would remain viable and adhe ren t to the s ten t af ter freezing, thawing, and coronary de- p loyment .

METHODS Endothelial cells. Human dermal microvascular endo-

thelial cells were transfected with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product and large T antigen. These cells can be pas- saged over 50 times and show no signs of senescence; they are virus and mycoplasma free and express the human leu- kocyte antigen (HLA) DR marker only on activation with -/-interferon. The cells exhibit the typical cobblestone morphologic characteristics when grown in m0nolayer culture, express and secrete von Willebrand factor, take up acetylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel, is

Seeding of stents with endothelial cells. Stents were sterilized by autoclaving at 121 ° C for 20 minutes. The

stents were then placed in six-well cell culture dishes. Fresh monolayers of immortalized human dermal microvascular endothelial cells (CDC/EU.HMEC-1 cell line) is were trypsinized and placed into the wells in concentrations of 2 x 105 cells/ml. The culture medium consisted of MCDB 131 (Clonetics, San Diego, Calif.), with 15% fetal bovine serum, 10 ng/ml epidermal growth factor, and 1/Lg/ml hy- drocortisone. The cell suspensions were added to each well with stents to attain a final volume of 4 ml/well. The plates were incubated at 37 ° C in 5% carbon dioxide. The plates were gently swirled every 15 minutes for the first 60 minutes of incubation and allowed to grow for 14 days. The growth medium was replaced every 4 days.

Stents. Tantalum wire coils and coronary stents (4.0 mm expanded diameter) were donated by the Cordis Corpora- tion (Miami Lakes, Fla.). After seeding with endothelial cells, the stents were placed onto a deflated PTCA balloon (4.0 mm inflated diameter; Cordis) by hand. The balloon was inflated to 6.0 atm with a hand-held inflation device (USCI, Billerica, Mass.) to expand the stent. The balloon was then deflated and withdrawn.

Freezing of endothelialized stents. Af ter a confluent layer of cells was grown onto the stents, several stents (n = 4) were removed from the six-well tissue culture plates and placed in Nalgene (Rochester, N. Y.) 2.0 ml cryovials containing equal parts of freezing mediums A and D in a sufficient volume to completely cover the stent. Medium A consisted of 483 ml of L-15 medium (Gibco, Grand Island, N. Y.), 16 ml of HEPES buffer (1 mol/L) (Gibco), 300 ml of fetal bovine serum (Hyclone, Logan, Utah), 200 ml of polyvinylpyrrolidine (100 ml/ml; US Biochemical), and 2 ml of gentamicin (50 mg/ml). Medium D consisted of 833 ml of L-15 medium (Gibco), 16 ml of HEPES buffer (1 mol/L) (Gibco), and dimethyl sulfoxide 151 ml (Sigma, St. Louis, Mo.). The media were sterilized by filtration using 0.2/~m filters. The vials were then put into a metal can used for freezing cells (Biotech Research Laboratories, Rockville Md.) and placed in a -70 ° C freezer. The frozen stents were thawed in a 37 ° C water bath and were washed once to re- move the freezing medium. Two of the stents were examined with scanning electron microscopy (SEM). The other two stents were trypsinized. The trypsinized cells were re- covered and inoculated into one well of a 24-well plate (Costar) and incubated at 37 ° C in 5% carbon dioxide to allow monolayer formation.

Coronary placement of endothelialized stents. Do- mestic Yorkshire swine were initially anesthetized with an intramuscular injection of ketamine (25 mg/kg)/acepro- mazine (0.1 ml/kg) and atropine (0.06 mg/kg). Intravenous access was established in an ear vein. After intravenous administration of Brevital 10 mg/kg, endotracheal intuba- tion was performed with a 7F endotracheal tube. General anesthesia was maintained with 1% to 2 % isofluorane. A cutdown on the right femoral artery was then performed. An 8F sheath (USCI, Billerica, Mass.) was inserted into the artery under direct vision. An 8F guide catheter (SciMed, Minneapolis, Minn.) with a hockey stick curve was ad- vanced to the ostium of the left coronary artery under flu- oroscopic guidance. After administration of intracoronary

May 1995 862 Scott et al. American Heart Journal

Fig. 1. SEM of bare tantalum wire (0.008-inch diameter) used in coronary stents.

Fig. 2. Higher-power image of tantalum wire.

nitroglycerine (0.6 #g/kg), coronary angiography was performed in orthogonal views. The endothelialized coronary stent was mounted onto a deflated PTCA balloon (3.5 mm inflated diameter) by hand. The balloon catheter that con- tained the stent was placed in the left anterior descending artery. The balloon was inflated to its nominal diameter with a handheld inflation device for a period long enough to allow full expansion of the stent (approximately 15 seconds). Three hours after stent deployment, the animal was killed with an overdose of intravenous barbiturate (Beutha- nasia 5, Ceteterinary Labs, Lenexa, Kan.; 0.25 mg/kg). The heart was rapidly removed and the left coronary perfused with heparinized lactated Ringer's solution at a pressure of 110 mm Hg for 20 to 30 seconds to clear the artery of blood. The vasculature was then fixed by perfusion with 2.5 % glutaraldehyde in 0.1 mol/L cacodylate, pH 7.4, at 110 mm Hg pressure and 37 ° C for 5 to 10 min. Both solutions were bubbled with 95% oxygen/5% carbon dioxide for at least 20 minutes before use. After at least 15 minutes of primary

fixation, the stented epicardial arterial segments were carefully dissected free from the heart, immersed in the same fixative, and stored at 4 ° C until analysis with scanning electron microscopy.

HLA Labeling. To differentiate between pig coronary and human HMEC-1 endothelial cells, we labeled both cell types with mouse monoclonal antibodies to HLA. After three washings with cold FACscan wash (10 mmol/L phos- phate buffered saline solution, 0.2% sodium azide, 0.1% bovine serum albumin, pH 7.4), 5 × 105 cells of each type were pelleted by centrifugation at 1000 rpm, 4 ° C, for 10 minutes. To label the cells, 25 ttL of a 1:5 dilution of mouse monoclonal antibody to HLA (ABC Class I, Pel-Freez, Browndeer, Wis.) was added to each pellet (except negative

controls) and vortexed. The reaction was incubated at 4 ° C for 30 minutes. The cells were then washed three times. Fluorescent (FITC)-labeled goat antimouse immunoglob- ulin G (25/A of a 1:50 dilution) was added to each cell pellet, including negative controls. The tubes were incubated at 4 ° C for 30 minutes and washed three times.

Stents that were completely covered with HMEC-1 cells were labeled by cutting the stent into smaller pieces, plac- ing the stent in 24-well plates, and washing the stent three times. Antibody to HLA was added to each well at a final dilution of 1:20 and incubated at 4 ° C for 30 minutes; the solution was gently swirled every 10 minutes. The stents were then washed three times and FITC-labeled goat antimouse antibody at a final dilution of 1:50 was added to some wells. The reaction was incubated at 4 ° C for 30 minutes, with gentle swirling every 10 minutes.

Scanning electron microscopy. Stents seeded with HMEC-1 cells were placed in fixative at 4 ° C for 24 to 48 hours. Each stent was carefully cut with scissors and razor into four longitudinal hemisections about 1 cm in length, while immersed in 0.1 mol/L cacodylate. They were post- fixed for 1 hour in 1% osmium tetroxide in 0.1 mol/L cacodylate and rinsed with distilled deionized water. After dehydration in graded ethanol, the stents were critical- point dried from liquid carbon dioxide by thermoregula- tion and flow monitoring. The stents were attached to alu- minum supports with silver paste and sputter coated with 15 to 30 nm 60/40 gold/palladium alloy. Morphologic doc- umentation was obtained with a scanning electron micro- scope equipped with a lanthanum hexaborate emitter op- erated at 10 to 15 kV accelerating voltage in the conven- tional secondary electron imaging mode.

RESULTS

Scanning electron microscopy of the tan ta lum wire coils used for stents revealed a surface consitent with tha t of a bare metal (Figs. 1 and 2). When these coils were placed in culture medium containing HMEC-1 endothelial cells, the cells formed a monolayer tha t covered approximately 75 % of the tan ta lum wire coil after 1 week and covered approximate ly 98 % of the coil after 2 weeks (Figs. 2 and 3). The monolayer had the characteristic cobblestone appearance frequently

Volume 129, Number 5 American Heart Journal SCOtt et al. 863

Fig. 3. Seeding of human microvascular endothelial cells onto uncoated stents. SEM image of tantalum wire that was placed in culture medium with human microvascular endothelial cells for 2 weeks. Note that endothelial cells formed monolayer that covered approximately 98% of stent. Monolayer has characteristic cobblestone appearance of endothelial cells.

Fig. 4. High-power image of stent wire seeded with microvascular endothelial cells.

associated with endothelial cells. When tantalum stents were seeded with the HMEC-1 endothelial cells and expanded in vitro with a PTCA balloon, the cells on the lateral aspect of the stent remained adherent. However, a significant number of the cells on both the luminal and outside surfaces were lost (Fig. 4). When these expanded stents were returned to the culture media, near-complete endothelialization of the stent was evident after 3 days (Fig. 5).

To determine whether stents seeded with endothelial cells could be frozen and their viability maintained after thawing, four balloon-expanded stents were initially placed in cell culture for 2 weeks and frozen at -70 ° C for at least 4 days. The stents were then thawed. Two stents were examined with SEM and the other two were placed directly into culture medium. After the stents were thawed, they were completely covered with endothelial cells. The ceils retained their characteristic cobblestone appearance. When the cell-seeded stents were placed in culture medium, a confluent monolayer on the culture dish was noted after incubation at 37 ° C for 7 days.

Three endothelialized balloon-expandable stents (3.5 mm expanded diameter) were placed in the left anterior descending coronary of three separate pigs. One stent had been seeded, frozen for 2 months, and thawed. Three to 4 hours after intracoronary deployment, the endothelialized stents retained a significant number of adherent cells, predominantly on their lateral aspect of the stent (Figs. 6 through 8).

Fig. 5. Balloon expansion of seeded stents (in vitro). Af- ter expansion of seeded stents with PTCA balloon in vitro, there was persistence of microvascular endothelial cells on lateral aspect of stent (Fig. 5). Cells on luminal aspect were probably sheared from stent by abrasive force of balloon. Cells on outside of stent were probably lost by handling stent when it was mounted on balloon.

There was no difference between the morphologic appearance of the stents that were maintained at 37 ° C and the stent that was frozen. To distinguish the human HMEC-1 cells from porcine coronary endothelial cells that may have attached to the stent, we first demonstrated the absence of HLA-ABC (type I) antigen that is specific to human cells on the surface of pig aortic endothelial cells and total labeling (>99 % ) of HMEC-1 cells in vitro. FACScan analysis with an HLA ABC (type I) antibody confirmed that the cells on the stents deployed in the pig coronary arteries were human.

May 1995 864 Scott et al. American Heart Journal

Fig. 6, High-power image of Fig. 5. Fig. 8. High-power image of endothelial cell-seeded stent implanted in coronary artery (high-power image of Fig. 7). Endothelialized stents retained adherent cells, predominantly on lateral aspect of stent. Platelet deposition on stent wire can also be noted.

Fig. 7. Coronary deployment of endothelial cell seeded stents. Balloon-expandable stents (3.5 mm expanded diameter) were seeded with human microvascular endothelial cells and placed on PTCA balloon catheter. Catheter was then placed in left anterior descending coronary of anesthetized pig. Four hours after intracoronary deployment, animal was killed and stented coronary segment was removed and imaged with SEM. Staining with antibody specific to human cells confirmed that endothelial cells on stent were human.

DISCUSSION

These results demonstrate that human endothelial cells can be seeded onto tantalum balloon-expandable stents that were not precoated with a fibronectin matrix. A number of the cells remain adherent after balloon expansion of the stent in vitro and in vivo, and complete coverage of the stent occurs after 3 days in culture. In addition, these cells retain their morphologic characteristics after freezing and intracoronary deployment of the stent.

Although other investigators have demonstrated seeding of stents with endothelial cells, 11,12 their methods were cumbersome and could not be readily adapted for rapid deployment of the stent when it is needed most (i.e., in the setting of an acute closure during or after PTCA). In addition, coating the stent with fibronectin, a thrombogenic substance, was re- quired before seeding. The present study demon- strates that immortalized human microvascular endothelial cells can be seeded onto an uncoated stent. Although Dichek et al. 12 were unable to seed bare metal stents with endothelial cells, successful seeding in our model was potentially the result of the utiliza- tion of microvascular cells rather than umbilical vein endothelium. Persistence of the cells on the lateral aspect of the stent was demonstrated after balloon expansion of the stent. These results corroborate those of Dichek et al. In addition, the present study documented near-complete regrowth of cells onto the stent after 3 days in culture medium:

An important factor for the clinical utility of endothelial cell-seeded stents will be their potential availability during an episode of acute closure during PTCA. Although subgroups of patients at risk for acute closure can be identified, 17 the predictive power of these indicators lacks adequate sensitivity or specificity to be of meaningful clinical use. Therefore it would be neither practical nor cost-effective to identify patients before their episode of acute closure and seed a stent with autologous endothelial cells in anticipation of an acute occlusion of the artery during a scheduled PTCA procedure.

In this study, we addressed these concerns by us-

Volume 129, Number 5 American Heart Journal Scof~t et al. 8 6 5

ing homologous endothelial cells. The use of a homo- geneous, well-characterized population of cells elim- inates the need to harvest the cells from each individual patient prior to the PTCA procedure. In addition, these endothelial cells do not require a fibronectin or collagen matrix for adherence to metal. The cells also maintain their morphologic characteristics after freezing and thawing. This issue is important because if this concept will ever be clinically useful, the stents can be seeded at a central facility, frozen, and shipped to cardiac catheterization laboratories. There the endothelial cell-seeded stents can be kept in a freezer until needed. At the time of an acute closure, the seeded stent can be thawed and deployed. If stent deployment can be accomplished without a significant loss of endothelial cells, the stent will not be thrombogenic and the patient will not require systemic anticoagulation. As a result, the morbid events accompanying stent thrombosis and systemic hemorrhage will be avoided. If these tech- niques are proved effective, an enormous decrease in the acute complications of PTCA and in the health care costs associated with their management could be achieved.

A potential obstacle that could curtail the use of these devices is the loss of cells during balloon expansion. Although a number of cells were lost on the luminal surface after balloon expansion, the endothelial cells on the lateral surfaces of the stent rapidly re-covered the bare metal. Because near- complete endothelialization occurs after approximately 3 days in culture, if the reendothelization process in vivo has similar kinetics, it is possible that the time necessary for systemic anticoagulation could be markedly decreased.

Although persistence of seeded endothelial cells onto balloon-expanded stents had been demonstrated in an artificial flow chamber, 13 this is the first study to prove that seeded endothelial cells can persist on the surface of a stent for up to 4 hours after intracoronary deployment. This in vivo experiment was performed solely to prove the feasibility of this concept. Long-term studies were not performed in this model because of the potentially significant species differences between the grafted cells and the host.

It should be noted that the cells used in these experiments were genetically altered by transfection of a viral genome. Although these cells have been shown to display a number of the characteristic functions of endothelial cells, it has not yet been shown that seeding a stent with these cells will inhibit thrombotic occlusion of the device. In addition, it is not known whether the homologous cells will persist on

the stent until the host cells can cover the device. The issues pertaining to the possible rejection of the cells and the ultimate fate of implanted immortalized cells will have to be addressed in detail before this project can be considered for potential clinical use. Never- theless, this study demonstrated the feasibility of the concept that endothelial cells can be seeded onto bare metal stents, stored by freezing, adhere to the stent after deployment, and persist in a coronary artery at least 4 hours.

REFERENCES

1. Detre KM, Holmes DR, Holubkov R, Cowley M J, Bourassa MG, Faxon DP, Dorros GR, Bentivoglio LG, Kent KM, Myler RK. Incidence and consequences of periprocedural occlusion. Circulation 1990;82:739-50.

2. Roubin GS, Cannon AD, Agrawal SK, Macander PJ, Dean LS, Baxley WA, Breland J. Intracoronary stenting for acute and threatened acute closure complicating percutaneous transinminal coronary angioplasty. Circulation 1992;83:916-27.

3. Hearn JA, King SBI, Douglas JSJ, Carlin SF, Lembo N J, Ghazzal ZMB. Clinical and angiographic outcomes after coronary artery stenting for acute or threatened closure following percutaneous transluminal coronary angioplasty. Initial results with a balloon-expandable, stainless steel design. Circulation 1993;88(part 1):2086-96.

4. Sigwart U, Fuel J, Mirkovitch V, Joffre F, Kappenberger L. Intravas- cular stents to prevent occlusion and restenosis after transluminal angioplasty. N Engl J Med 1987;316:701-6.

5. De Feyter P J, DeScheerder I, van den Brand M, Laarman GJ, Surypra- nata H, Serruys PW. Emergency stenting for refractory acute coronary arte13~ occlusion during coronary angioplasty. Am J Cardiol 1990;66: 1147-50.

6. Schatz RA, Bairn DS, Leon M, Ellis SG, Goldberg S, Hirshfeld JW, Cleman MW, Cabin HS, Walker C, Stagg J, Buchbinder M, Teirstein PS, Topol E, J., Savage M, Perez JA, Curry RC, Whitworth H, Sousa JE, Tio F, Almagor Y, Ponder R, Penn IM, Leonard B, Levine SL, Fish RD, Palmaz JC. Clinical experience with the Palmaz-Schatz coronary stent. Initial results of a multicenter study. Circulation 1991;83:148-61.

7. Haude M, Erbel R, Straub U, Dietz U, Meyer J. Short- and long-term results after intracoronary stenting in human coronary arteries: mono- cetre experience with the balloon-expandable Palmaz-Schatz stent, Br Heart J 1991;66:337-45.

8. Triana JF, Voorhees W, Fearnot NE, George B, Kleiman NS, Raizner AE. Intracoronary stent thrombosis: a major contributor to an adverse long-term outcome [Abstract]. Circulation 1992;86(suppl I):I-113.

9. Agrawal SK, Hearn JA, Liu MW, Cannon AD, Bilodeau L, Iyer SS, Baxley WA, Dean LS, Roubin GS. Stent thrombosis and ischemic complications following coronary artery stenting [Abstract]. Circulation 1992;86(Suppl I):t-113.

10. Palmaz JC. Intravascular stents: tissue-stent interactions and design considerations. Am J Roentgenol 1993;160:613-8.

11. Van der Giessen WJ, Serruys PW, Visser WJ, Verdouw PD, Van Schalkwijl~ WP, Jongkind JF. Endothelialization of intravascular stents. J Intervent Cardiol 1988:1:109-20.

12. Dichek DA, Neville RF, Zwiebel JA, Freeman SM, Leon MB, Anderson WF. Seeding of intravascular stents with genetically engineered endothelial cells. Circulation 1989;80:1347-53.

13. Flugelman MY, Virmani R, Leon MB, Bowman RL, Dichek DA. Genetically engineered endothelial cells remain adherent and viable after stent deployment and exposure to flow in vitro. Circ Res 1992;70:348- 54.

14. Budd JS, Allen KE, Bell PR, James RF. The effect of varying fibronectin concentration on the attachment of endothelial cells to polytet- rafluoroethylene vascular grafts. J Vasc Surg 1990;12;126-30.

15. Greisler HP, Klosak JJ, Steinam S J, Lam TM, Burgess WH, Kim DU. Effects of class I heparin binding growth factor and fibronectin on platelet adhesion and aggregation. J Vasc Surg 1990;11:665-74.

May 1995 Scott et al. American Heart Journal

16. Kempczinsski RF, Ramalanjoana GR, Douville C, Silberstein EB. Thrombogenicity of a fibronectin-coated, experimental polytetrafluo- roethylene graft. Surgery 1987;101:439-44.

17. Ellis SG, Roubin GS, King SBI, Douglas JSJ, Weintraub WS, Thomas RG, Cox WR. Angiographic and clinical predictors of acute closure after native vessel coronary angiop!asty. Circulation 1988;77:372-9.

18, Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, Lawley TJ. HMEC-I: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol 1992;99:683-90.

Comparison of the thrombogenicity of stainless steel and tantalum coronary stents

This study was designed to compare the thrombogenicity of stainless steel and tantalum coronary stents of the same design. Stainless steel and tantalum coronary stents are being evaluated for their utility in treating acute closure and restenosis. A major disadvantage of stainless steel stents is radiolucency. To determine whether radioopaque tantalum stents may be safely substituted for stainless steel stents, we compared the relative thrombogenicity of these materials in stents of identical design. Total platelet and fibrin deposition on the stents were determined from measurements of indium 111-labeled platelet and iodine 125-labeled fibrinogen accumulation after deployment into exteriorized chronic arteriovenous shunts in seven untreated baboons. In another series of experiments, 1111n-plateiet deposition was compared 2 hours after stent implantation in coronary arteries of pigs. In baboons, platelet tbrombus formation on stainless steel and tantalum stents was equivalent and plateaued at approximately 2.5 × 109 platelets after 1 hour (p > 0,05). Fibrin deposition averaged approximately 1 mg/stent and did not differ between the stainless steel and tantalum stents (p > 0.05), in the porcine coronary model there was no significant difference in 111In-labeled platelet deposition between the stainless steel and tantalum stents (p > 0.05). This result was confirmed by scanning electron microscopic analysis of the coronary stents. Based on these two models, w e conclude that there is no significant difference in the thrombogenicity of stainless steel and tantalum wire coil stents. (AM HEART J 1995;129:866-72.)

Neal A. Scott, MD, PhD, a Keith A. Robinson, PhD, a Gilberto L. Nunes, MD, a

Clifford N. Thomas, MBBS, a Kevin Viel, BS, a Spencer B. King III, MD, a Laurence A. Harker, MD, b Steven M. Rowland, PhD, e Ike Juman, PhD, e

Gustavo D. Cipolla, DVM, a and Stephen R. Hanson, PhD b Atlanta, Ga., and Miami, Fla.

Intracoronary stenting has proved to be an effective therapy for treating dissections and acute occlusions

From the aAndreas Gruentzig Cardiovascular Center, Emory University Hospital, and the bDivision of Hematology and Oncology, Emory University School of Medicine, Atlanta, and the CCordis Corporation, Miami.

Dr. Scott is a fellow of the Robert Wood Johnson Foundation for Minority Faculty Development. This work was supported by research grants HL41619 and HL31469 from the National Institutes of Health and part by National Institutes of Health grant RR-00165, awarded to the Yerkes Regional Pri- mate Center, which is fully accredited by the American Association for Ac- creditation of Laboratory Animal Care.

Received for publication Nov. 17, 1993; accepted Aug. 15, 1994.

Reprint requests: Neal A. Scott, MD, PhD, Andreas Gruentzig Cardiovas- cular Center, Emory University Hospital, Suite F-606, 1364 Clifton Rd., Atlanta, GA 30322.

Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$3.00 + 0 4/1/61641

that occur during percutaneous transluminal coronary angioplasty. 1-3 In selected cases, coronary stenting may also decrease the incidence of restenosis. 4, 5 However, thrombotic occlusion of stented vessels has significantly limited the use of this device. Thus, despite the concomitant use of heparin, warfarin, aspirin, and dipyridamole, the incidence of thrombotic occlusion is 2 % to 25 % .1, 6-10 Not surprisingly, hemorrhagic complications related to the anticoagulation regimen also are frequently seen. 2, 7 Issues relating to stent thrombogenicity are therefore of considerable interest.

Most stents currently under investigation are com- posed of stainless steel. 11-14 However, platelets and fibrinogen adhere to stainless steel on contact with

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seeding of intracoronary stents with immortalized human microvascular endothelial cells

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