Lasers in Surgery and Medicine 12:569-575 (1992)

Initial Results of Endothelial Cell Seeding Following Argon Laser Carotid Endarterectomy Alan P. Sawchuk, MD, Malcolm B. Herring, MD,and Michael C. Dalsing, MD Department of Surgery, Division of Vascular Surgery, Indiana University Medical Center, Indianapolis, Indiana 46202

This study evaluates the initial results of endothelial cell (EC) seeding following argon laser carotid endarterectomy. Venous endothelial cells were harvested from 12 dogs and cultured. A laser endarterectomy was performed on both carotids of each dog. One side was seeded with endothelial cells. Six dogs had both carotids harvested 1 hour after restoring blood flow. The others were harvested in 24 hours. The percentage of lumen covered with EC was evaluated by scanning electron microscopy. At 1 hour, the seeded arteries demonstrated 35 -t 3 percent EC coverage, whereas the unseeded arteries had no EC coverage (P = 0.0002). At 24 hours, the seeded arteries had 58 rt 15 percent EC coverage, whereas the unseeded arteries had no coverage (P = 0.01). Significant gross thrombus developed only in unseeded arteries (P = 0.0471, two of which were occluded at 24 hours. EC seeding is beneficial following argon laser carotid endarterectomy resulting in improved patency and less surface thrombogenicty. o 1992 Wiley-Liss, Inc. Key words: argon laser, carotid endarterectomy, endothelial cell seeding

INTRODUCTION

Argon laser carotid endarterectomy is currently being evaluated clinically because it can securely weld smooth muscle cells and intimal flaps and may prevent cerebral embolization [ll. Arteries exposed to laser energy may have an increased rate of acute thrombosis and later development of neointimal hyperplasia. Endothelial cell (EC) seeding may minimize these adverse effects because endothelial cells secrete substances with anticoagulant and antiproliferative properties. This study evaluates the early effects of endothelial cell seeding on canine carotid arteries following argon laser carotid endarterectomy in order to determine if endothelial cell seeding is effective (i.e., the cells remain) and if it improves arterial patency.

on a ventilator. The external jugular veins were harvested by a “no touch” technique through longitudinal incisions. Each of the vein segments were inverted on a metal rod and placed in a tube containing phosphate buffered saline (PSB). The PBS was then drained and a solution of 0.1 percent Worthington type I1 collagenase in PBS was pipetted into the tube such that it covered the entire vein. The collagenase was allowed to digest endothelial cell attachment sites for 20 minutes. The metal rod containing the vein was removed from the tube. A 20-ml syringe filled with PBS was attached to a 20-gauge needle and was used

Accepted for publication August 13, 1992.

MATERIALS AND METHODS

Address reprint requests to Alan P. Sawchuk, Assistant Professor, Department of Surgery, Section of General Vascular Surgery, Indiana University Medical Center, 1001 West 10th Street, Indianapolis, IN 46202.

Twelve mongrel dogs (20-30 kg) were anesthetized with pentabarbital(25 mg/kg) and placed

This work was presented at the American Society for Laser Medicine and Surgery Twelfth Annual Meeting, Orlando, FL, May 17-19, 1992.

0 1992 Wiley-Liss, Inc.

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t o gently wash the endothelial cells (EC) from the vein into a 50-ml centrifuge tube filled with 5 ml of Supreme Medium (SM), which contains Fetal Bovine Serum (FBS), which suppresses the action of the collagenase. This tube was then centrifuged for 10 minutes on a clinical centrifuge (IEC) with a 17-cm rotor at 220 g. The supernatant was decanted from each tube leaving a pellet of cells in the bottom. The cells were resuspended in 10 cc of SM and centrifuged for 10 minutes on a clinical centrifuge with a 17-cm rotor at 90 g. The cells were resuspended in 2.5 cc of SM and plated on a gel-coated T-35 culture dish. The endothelial cells were cultured at 37°C in a 5 percent CO,, 100 percent humidity environment, and were fed twice weekly. When confluence was attained as demonstrated by inverted phase contrast microscopic evaluation (in approximately 1 week), the cells were harvested with 0.75 ml of 0.17 percent trypsin after two washes with 2 ml of PBS. The cells were then resuspended in 6 cc of SM and plated on T-60 culture dishes after salvaging two 0.5-ml aliquots for Factor VIII-related antigen verification. Similarly, upon reaching confluence in the T-60 culture dishes, the cells were harvested with 1ml of 0.17 percent trypsin and resuspended in 12 ml of SM. They were then inoculated on T-75 flasks and cultured again until confluent. After EC reached confluence on the T-75 flasks (-14 days after their initial harvest), they were harvested with 2.0 ml of 0.17 percent trypsin. Following a final wash, the cells were resuspended in SM t o a concentration of lo6 cells/ml. This concentration is known to result in high density seeding on graft material [2]. At the time of EC harvest, both carotid arteries of the dog from whom the cells were cultured were exposed through a midline neck incision. Heparin, 100 units per kg, was administered intravenously and allowed to circulate for 5 minutes. The carotid arteries were then clamped proximally and distally and a 2-cm-long arteriotomy was made in each of the arteries; 1-cm-long argon laser endarterectomies were performed bilaterally using a 300-pm quartz fiber t o deliver laser power set at 1.0 watt. The intima was retracted and the laser beam was directed at the cleavage plane just beneath the internal elastic lamina. The endarterectomy site was then held open using an atraumatic plexiglass holder. One artery in each dog was randomly chosen for endothelial cell seeding. This artery was covered with the EC suspension for 1 hour while the other ar-

tery was covered with SM. EC suspension or SM was added as needed t o keep the endarterectomy surfaces covered. The arteriotomies were then closed with running 6-0 prolene sutures and blood flow was restored. Twelve carotid arteries in six of the dogs were harvested 1 hour after restoration of blood flow. They were opened longitudinally, examined for any gross thrombus formation, and fixed in 2.5 percent glutaraldehyde (diluted in sodium cacodylate buffer) for 24 hours. A longitudinal strip was then prepared for scanning electron microscopy (SEM) by dehydration in ethanol, criticalpoint drying, and gold-palladium coating. Electron micrographs were assessed by planimetry. In six of the dogs, the neck incisions were closed in multiple layers. These arteries were then harvested 24 hours after the restoration of blood flow. They were examined for thrombosis and prepared for SEM as described above. These electron micrographs were also assessed by planimetry . The percentage of the lumen covered with EC was compared between the seeded and unseeded arteries using the T-test to separately evaluate the 1-hour and 24-hour perfusion groups. The formation of gross intraluminal thrombus formation or arterial thrombosis was compared between the seeded and unseeded arteries using a one-tailed Fisher’s exact test. RESULTS

The seeded arteries harvested 1 hour after the reestablishment of blood flow had 35 ? 3 percent of their intraluminal surface covered with EC, whereas the unseeded arteries had no EC coverage (P = 0.0002) (Figs. 1, 2). The seeded arteries harvested 24 hours after the reestablishment of blood flow had 58 +- 15 percent of their intraluminal surface covered with EC, whereas the unseeded arteries had no EC coverage (P = 0.01) (Figs. 3,4). The endothelial cells were found to be relatively uniformly distributed on the luminal surface. The visibly exposed smooth muscle cells appeared to be intact with some surface irregularity. In four of the 12 dogs, unseeded arteries developed significant gross intraluminal thrombus, whereas none of the contralateral, seeded arteries had significant thrombus formation (P = 0.047). Two of the unseeded arteries were occluded at 24 hours, and two of the unseeded arteries were nearly occluded with fresh thrombus at 1 hour. The seeded arteries were all widely

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Fig. 1. SEM micrograph showing an unseeded arterial luminal surface 1 hour following argon laser carotid endarterectomy. No endothelial cells are visible and there are many adherent red blood cells and platelets present (magnification x 600).

patent with minimal visible thrombus. Qualita- the arterial surface following conventional endartively the seeded arteries had less adherent red terectomy and may be more prone to develop blood cells and platelets at 24 hours compared to neointimal hyperplasia [1,51. Pollock et al. [61 studied the surface thromthe unseeded arteries (Figs. 3, 4). bogenicity of normal rabbit aorta, atherosclerotic rabbit aorta, and atherosclerotic rabbit aorta DISCUSSION treated with laser angioplasty, laser endarterecLaser endarterectomy is a new technique tomy, or conventional endarterectomy. The argon that holds great promise. It is currently being in- laser was used t o perform the laser surgery. The vestigated in the clinical setting [31. It can seal shorter clotting times of arterial surfaces followintimal flaps and medial smooth muscle cells, ing both conventional and laser endarterectomy which can dissect or embolize following conven- demonstrated that these surfaces were signifitional endarterectomy [ll. Laser endarterectomy cantly more thrombogenic than normal arteries results in a smoother surface than conventional or atherosclerotic arteries with an intact intima. endarterectomy [l]. Since this technique does not A study by McViker et al. [71 demonstrated that leave a distal intimal flap, it can be used to repair the level of prostacycin metabolites was lower in stenoses, which cannot be reached by conven- arteries following a carbon dioxide laser endartional endarterectomy such as those in the distal terectomy than in control arteries or in arteries internal carotid artery 141. However, clinical and following conventional surgical endarterectomy. Cikrit et al. [11 experimentally used g-lasing experimental studies with laser endarterectomy have shown that this technique may result in an to repair intimal flaps in the distal canine carotid arterial surface that is more thrombogenic than artery following intimectomy. G-lasing refers to

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Fig. 2. SEM micrograph showing an endothelial cell seeded arterial luminal surface 1hour following argon laser carotid endarterectomy. There is extensive endothelial cell coverage present. The arrow points to an endothelial cell (magnification x 600).

mize these adverse developments. Autogenous artery denuded of its endothelial monolayer by laser endarterectomy might be the ideal substrate for endothelial cell seeding. If endothelial cell seeding restored the arterial surface to its prediseased state, laser endarterectomy might have all of its advantages without its potential disadvantages. Data from animal studies has convincingly shown that endothelial cell seeding improves vascular graft patency [10,111. Seeding prostheses with autologous endothelial cells promotes endothelialization of the luminal surface [10,121. Endothelial cell seeding can improve blood flow and graft patency in experimental arterial grafts [13,141. A number of groups have shown that immediate endothelial cell seeding combined PI. Following the argon laser carotid endar- with antiplatelet therapy enhances the cumulaterectomy, the arterial luminal surface may have tive patency rates of small caliber prosthetic or develop increased surface thrombogenicity, de- grafts in the canine model at 4-28 weeks [13,151. creased prostacyclin synthesis, neointimal and Seeded endothelial cells can produce a nummyointinial hyperplasia, and increased lipid ac- ber of products with anticoagulant and antiprolifcumulation. Endothelial cell seeding may mini- erative properties that may theoretically improve the process of glazing the roughened intimectomy surface with a continuous laser beam to smooth out any roughened areas or loose tissue. Vessel patencies were 82.4 percent in arteries treated with g-lasing compared to 88.2 percent in arteries undergoing intimectomy only. Arteries treated by laser or conventional surgical endarterectomy which do not thrombose, undergo reendothelialization within a few weeks in several species [81. However, the exposure of smooth muscle in the media to platelets, plateletderived growth factor, and leukocytes may lead t o neointimal hyperplasia [ l ] as well as to recurrent arteriosclerosis with an increased accumulation of lipoprotein and cholesterol in the arterial wall

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Fig. 3. SEM micrograph showing a n unseeded arterial luminal surface 24 hours following argon laser carotid endarterectomy. No endothelial cells are visible and there are many adherent red blood cells and platelets present (magnification x 500).

patency. Anticoagulant cell products include heparinlike glycosaminoglycans and certain growth factors [161. Lining grafts with endothelial cells has many beneficial effects. The accumulation of platelets on the luminal surface of the graft is reduced compared to unseeded graft surfaces when measured several weeks after implantation [171. Seeded endothelial cells can improve platelet survival in the blood, increase the clot free surface area of the graft, lower the whole blood clotting time on the surface of the prosthesis, and return platelet serotonin levels toward normal [MI. Endothelial cell seeding can diminish thromboxane and increase prostacyclin production at the graft surface [151. Bush et al. [191studied thrombosis occurring after conventional surgical endarterectomy in a canine model with and without endothelial seeding. At 6 weeks, patency of the endarterectomized carotid arteries was 88 percent in the endothelial cell seeded group and only 35 percent in the con-

trol group (P < 0.01). These authors did not investigate laser endarterectomy. The present study demonstrates that endothelial cells can be seeded in adequate numbers on the carotid artery luminal surface following an argon laser carotid endarterectomy in a canine model. The endothelial cell coverage is retained on the luminal surface and increases due to cell flattening and/or dividing. It significantly decreases the acute thrombosis rate following argon laser carotid endarterectomy, and qualitatively there are fewer adherent platelets and red blood cells on the luminal surface at 24 hours. In the absence of a conventional surgical endarterectomy control, no conclusions can be reached regarding the relative thrombogenicity of argon laser carotid endarterectomy vs. conventional surgical endarterectomy. This is the first study demonstrating that endothelial cells can be seeded and retained on the laser carotid endarterectomy surface. The endo-

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Fig. 4. SEM micrograph showing a n endothelial cell seeded arterial luminal surface 24 hours following argon laser carotid endarterectomy. The luminal surface is nearly covered with endothelial cells and there are few adherent red blood cells and platelets present. The arrow points to an endothelial cell (magnification x 500).

thelial cells decrease acute arterial thrombosis following argon laser carotid endarterectomy in a canine model. Additional studies are needed t o determine whether the endothelial cells function normally following seeding. Further studies are needed t o evaluate the effects of endothelial cell seeding on the subsequent development of neointimal hyperplasia and arteriosclerosis following argon laser carotid endarterectomy. If further studies in animal models continue to show the beneficial effects of endothelial cell seeding following argon laser carotid endarterectomy, controlled clinical trials may be warranted. ACKNOWLEDGMENTS

This study was supported by an American Heart Association Grant, Indiana Affiliate.

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Initial results of endothelial cell seeding following argon laser carotid endarterectomy.

This study evaluates the initial results of endothelial cell (EC) seeding following argon laser carotid endarterectomy. Venous endothelial cells were ...
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