Influence of endothelial cell seeding on platelet deposition and patency in small-diameter Dacron arterial grafts

Serial platelet deposition, surface topography, and patency were evaluated in control (N = 28) and endothelial cell-seeded (N = 28) small-diameter (4 mm inner diameter) USCI Dacron grafts implanted in the carotid and femoral arteries of dogs. All dogs received aspirin (325 mg) daily for 2 weeks starting 24 hours prior to graft implantation. Endothelial cell seeding was performed by mixing suspensions of autologous endothelial cells that had been enzymatically harvested from segments of external jugular vein with blood that was used to preclot the prostheses. The platelet deposition on each graft was quantitated by means of indium 111-labeled platelets and technetium 99m-labeled red cells in a dual-isotope platelet-imaging technique. Platelet deposition on seeded grafts 24 hours after implantation was significantly higher than on the controls (p less than 0.05). Two weeks after implantation platelet deposition on seeded prostheses had decreased to a level significantly lower than that on the controls and continued to decline on serial studies up to 7 months. In contrast to seeded grafts, platelet accumulation on control grafts dramatically increased after the withdrawal of aspirin therapy and was associated with a sharp rise in control graft thromboses. Gross and scanning electron microscopic evaluation of endothelial cell-seeded grafts after 1 month indicated complete neointimal coverage, whereas none of the control grafts explanted at 1 month or later exhibited a continuous neointimal lining. Cumulative 7-month patency for seeded prostheses was significantly higher than for the controls (96% and 29%, respectively; p less than 0.001). We conclude that endothelial cell seeding in combination with short-term aspirin therapy is a simple, reliable diameter Dacron prostheses. Abrupt withdrawal of aspirin therapy may be contraindicated in nonseeded control grafts because it results in increased platelet deposition and thrombosis.     

Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts

A possible approach to the low seeding efficiency of endothelial cells into prosthetic grafts is to increase the number of cells to be seeded in cell culture and improve seeding efficiency by graft precoating with fibronectin. The effect of cell culture on cell adhesion is unknown, however, and fibronectin also binds fibrin, which may increase the thrombogenicity of the graft luminal surface. To investigate these questions, freshly harvested canine jugular vein endothelial cells from six animals and similar cells harvested from six primary and eight secondary cell cultures were labeled with 111Indium and seeded into 5 cm, 4 mm PTFE grafts coated with fibronectin, using similar uncoated PTFE grafts as controls. Platelet accumulation and distribution on six similar coated and uncoated grafts placed in canine carotid, external jugular arterial venous shunts for 2 hr were also determined using autogenous 111Indium-labeled platelets. Significant differences between group means were determined using the paired Student's t test. Results reveal that seeding efficiency is significantly better in all groups of coated grafts compared to uncoated grafts (P less than 0.01). Cells derived from cell culture also had significantly higher seeding efficiencies than freshly harvested cells when seeded into coated grafts (P less than 0.05) and tended to have higher seeding efficiencies than harvested cells when seeded into uncoated grafts (P = 0.53). Fibronectin coating increased mean platelet accumulation on the entire graft luminal surface, but not to a statistically significant degree (P greater than 0.1). Whether this increased seeding efficiency will improve graft endothelialization remains to be investigated.     

The association of in vitro arachidonic acid responsiveness and plasma thromboxane levels with early platelet deposition on the luminal surface of small-diameter grafts

The response of canine platelets to arachidonic acid (AA) stimulation was studied as a predictor of thrombotic potential. Fifty mongrel dogs underwent in vitro platelet aggregation studies with adenosine diphosphate (ADP), collagen, and AA used as inducing agents. Thirty-two dogs were selected on the basis of their response to AA stimulation. Platelet aggregation in response to AA stimulation occurred in 16 (responders) and 16 showed no aggregatory response (nonresponders). The animals were divided into four groups. Group I received no antiplatelet agents (control); group II received U-63,557A, a specific thromboxane synthetase inhibitor (TSI); group III received aspirin; and group IV received aspirin and TSI. Polytetrafluoroethylene grafts were implanted in the carotid and femoral arteries of the dogs in all four groups. Plasma thromboxane (TxB₂) levels were drawn before drug treatment and 4 weeks after surgery. Platelet deposition on the luminal surface of the implanted grafts was studied in vivo with a technique that uses both ¹¹¹In-labeled platelets and ^99mTc-labeled red blood cells and was expressed as percentage of indium excess (%IE). Group I (control) dogs whose platelets aggregated in response to AA stimulation had significantly higher 24-hour platelet deposition (%IE) on the luminal surface of implanted grafts (p < 0.02), lower 4-week graft patency (p < 0.002), and higher plasma TxB₂ levels (p < 0.01) than those dogs whose platelets did not aggregate. In contrast to the results of AA stimulation, neither ADP nor collagen responsiveness was discriminatory for the thrombotic potential of canine arteries as measured by 24-hour platelet deposition (%IE), 4-week graft patency, or plasma TxB₂ levels. Pharmacologic platelet inhibition by use of aspirin (groups III and IV) effectively transformed AA responders into AA nonresponders as manifested by lowering the 24-hour platelet deposition (%IE) and increasing the 4-week patency to a value similar to AA nonresponders. This study confirms that the in vitro platelet response to AA stimulation is the best method for determining the endogenous thrombotic potential of canine arteries and correlates well with plasma TxB₂ levels and 24-hour in vivo platelet deposition studies. (J VASC SURG 1988;7:554-61.)     

Kinetics of endothelial cell seeding

Endothelial cell seeding improves patency of small-diameter Dacron grafts and facilitates the development of a complete endothelial flow surface. However, the ideal number of cells relative to the length of graft to be seeded has not been determined. With a canine model previously shown to result in a well-endothelialized graft within 4 to 6 weeks, this study measured the quantity of autogenous endothelial cells labeled with indium 111-oxine that initially adhered to 10 cm long, experimental, porous 4 mm I.D. polytetrafluoroethylene grafts and then calculated their subsequent disappearance following implantation as carotid interposition grafts. Graft radioactivity was monitored with a gamma camera and compared with that of control vials of indium 111 implanted in the same animals. Counts were measured immediately at implantation and for up to 72 hours following restoration of flow. Data were analyzed by linear regression. The mean number of harvested endothelial cells was 6.2 X 10(5). A mean of 19.8% of the harvested cells were adherent to the grafts initially after seeding. In the first 30 minutes following restoration of flow, there was a rapid loss of these cells to a mean value, which was 70.2% of those initially present. From 30 minutes to 24 hours, cell losses continued at a constant rate of 3.7%/hr (r = -0.922, p less than 0.001). Beyond 24 hours, further loss was insignificant. Consequently, approximately 2.72 X 10(4) cells, or only 4.4% of all cells originally harvested, appear adequate to seed 12.5 cm2 of graft.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved patency of collagen-impregnated grafts after in vitro autogenous endothelial cell seeding

Currently available prosthetic vascular grafts remain sufficiently thrombogenic to preclude their use as small-caliber arterial substitutes. However, thrombogenicity may be significantly reduced by the presence of an endothelial monolayer on the luminal surface. The present study was undertaken to test the efficacy of lining a small-caliber prosthesis with autogenous endothelial cells in vitro so that the graft may subsequently be implanted with an established confluent endothelial lining. For this purpose, cells were obtained from canine external jugular vein, harvested enzymatically, and passaged in culture. Dacron grafts (4 X 150 mm) were then seeded in vitro and maintained for 48 to 72 hours before implantation in the femoral position of the same animal. Seeded grafts were implanted contralateral to unseeded control grafts and explanted after 1 month. Seeded grafts demonstrated an 86% patency rate at explanation in contrast to the significantly lower 14% patency rate of the unseeded control grafts. This study justifies further investigation directed toward the feasibility of endothelializing intravascular prostheses in vitro before implantation.     

In vivo evaluation of the effects of a new ice-free cryopreservation process on autologous vascular grafts.

Conventionally cryopreserved vascular grafts have performed poorly as arterial grafts. One possible mechanism that causes the poor function is the extracellular ice damage in tissue. We used a novel new ice-free cryopreservation (namely, vitrification) method for prevention of ice formation in cryopreserved venous grafts. This study was designed to evaluate the in vivo effects of the vitrification process on autologous vascular grafts using a short-term transplantation model and to examine the morphology and patency of vitrified grafts in correlation with control grafts. New Zealand White rabbits underwent a right common carotid interposition bypass graft. Fresh and vitrified reversed ipsilateral external jugular veins were used as autologous grafts. Animals were sacrificed at either 2 or 4 weeks after implantation, and fresh and vitrified vein grafts were harvested for histology studies. The results, comparing the patency of fresh and vitrified grafts, demonstrated similar short-term patency rates (approximately 90%). There were no signs of media disruption, aneurysm, or graft stenosis in vitrified vein grafts. Vitrification had not altered the pathophysiological cascade of events that occur when a vein graft is inserted into the arterial system. The vitrification process had no adverse effects locally or systemically in vivo. In addition, vitrification has preserved endothelial cell and smooth muscle cell integrity posttransplantation. In conclusion, this study, using an autologous animal model, clearly demonstrated a significant benefit of vitrification for preservation of graft function, and vitrification may be an acceptable approach for preservation of blood vessels or engineered tissue constructs.     

Platelet deposition on vascular grafts. The accuracy of in vivo quantitation and the significance of in vivo platelet reactivity.

An in vivo platelet imaging system utilizing indium-111-labeled platelets and technetium-99m-labeled red cells was used to serially study and compare platelet deposition on autologous external jugular vein grafts, autologous arterial grafts, polytetrafluoroethylene (Gore-tex) and two Dacron (Meadox and USCI) small diameter (4mm) vascular grafts implanted end-to-end in canine carotid and femoral arteries. This method of quantitating platelet deposition was validated by correlating deposition measured in vivo with deposition measured directly on explanted grafts (r = 0.94, p less than 0.01). Platelet accumulation on all grafts was greatest immediately after implantation and declined over time. None of the artery or vein grafts thrombosed, and they had the lowest level of platelet deposition at all times. Platelet deposition on Gore-tex grafts was significantly less than on USCI Dacron grafts from 24 hours to 1 month after implantation. There was no statistical difference in 1-month patency among the synthetic graft groups. Synthetic grafts that thrombosed during the first month accumulated significantly more platelets immediately after operation than did those grafts that remained patent. Patent Dacron grafts with low levels of platelet deposition had less thrombotic debris at explantation on the luminal surface than did those grafts with high levels of platelet deposition. Differences in initial platelet deposition appeared to be more a function of platelet reactivity within each dog rather than the material used in graft construction.     

Rapid,arteriovenous graft failure due to intimal hyperplasia: a porcine,bilateral, carotid arteriovenous graft model

BACKGROUND: The loss of patency constitutes the major complication of arteriovenous (AV) polytetrafluoroethylene hemodialysis grafts. In most cases, this graft failure is due to intimal hyperplasia at the venous outflow tract, including proliferation of vascular, smooth muscle cells and fibroblasts with deposition of extracellular matrix proteins. Thus far, procedures developed for improving patency have proven unsuccessful, which can be partly explained by the lack of relevant animal models. For this purpose, we developed a porcine model for AV graft failure that will allow the assessment of promising therapeutic strategies in the near future. MATERIALS AND METHODS: In 14 pigs, AV grafts were created bilaterally between the carotid artery and the jugular vein using expanded polytetrafluoroethylene. Two, 4 or 8 weeks after AV shunting, the grafts and adjacent vessels were excised and underwent histologic analysis. RESULTS: From 2 weeks onwards, a thick neo-intima developed at the venous anastomosis, predominantly consisting of alpha-actin-positive vascular smooth muscle cells (VSMC). Intimal area increased over time, coinciding with a decreased graft flow. Grafts remained patent for at least 4 weeks. At 8 weeks, patency rates declined to less than 50% due to thrombus formation superimposed on progressive neo-intima formation. CONCLUSIONS: Implantation of an AV graft between the carotid artery and jugular vein in pigs causes a rapid neo-intimal response, accompanied by a loss of patency of 50% at 8 weeks after surgery. This model offers a suitable tool to study local interventions aimed at the improvement of AV graft patency rates.     

Long-term effects of extraluminal dilatation with a rigid circle on arteriovenous anastomotic line in venous grafts transposed into arterial defects

Postanastomotic narrowing resulting from subintimal hyperplasia is a well-known phenomenon. In the current study the authors compared a metallic circle and conventional suture technique in anastomoses performed in two ends of external jugular vein grafts interposed in carotid arteries of rabbits. They recorded the patency rates, fluid flow rates, and histological effects of the circle on the anastomotic line and compared them with conventional suture anastomoses. In 16 rabbits (experimental group) a standard suture was used in both ends of the jugular vein graft transposed to the carotid arteries on one side. On the other side, circle anastomoses were performed on both ends of the vein graft. In an additional 8 rabbits (control group), the anterior jugular veins and carotid arteries were dissected on both sides and left. During postoperative week 12, in 8 rabbits of the experimental group, the flow rates of carotid arteries were measured in vitro, and intraluminal silicone casts were prepared. In the remaining 8 experimental rabbits, carotid angiographies were performed and anastomotic segments were harvested for histological examination. Flow rates were also measured in the control group, and artery and vein segments were harvested. The patency rates of the vein grafts with metallic circle anastomoses were 100%, whereas conventional suture patency was 75% at week 12. Flow rates were significantly higher in the metallic circle-anastomosed vein grafts (74 ml per minute vs. 123 ml per minute, mean values; p < 0.05). Histological examination revealed reduced intimal thickness in the metallic circle anastomoses compared with conventional suture anastomoses. Dilatation of the arteriovenous end-to-end anastomotic line by a rigid circle prevents anastomotic narrowing in the long term.     

Increased platelet deposition on polytetrafluoroethylene grafts after balloon catheter thrombectomy

Prosthetic graft rethrombosis after thrombectomy may be potentiated by increased thrombogenicity of the restored flow surface. This experiment compared platelet deposition on polytetrafluoroethylene (PTFE) grafts after balloon catheter thrombectomy with deposition on new, nonthrombosed grafts. Three models of graft thrombosis were studied in eight dogs with 4 mm diameter by 7 cm PTFE graft segments: (1) in vitro model: grafts filled with blood, stored in 37 degrees C saline solution; (2) in vivo model: blood-filled grafts stored in subcutaneous tissue; and (3) in situ model: one end of grafts anastomosed to femoral or carotid artery as a blind tube. Duration of thrombosis (1, 2, and 3 weeks) was studied by initiating one graft of each type per week in each dog. After 3 weeks, nine thrombosed grafts per dog were harvested and graft thrombectomy was performed with a 3F balloon catheter. An ex vivo flow-controlled perfusion circuit was then created in each dog and platelet deposition was measured during the initial 20 minutes of graft perfusion after 111In platelet labeling. Thrombectomized grafts were compared with new, control grafts not previously exposed to blood, as well as with grafts exposed for 1 hour to blood or plasma. Compared with control grafts, platelet deposition was significantly increased on in vivo (3.7 times control; p less than 0.01), in situ (2.6 times control; p less than 0.05), and in vitro thrombosed grafts (2.0 times control; p less than 0.05). Age of thrombus was not a significant source of variation. Blood or plasma exposure alone did not significantly increase platelet deposition. These data suggest that antiplatelet therapy may be important at the time of PTFE graft thrombectomy.     

Cryopreservation affects endothelial and smooth muscle function of canine autogenous saphenous vein grafts

Cryopreserved veins used as arterial grafts may be affected by both rejection and the cryopreservation process. Experiments were designed to study changes in endothelial and smooth muscle function after cryopreservation but independent of rejection. One saphenous vein from each of eight dogs was cryopreserved for subsequent use as autografts. After 3 weeks one cryopreserved and one freshly harvested autogenous saphenous vein were implanted as bilateral femoral arterial interposition grafts. Platelet deposition was studied in vivo with indium 111-labeled platelets. At 4 weeks the autografts were removed, and the functional characteristics of the grafts were studied in organ chambers; and the ability of nerve terminals to uptake transmitter was studied with 3H-norepinephrine. Neither patency rates, blood flows, nor platelet deposition were significantly different between freshly harvested and cryopreserved grafts. Uptake of 3H-norepinephrine was significantly reduced in both grafts as compared to unoperated veins. The smooth muscle of the cryopreserved and fresh grafts contracted comparably to alpha-adrenergic agonists and endothelin. In cryopreserved grafts, the maximal tensions that developed to KCl, prostaglandin F2 alpha, and endothelin were greater when the endothelium was present compared to that developed by the smooth muscle alone. Calcium ionophore A23187 caused relaxations only in rings with endothelium; these were not significantly different between graft types. However, relaxations of the smooth muscle to nitric oxide were decreased in the cryopreserved grafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Celiprolol reduces the intimal thickening of autogenous vein grafts via an enhancement of nitric oxide function through an inhibition of superoxide production

BACKGROUND: Beta-adrenoceptor antagonist celiprolol has been widely used as an effective antihypertensive agent. Some studies reported that celiprolol enhances nitric oxide production. The purpose of the present study is to examine the effects of celiprolol on vein graft intimal hyperplasia and endothelium-dependent nitric oxide (NO)-mediated relaxation. METHODS: Japanese white rabbits were randomized to a control group that was fed regular rabbit chow or to a celiprolol group that was fed regular rabbit chow supplemented with 100 mg/body celiprolol sodium. The reversed jugular vein was implanted into the carotid artery. At 2 and 4 weeks after the operation, vein grafts in both groups were harvested, and intimal hyperplasia of the vein grafts was assessed. At 4 weeks after the operation, harvested vein grafts from both the groups were examined on the endothelium-dependent relaxation by application of Ach and were examined to detect for endothelial NO synthase (eNOS) expression and superoxide anion production. RESULTS: Celiprolol inhibited intimal hyperplasia of carotid interposition-reversed jugular vein grafts 4 weeks after implantation (Intima/media index of celiprolol group, 0.48 +/- 0.01 vs control group, 1.07 +/- 0.08, P < .05) and suppressed cell proliferation in the neointima 2 weeks after implantation. In addition, celiprolol significantly enhanced endothelium-dependent NO-mediated relaxation in the vein graft with no change in eNOS expression and a reduction in superoxide production. CONCLUSIONS: These novel findings clearly demonstrate that beta-adrenoceptor antagonist celiprolol can suppress intimal hyperplasia of the vein graft, which may be due to the enhancement of nitric oxide function through an inhibition of superoxide production. These results strongly support the clinical usefulness of celiprolol administration for preventing intimal hyperplasia of the vein graft after bypass grafting.     

Intraarterial 9-beta-methyl carbacyclin improves canine polytetrafluoroethylene graft patency

This study examined the effect of 9-beta-methyl carbacyclin, a synthetic, stable prostacyclin analog, on canine polytetrafluoroethylene (PTFE) graft patency. Twenty-five dogs had 4 mm x 7 cm PTFE grafts implanted bilaterally into the femoral arteries. A subcutaneous infusion pump was used to deliver either saline solution (control) or 9-beta-methyl carbacyclin (Ciprostine) at 100 (CARB-100) or 200 ng/kg/min (CARB-200) through a femoral artery branch just proximal to one of the femoral grafts, with the contralateral graft serving as a noninfused control. Graft-platelet deposition (with 111In-labeled platelets) was measured between the fifth and seventh days, with patency determined on the seventh day. Dogs were classified as aggregators (AGG [+]) if the preoperative epinephrine-enhanced sodium arachidonate platelet aggregation was greater than 20%. CARB-200 infusion significantly improved ipsilateral graft patency (80%) compared with noninfused grafts (50%, p less than 0.05), or grafts in control and CARB-100 dogs (43%, p less than 0.05). Anastomotic platelet deposition was decreased bilaterally in CARB-200 dogs by 45% to 59% compared with CARB-100 and control dogs (p less than 0.05). With the exception of grafts infused with CARB-200, AGG (+) dogs had significantly lower graft patency (26%) than nonaggregator AGG (-) dogs (71%, p less than 0.01). CARB-200 infusion significantly improved graft patency in AGG (+) dogs (71%), compared with control and CARB-100-infused grafts (19%, p less than 0.025). Intra-arterial 9-beta-methyl carbacyclin improved early PTFE graft patency and inhibited platelet deposition in a severe canine model, independent of baseline platelet aggregation status, which also had an important effect on graft patency.     

Hydrophilic statin suppresses vein graft intimal hyperplasia via endothelial cell-tropic Rho-kinase inhibition

BACKGROUND: Recent studies suggest that statins can protect the vasculature in a manner that is independent of their lipid-lowering activity through inhibition of the small guanosine triphosphate-binding protein, Rho, and Rho-associated kinase. Little information is available on the inhibitory effect of statins on vein graft intimal hyperplasia, the main cause of late graft failure after bypass grafting. We therefore examined the effects of a hydrophilic statin on vein graft intimal hyperplasia in vivo and Rho-kinase activity in vitro. METHODS: In the first experiment, rabbits were randomized to a control group (n = 7) that was fed regular rabbit chow or to a pravastatin group (n = 7) that was fed regular rabbit chow supplemented with 10 mg/kg pravastatin sodium. The branches of the jugular vein were ligated and an approximately 3-cm segment of the jugular vein was taken for an autologous reversed-vein graft. The carotid artery was cut and replaced with the harvested autologous jugular vein. At 2 and 4 weeks after the operation, vein grafts in both groups were harvested, and intimal hyperplasia of the vein grafts was assessed. In the second experiment, human umbilical vein endothelial cells and vascular smooth muscle cells were cultured and then treated with 1 micromol/L and 30 micromol/L pravastatin for 24 hours and harvested. Immunoblotting was performed on the resulting precipitates. Quantitative evaluation of phosphorylated myosin binding subunit and endothelial nitric oxide synthase was performed by densitometric analysis. RESULTS: We demonstrated that oral administration of the hydrophilic statin pravastatin to normocholesterolemic rabbits inhibited intimal hyperplasia of carotid interposition-reversed jugular vein grafts 4 weeks after implantation (pravastatin group, 39.5 +/- 3.5 microm vs control group, 64.0 +/- 7.1 microm; n = 7; P < .05) and suppressed cell proliferation and apoptosis in the neointima 2 weeks after implantation. In addition, we found that pravastatin inhibited Rho-kinase activity and accelerated endothelial nitric oxide synthase expression in human umbilical vein endothelial cells but did not inhibit Rho-kinase activity in vascular smooth muscle cells. CONCLUSIONS: These novel findings clearly demonstrate that a hydrophilic statin can suppress intimal hyperplasia of the vein graft in vivo and also show endothelial cell-tropic inhibition of Rho-kinase in vitro. Furthermore, these results strongly support the clinical use of hydrophilic statins to prevent intimal hyperplasia of the vein graft after bypass grafting. CLINICAL RELEVANCE: Late graft failure caused by neointimal hyperplasia limits the efficacy of vein grafting. Various treatments were examined to reduce neointimal hyperplasia, but a standard clinical treatment has not yet been established. We report here the inhibitory effect of pravastatin on the development of vein graft intimal hyperplasia. In addition, we demonstrate that pravastatin showed endothelial cell-tropic benefits through both the inhibition of Rho-kinase activity and acceleration of eNOS expression in vitro. Because the clinical benefits and safety of pravastatin have been established to a certain extent through long-term clinical usage, pravastatin may soon become standard treatment after vein bypass grafting.     

Cellular remodeling of depopulated bovine ureter used as an arteriovenous graft in the canine model

BACKGROUND: One of the greatest challenges in hemodialysis access surgery is improving the durability of prosthetic grafts caused by structural deterioration. The depopulated bovine ureter SynerGraft (SG) (CryoLife, Inc) is a tissue-engineered vascular graft processed to remove the xenograft cells while maintaining an unfixed connective tissue matrix capable of autologous cell repopulation by the recipient. STUDY DESIGN: Nineteen 6-mm diameter bovine ureter SG conduits were implanted in 12 dogs as arteriovenous grafts between the carotid artery and jugular vein (n = 11) or between the femoral artery and vein (n = 8). Performance of these biologic conduits was compared with that of 15 IMPRA (Bard) ePTFE grafts implanted in 9 dogs, including 9 arteriovenous grafts between the carotid artery and jugular vein and 6 femoral artery to femoral vein grafts. After 14 days, the grafts were accessed once weekly. Histologic and immunohistochemical analyses were performed on grafts explanted between 10 to 60 weeks. RESULTS: The 6- and 12-month primary patency rates of the bovine SG were 72.6% and 58.6%, respectively, compared with 6- and 12-month primary patency for ePTFE conduits of 57.4% and 57.4%, respectively. None of the bovine SG grafts became infected, but synthetic conduits became infected within 54 days of implantation. At 10 weeks, bovine ureter SG conduit showed fibroblast cell migration and proliferation with incorporation into the surrounding subcutaneous tissue, and elongated cells expressing the contractile protein smooth muscle actin were also observed. After 24 weeks, procollagen synthesis was demonstrated in the fully colonized graft matrix. The ePTFE grafts had no evidence of cellular ingrowth and an absence of endothelium. CONCLUSIONS: The bovine SG was appropriately remodeled to its host environment through an organized process of recellularization and neovascularization. The absence of infection, similar patency rates, and cell repopulation of the matrix warrant further investigation.     

Impact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft

Previous reports have demonstrated that endothelial cell seeding of polytetrafluoroethylene (PTFE) grafts enhances short-term patency. This experiment was undertaken to study its impact on the long-term patency of a highly porous, experimental PTFE graft and to determine whether increasing the internodal distance of the graft material resulted in increased proliferation of the subendothelium. Ten centimeter long, 4 mm internal diameter segments of an unreinforced, experimental PTFE graft were implanted into 36 mongrel dogs as carotid interpositions. In each animal, one graft was seeded with autologous endothelial cells, enzymatically derived from the external jugular veins, whereas the contralateral graft was treated in identical fashion except that endothelial cells were not added to the preclot mixture. Nineteen animals were killed at 12 weeks; six at 22 weeks; eight at 26 weeks; and three at 52 weeks. The mean follow-up period was 20.1 weeks. The overall patency rate was 58.3% (21 of 36 grafts) for seeded grafts vs. 27.8% (10 of 36 grafts) for control grafts (p less than 0.01). The thrombus-free area was planimetrically measured at 83.4% +/- 4.5% in seeded grafts vs. 55.1% +/- 9.7% in control grafts (p less than 0.05). Scanning electron microscopy confirmed the presence of a confluent cellular monolayer in seeded grafts, whereas control grafts exhibited a variable coagulum of fibrin, platelets, and endothelial cells. The thickness of the subendothelial layer varied from 56 to 95 micron with no progressive increase in thickness between 12 and 52 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of Vascular Intimal-Medial Hyperplasia in Polytetrafluoroethylene Arteriovenous Grafts via Expression of an Inhibitor of G Protein Signaling

Polytetrafluoroethylene (PTFE) arteriovenous (AV) grafts are performed routinely for vascular access. The limited life span of PTFE grafts is a major cause of morbidity. Graft failure is attributed to venous outflow tract vascular smooth muscle (VSM) hyperplasia, which is linked to heterotrimeric G protein signaling. We proposed that expression of a peptide inhibitor of G_βγ signaling (βARKct) in the venous outflow of PTFE grafts would reduce hyperplasia and prolong graft patency. Left carotid to right external jugular vein PTFE AV grafts were placed in swine. The isolated external jugular vein was treated with an adenovirus encoding βARKct, empty adenovirus, or phosphate-buffered saline for approximately 25 min. After 7 or 28 days, flow probe analysis was performed and the vein was harvested and analyzed for cross-sectional area comparison. After both 7 and 28 days, when compared to controls, treated animals demonstrated a statistically significant reduction in VSM hyperplasia with a reduction in cross-sectional intimal and medial areas of >40% (p < 0.05). Flow was maintained in treated grafts, while control groups demonstrated a >50% reduction (p < 0.05) at 7 days. Further, treated grafts demonstrated significant improvement in graft patency at 28 days (100% vs. 12% for treated and untreated grafts, respectively). The inhibition of G_βγ signaling reduces intimal-medial hyperplasia and prolongs graft patency in PTFE AV grafts. This represents a novel molecular therapeutic strategy for improving the patency of vascular access grafts.Part of this work was presented at the American Heart Association Annual Meeting, Chicago, IL, September 2002.     

The natural history of endothelial structure and function in arterialized vein grafts

When the saphenous vein is used in the in situ position for arterial bypass surgery, it is associated with more optimal preservation of the endothelial lining and with improved graft patency compared with reversed vein grafts. However, it is not clear whether preservation of endothelial integrity persists after arterialization. The goal of this study was to establish whether preservation of the endothelium before arterialization is a critical factor in the development of late functional and morphologic abnormalities of autogenous vein grafts. Paired reversed and in situ vein grafts were created in 75 mongrel dogs. Veins to be used in the reversed position were excised and stored in either heparinized whole blood at 37 degrees C or saline solution at 4 degrees C. Veins were studied before and after arterialization. The veins were arterialized by anastomosis to the carotid artery and excised at intervals of 1 day to 12 weeks for studies of the luminal production of prostacyclin and thromboxane A2 in addition to luminal morphology. Before arterialization, normothermic whole blood preserved biochemical function of the endothelium significantly better than hypothermic saline solution, but not as well as the in situ vein procedure. Soon after arterialization, all three vein grafts showed significant functional and morphologic abnormalities consistent with injury of the vein graft. Morphologic healing of the endothelial monolayer progressed slowly back to normal; however, the biochemical capacity of the vein graft never matched that of the prearterialized vein, nor that of normal host arteries. Regardless of surgical technique, all vein grafts exhibited a period of abnormal structure and function, which exposed them to the risk of thrombogenesis. This period of potential leukocyte or platelet interaction with the vein wall could lead to release phenomena as well as proliferative changes in the vessel wall.     

Reduction of vein graft intimal hyperplasia and preservation of endothelium-dependent relaxation by topical vascular endothelial growth factor

Purpose: Recent evidence suggests that vascular endothelial growth factor (VEGF), in addition to stimulating angiogenesis, also serves a repair/maintenance or survival function, modulating various aspects of endothelial cell function. This study was designed to examine the effect of VEGF pretreatment in a model of vein graft intimal hyperplasia. Methods: Reversed jugular vein–to–common carotid artery interposition grafts were constructed in New Zealand White rabbits. Vein conduits were immersed in solution containing 500 mg rhVEGF165 or saline solution for 20 minutes before implantation. Twenty-eight days later the vein grafts and contralateral control jugular veins were harvested for either histologic or isometric tension studies. Results: VEGF-treated vein grafts showed a 23% reduction in intimal area (0.76 ± 0.07 mm² vs 0.98 ± 0.06 mm²; p = 0.028) and a 30% reduction in intimal thickness (62 ± 6 μm vs 89 ± 5 μm; p = 0.001) when compared with control grafts. After precontraction with norepinephrine, the maximal relaxation to acetylcholine (endothelium-dependent, receptor-mediated agonist) for control vein grafts was 0%, whereas for VEGF-treated vein grafts it was 25% ± 9% (p < 0.05 vs control grafts). The maximal relaxation to the calcium ionophore A23187 (endothelium-dependent, receptor-independent agonist) was also greater in VEGF-treated grafts than in control grafts (172.3% ± 19.4% vs 122.5% ± 13.7%; p < 0.05). There was no difference in the response to sodium nitroprusside (endothelium-independent agonist) between the two groups. Conclusions: A single topical application of VEGF before implantation reduces intimal hyperplasia and improves endothelial function in a rabbit vein graft model. Further evaluation of this simple strategy to improve vein graft patency appears warranted. (J Vasc Surg 1998;27:167-73.)     

骨髓种植人工血管在静脉系统的实验研究

目的研究骨髓种植的人工血管在静脉系统的应用 ,旨在探索一种新的、更理想的静脉代用品。方法选北京地区杂种犬 8条 ,实验组和对照组各 4条。实验组采用自体骨髓种植的涤纶双绒人工血管置换肾下下腔静脉 ,对照组则采用单纯自体血浆预凝人工血管。术后 10d获取人工血管标本 ,观察其通畅率 ,行光镜、电镜检查 ,比较新生内膜厚度、新生内膜表面内皮化情况 ,并通过检测6 keto PGE1α和TXB2水平 ,比较分析实验组和对照组抗血栓能力。结果实验组全部移植血管均通畅 ,对照组 2 / 4条通畅 ,光镜下实验组的内膜厚度明显薄于对照组 (P <0 0 1) ,电镜发现实验组人工血管达到完全内皮化 ,而对照组表面则无成片内皮细胞存在。实验组 6 keto PGF1α水平明显高于对照组 (P <0 0 1) ,而TXB2水平实验组低于对照组 (P <0 0 5 )。结论骨髓种植的人工血管在静脉系统 10d时能实现人工血管腔面的快速完全内皮化 ,新生内皮细胞具有抑制新生内膜增生和抗血栓形成的能力。