Malcolm B. Herring

Seeding arterial prostheses with vascular endothelium. The nature of the lining.

Arterial prostheses seeded with autogenous vascular endothelium demonstrate a well-organized, cellular, inner lining. To determine the nature of the lining cells, six animals underwent replacement of the infrarenal aorta with Dacron® prostheses. During the preparation of three such grafts, endothelium was scraped from the saphenous vein with a steel wool pledget, suspended in chilled Sacks solution, and mixed with blood used to preclot the graft. This suspension was omitted from the three control grafts. After six weeks, the grafts were removed, rinsed and examined. Fluorescent Factor VIII related antigen (F VIIIRA) strongly stained the lining cells. Silver nitrate Haütchen and electron microscopy preparations revealed a lining pattern characteristic of vascular endothelium. Endothelial cell-specific Weibel-Palade bodies were identified in the lining cell cytoplasm. Massons trichrome staining revealed a relatively collagen-poor connective tissue within the seeded fabric. Transmission electron microscopy disclosed vascular smooth muscle cells between the seeded graft fabric and the lining cells. Vasa vasorum, arising from the outer capsule, penetrated the fabric to supply the inner capsules of the seeded grafts. It is concluded that the cells lining seeded canine arterial prostheses are true vascular endothelium supported by vascular smooth muscle cells, that the lining contains minimal connective tissue, and that vasa vasorum develop. Unseeded control grafts lacked these features.

Seeding human arterial prostheses with mechanically derived endothelium *: The detrimental effect of smoking

Endothelial healing of Dacron arterial prostheses can be hastened in dogs by seeding autogenous venous endothelium onto the prostheses in a single-staged operation. To determine whether this technique enhances the patency of human grafts, we studied the results of 186 operations on 161 patients performed between February 23, 1978, and December 1, 1982. Alternately allocating patients to treatment with seeded and unseeded Dacron knitted prostheses, we performed axillary-femoral and axillary-femoral-femoral bypasses in 11 patients (six seeded and five unseeded) and femoral-femoral bypasses in 28 (13 seeded and 15 unseeded). By a randomized block method of treatment allocation, femoral-popliteal grafts were installed in 147 limbs (112 vein, 18 seeded, and 17 unseeded). Patency was analyzed by the life-table method. Overall, femoral-femoral and femoral-popliteal bypasses demonstrated no difference between the seeded and unseeded grafts. Patency was somewhat better in seeded than unseeded axillary-femoral bypasses. Nevertheless, nonsmokers with seeded femoral-popliteal Dacron grafts enjoyed a significantly better graft patency than those with unseeded grafts (p = 0.035), whereas a substantial deterioration of seeded Dacron grafts was observed in those patients who smoked (p = 0.008 at 6 months). Vein grafts performed better than either seeded or unseeded Dacron prostheses (p = 0.016). Serum beta-thromboglobulin (BTG) levels varied widely and did not differ among any of the treatment groups. We concluded that endothelial seeding improved the patency of human arterial prostheses but that results were worse if the patient was a smoker. BTG was not a useful measure of the platelet activation induced by an arterial prosthesis.

Endothelium develops on seeded human arterial prosthesis: A brief clinical note

Despite a considerable and growing body of evidence that endothelial cell seeding accelerates the healing of arterial prostheses in laboratory animals, there has been no histologic evidence thus far to indicate that a similar process occurs in human beings. A case is reported of histologically confirmed, extensive endothelial healing on a polytetrafluoroethylene femoropopliteal bypass graft 90 days after it was seeded and implanted.

Endothelial cell seeding.

Endothelial cell seeding is the transplantation of vascular endothelial cells to denuded vascular surfaces. Seeding theoretically reduces the probability of graft or vessel thrombosis and of neointimal fibrous hyperplasia. Thus far, clinical seeding trials disclosed modest improvements in patency and the development of hyperplastic anastomotic lesions in failed grafts. Seeding inefficiency theoretically contributes to anastomotic hyperplasia. The inefficiency is linked to two steps in the seeding process, namely harvesting and cell retention. Of these, cell retention on the seeded surface is the more critical. Priorities for future research should be set first on the retention of seeded endothelium in vitro and second on improved and standardized methods of cell harvesting.

Seeding endothelium on canine arterial prostheses--the size of the inoculum.

Nineteen dogs were studied to determine the minimum length of saphenous vein which will yield sufficient endothelium to line arterial prostheses interposed in the infrarenal aorta. Knitted Dacron grafts preclotted by the Sauvage technique were seeded by endothelial cells mechanically harvested from lengths of autogenous saphenous vein varying from 3 to 150 mm. After 1 month, the grafts were removed; the percentage of clot-free surface was determined; the inner surface area of the grafts was calculated (Ag), and the linings were examined histologically. The inner surface areas of the donor vein (Av) and the installed graft (Ag) were computed. The percentage of clot-free surface was 87.6 ± 9.4 in dogs in which the ratio of the area of the vein to that of the graft (Av/Ag) was greater than 0.425 as compared to 25.9 ± 25.9 in others (P 0.425 had thinner inner capsules (217 ± 99 versus 480 ± 222 μm, P < 0.0005). We conclude that the amount of venous surface necessary to provide endothelium to line an arterial prosthesis using these techniques is slightly less than half the area of the graft to be lined. The size of undistributed venous endothelial cells was used to calculate the expected cell yield. In nine additional dogs the number of endothelial cells in the suspensions were compared to Av. If all cells were harvested and seeded successfully, the Av/Ag ratio might be lowered from 0.425 to 0.031.

Inhibition of Neutrophil Superoxide Production by Adenosine Released from Vascular Endothelial Cells

To investigate the inhibitory effect of adenosine released by endothelium on neutrophil Superoxide (O2−) production, we treated confluent monolayers of cultured human umbilical vein endothelial cells with the enzyme adenosine deaminase, and then added human neutrophils. Superoxide (O2−) production by human neutrophils stimulated with 10−6 M formyl-methionyl-leucyl-phenylalanine was inhibited by 49% in the presence of a confluent monolayer of human umbilical vein endothelial cells (5.1 ± 0.1 versus 2.6 ± 0.3 nmols O2−/106 neutrophils). Addition of 0.25 U/ml adenosine deaminase to neutrophils plus endothelial cells restored formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil Superoxide production to the level seen with neutrophils alone. Deoxycoformycin (10−4 M), an inhibitor of adenosine deaminase activity, prevented the increase in Superoxide production associated with adenosine deaminase addition. The adenosine analogue 5′-(N-ethylcarboxamido)-adenosine (3 × 10−4 M) caused increased inhibition of formyl-methionyl-leucylphenylalanine-stimulated superoxide release by neutrophils in the presence of endothelial cells and prevented neutrophil-mediated endothelial cell damage, as measured by release of3H-2-deoxy-D-glucose. Pairing 2-chloroadenosine (10−5 M) or 5′-(N-ethylcarboxamido)-adenosine (3 × 10−4 M) with a cyclic adenosine monophosphate phosphodiesterase inhibitor, 3-isobutyl-l-methyl-xanthine (10−4 M), produced greater inhibition of neutrophil superoxide production than occurred with either compound alone. The results support the hypothesis that vascular endothelial cells protect themselves from neutrophil attack by releasing adenosine to inhibit superoxide production.

Acellular vascular matrix: A natural endothelial cell substrate

A preliminary assessment was made of the acellular vascular matrix graft as a substrate for endothelial cell seeding, with respect to surface pretreatment (none versus fibronectin and/or serum) and presence of exogenous growth factor. Arteries were harvested from greyhounds and exposed to a sequential detergent extraction process to produce the acellular vascular matrix. Human umbilical vein endothelial cells were grown in tissue culture, harvested in first passage, then seeded at 10(5) cells/cm2 on sections of acellular vascular matrix and on gel-coated polystyrene positive controls. After 18 hour incubation, endothelial cell-seeded acellular matrices were fixed and processed for histologic and planimetric analysis; control wells were fixed and endothelial cells were counted by planimetry. Pretreatment of the acellular vascular matrix was found to have no effect on the percentage of endothelial cell coverage of the matrix. There was significantly better endothelial cell coverage of the acellular matrix than on matched gel-treated polystyrene control wells. Withdrawal of growth factor resulted in a significant reduction in endothelial cell coverage for all acellular vascular matrix groups. Growth factor withdrawal also significantly reduced attachment of endothelial cells on gel-treated polystyrene. Cell surface area was significantly smaller when growth factor was withdrawn from all groups except from the acellular vascular matrix without pretreatment. We conclude that: (1) the acellular vascular matrix is conductive to endothelial cell adherence and spreading even without pretreatment; and (2) sudden withdrawal of exogenous growth factor may impair early coverage of substrates by endothelial cells due to an effect on their adherence or spreading.

Patency in canine inferior vena cava grafting: Effects of graft material, size, and endothelial seeding

We studied 117 inferior vena cava (IVC) replacements in dogs to determine the effects of graft material, graft size, endothelial seeding, and cultured endothelial linings on graft patency. As a control, the IVC was removed and reimplanted in 11 dogs. Dacron (n = 7) and expanded polytetrafluoroethylene (e-PTFE) grafts (n = 12) were seeded immediately with the use of enzymatically derived autogenous jugular vein endothelium. Cultured linings were prepared for e-PTFE grafts (n = 9) by inoculating the graft with jugular endothelium and nurturing the lining in tissue culture for 14 to 30 days before implantation. Unseeded grafts (n = 27) were prepared according to the manufacturers recommendations. These six methods of preparation were tested in grafts measuring 6 mm I.D. and 60 mm in length. Other sizes were tested with a Latin square study design. After 30 to 60 days the grafts were perfusion fixed and studied with light and transmission electron microscopy. Patency was determined by contrast cavography after 7 and 30 days. Patency in the IVC reimplantation was 100% compared with 28.0% of the e-PTFE (p = 0.001) and none of the Dacron grafts that measured 6 mm I.D. and 60 mm long. e-PTFE and Dacron graft patency also differed significantly (p = 0.035). Seeded and culture-lined e-PTFE grafts in that same size were patent in 31.6% compared with 16.7% of unseeded e-PTFE. With grafts measuring 80 mm long, three of the five e-PTFE grafts were patent between 3 and 7 days. All progressed to occlusion by 30 days and compared poorly with all other graft sizes tested (2.6% progression to occlusion [p = 3 X 10(-8)]). Recanalization was not seen in 10 occluded grafts that were followed for 60 days. The histologic features of seeded grafts differed remarkably from grafts previously studied in the arterial circulation and from culture-lined and unseeded venous prostheses in that 60% had prominent large, random, endothelium-lined channels within the inner capsule. Larger graft diameters (p = 0.009) and the omission of an endothelial surface treatment (p = 0.004) were associated with anastomotic subendothelial fibrous hyperplasia. We conclude that graft material is the major determinant of patency in IVC replacements, that an extensive endothelial surface promotes patency, but that simply seeding e-PTFE or Dacron grafts with 10(5) endothelial cells does not provide sufficient endothelium to alter early patency.(ABSTRACT TRUNCATED AT 400 WORDS)

Leukocyte depletion enhances cultured endothelial retention on vascular prostheses

Approximately 90% of endothelial cells that are seeded or cultured onto vascular prostheses are lost from the flow surface within 24 hours of implantation. To determine the contribution of leukocytes to endothelial cell loss, 111In-labeled, cultured canine jugular venous endothelial cells were grown to confluence on fibronectin-coated polyester elastomer tubes measuring 4 mm inner diameter and 30 mm in length. Autogenous cell-lined tubes were implanted as bilateral carotid replacement grafts in six dogs made leukopenic by cyclophosphamide. Similar unilateral grafts were placed in 12 control dogs. Grafts were removed and perfusion-fixed from six control animals after 2 hours of in vivo arterial perfusion and from the other six animals after 6 hours of perfusion. One graft was removed and perfusion-fixed from each leukopenic animal after 2 hours of implantation and the other after 6 hours. Attachment of endothelial cells to the grafts was measured by indium-labeling technique. Retention of endothelium on grafts removed after 2 hours was measured by planimetric counting with scanning electron microscopy and on those removed after 6 hours by radioisotope quantification. Endothelial cell retention after 2 hours was 37.6% +/- 27.0% in control dogs and 97.0% +/- 3.4% in leukopenic animals (p less than 0.0007). After 6 hours retention was 35.9% +/- 23.2% in control animals and 86.5% +/- 6.0% in leukopenic animals (p less than 0.0009). Leukocyte surface activity was present in less than 1% of the leukopenic dogs compared with 8.5% of the other in vivo midgrafts after 2 hours. These results suggest that leukocytes play a significant role in the loss of seeded endothelium from vascular prostheses.

Graft material, length, and diameter determine the patency of small arterial prostheses in dogs

Abstract Thirty-two dogs were studied to determine the effects of graft material, length (L), and diameter (i.d.) on the patency of small arterial prostheses. Knitted Dacron (n = 15) and expanded polytetrafluoroethylene (e-PTFE, n = 17) were installed in the common carotid and common femoral arteries of dogs. The grafts were removed one month after implantation and their patency noted. Two of three Dacron grafts measuring 4 mm i.d. and 20 mm in length were patent, whereas none of the eight measuring 40 mm or more in length remained open (P = 0.01). With 4-mm e-PTFE grafts, 80% of the 40-mm and none of the 60-mm lengths of graft remained patent (n = 8, P = 0.03). When the grafts measured 4 mm i.d. and 40 mm in length, no Dacron grafts (n = 4) and 80% of the e-PTFE grafts (n = 5) remained patent (P = 0.016). With 3-mm grafts, the lengths had to be much shorter to insure that any grafts remained open, and even small differences approached significance: 60% of those 4 mm long were patent compared to 14% of those 8 mm long (P = 0.07). There was no difference between Dacron and e-PTFE. Given the clinical observation that much longer grafts made of both of these materials generally remain patent when i.d. = 6, equations predicting the maximum critical length consonant with a reasonable incidence of graft patency may be derived for each material. For e-PTFE: L c = r 4 (2.41 - 1.05r) , and for knitted Dacron: L c = r 4 (1.60 - 0.53r) , where Lc = maximumcriticallength of a particular graft and r = radius. We conclude that the patency of small arterial prostheses in dogs is a function of their length, the fourth power of their radius, and the material from which they are constructed.