Increased platelet-derived growth factor production and intimal thickening during healing of Dacron grafts in a canine model*

Increased platelet-derived growth factor production and intimal thickening during healing of Dacron grafts in a canine model* David A. Margolin, M D , Bram R. Kaufman, M D , Dennis J. DeLuca, PhD, Paul L. Fox, PhD, a and Linda M. Graham, M D , Cleveland, Ohio

Purpose: Growth factor production by endothelial cells on grafts may play a role in the development of intimal hyperplasia and subsequent graft failure. Methods: To study the relationship between platelet-derived growth factor production and graft healing, 26 beagles underwent placement of 20 cm long, 6 mm internal diameter, knitted Dacron thoracoabdominal grafts, either seeded with autologous endothelial cells (n = 14) or tmseeded controls (n = 12). The grafts and adjacent arteries were removed 4 or 20 weeks after implantation for measurement of platelet-derived growth factor production in organ culture, endothelial cell coverage, and intimal thickness. Results: Midgraft platelet-derived growth factor production by seeded graft segments increased from 41 - 6 to 148 --. 27 pg/cm2/72 hr (p < 0.002) between 4 and 20 weeks. This was accompanied by a significant increase in inner-capsule thickness. Platelet-derived growth factor production by control graft segments also increased from 58 + 21 to 163 -+ 42 pg (p < 0.05) and was similar to that of seeded grafts despite more rapid endothelialization of seeded grafts. The increase in growth factor production by Dacron grafts was greater than that of the expanded polytetrafluoroethylene grafts studied previously despite similar endothelial cell coverage. Conclusion: This increase corresponded with the rapid appearance of smooth muscle cells in the pseudointima of Dacron grafts, suggesting that these cells may be responsible for the observed increase in platelet-derived growth factor production. (J VASC SURG 1993;17:858-67.)

Intimal hyperplasia at the anastomoses of prosthetic grafts, characterized by smooth musde cell (SMC) accumulation and matrix deposition, is a recognized cause of late graft failure in the clinical setting. The cause of this lesion appears to be multifactorial and includes such factors as platelet activation with release of growth factors, flow disturbances at anastomoses, and compliance misFrom the Departmentof Surgery, CaseWestern ReserveUniversity,the DepartmentofVeteransAffairsMedicalCenter,and the Department of Vascular Cell Biology and Atherosclerosis Research, ClevelandClinic ResearchInstitute. Supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL-41178 and HL-40352) and the Department of Veterans Affairs. Presented at the SixteenthAnnual Meeting of the Midwestern Vascular SurgicalSociety,Cleveland,Ohio, Sept. 11-12, 1992. Reprint requests: LindaM. Graham,MD, SurgeryService112W, VA Medical Center, 10701 E. Blvd., Cleveland,OH 44106. ~EstablishedInvestigatorof the AmericanHeart Association. *Winner of the 1992 Charles C. Guthrie Award. Copyright © 1993 by The Society for Vascular Surgery and InternationalSocietyfor CardiovascularSurgery,North American Chapter. 0741-5214/93/$1.00 + .10 24/6/45118

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match. In addition, mitogens released from leukocytes, SMCs, and endothelial cells (ECs) also may contribute to the development ofintimal hyperplasia. Intimal thickening is most severe adjacent to the anastomoses of prosthetic grafts, the very areas first covered with endothelium, suggesting that ECs may contribute to its development. Supporting this hypothesis are studies by Clowes et al},2 on healing of expanded polytetrafluoroethylene (ePTFE) grafts in baboons. In these grafts, SMC proliferated only under ECs, at the anastomoses, and in areas of transmural tissue ingrowth where SMCs followeC~ ECs migrating through the interstices of the prosthesis. These SMCs did not bccome quiescent but continued to proliferate at elevated rates 1 year after graft placement? One explanation for ongoing SMC proliferation beneath graft endothelium is the production of mitogens by ECs. One of these mitogens, plateletderived growth factor (PDGF), is mitogenic and chemotactic for SMCs and fibroblasts. 4 Golden et al.s demonstrated that endothelialized ePTFE grafts

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produced more mitogenic activity and expressed higher levels of PDGF A-chain m R N A than the adjacent artery. However, in these studies the amount of PDGF secreted was not measured and the cell type producing the PDGF was not identified. Studies in our laboratory demonstrated greater PDGF production by EC-seeded ePTFE grafts than by control grafts. 6 In addition, PDGF production correlated with EC coverage 4 weeks after graft implantation. With longer implantation times, PDGF production by EC-seeded ePTFE grafts ig[i:reased, particularly at the anastomoses, suggesting a role for PDGF in the development of intimal hyperplasia and subsequent graft failure. 7 However, at the later time points in that study, the correlation between PDGF production and EC coverage was weaker, suggesting that other cells also contributed to PDGF production. The accumulation of SMCs in the inner capsule of ePTFE grafts adjacent to the anastomoses suggested that SMCs themselves might be a source of this mitogen. The object of this study was to investigate PDGF production during healing of Dacron grafts, which, in a canine model, is characterized by the accumulation of SMCs in the inner capsule or pseudointima of the graft. MATERIAL A N D M E T H O D S Adult female beagles, aged 44 to 218 weeks and w,o~ighing 7.0 to 12.0 kg, underwent placement of knitted Dacron thoracoabdominal grafts. Grafts in 14 dogs were seeded with autologous jugular vein ECs. Unseeded grafts in 12 dogs served as controls. Seven seeded and six control grafts were removed 4 weeks after implantation and the remaining seven seeded and six control grafts were removed after 20 weeks. The mean age of dogs whose grafts were removed at 4 weeks and 20 weeks was similar: 115 + 12 (mean + SEM) and 117 + 9 weeks, respectively. Animal studies followed a protocol approved by the institutional committee on animal use, and care complied with the "Principles of Laboratory Animal Care" and "Guide for the Care and Use of -~aboratory Animals" (National Institutes of Health publication No. 80-23, revised 1985). Graft seeding and implantation. Anesthesia was induced with intravenous thiamylal sodium, 20 mg/kg (Parke-Davis, Professional Medical Products, Inc., Greenwood, S.C.), and an endotracheal tube was placed. Anesthesia was maintained with 1% to 2% isoflurane and oxygen, and mechanical ventilation was instituted during the thoracotomy. Each animal Jreceived 450,000 units penicillin G benzathine and 450,000 units penicillin G procaine (Pen

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BP-48; Pfizer Inc., New York, N.Y.) subcutaneously before the operative procedure. Hydration was maintained by intravenous infusion of 10 ml/kg/hr lactated Ringer's solution th~:oughout the procedure. For EC harvest, external jugular veins were carefully isolated and everted over stainless-steel rods. The ECs were removed by sequential incubations in 0.05% trypsin (Sigma Chemical Co., St. Louis, Mo.) for 5 minutes and 630 units/ml collagenase A (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) for 16 minutes. The cells were washed and resuspended in 1 ml medium 199 tissue culture medium (Sigma Chemical Co.) for immediate graft seeding. The homogeneity of EC harvests was verified by immunocytochemistry with antibody to factor VIII-related antigen (Chemicon International, Temecula, Calif.) and antibody HHF-35 to SMC ot-actin (Enzo Biochern, Inc., New York, N.Y.). Each 22 cm long, 6 m m internal diameter Cooley double-velour knitted Dacron graft (donated by Meadox Medicals Inc.; Oakland, N.J. ) was preclotted in three steps with 5 ml aliquots of venous blood. In all cases 1 ml medium 199 was added to the first aliquot of blood. For seeded grafts the medium 199 contained the harvested EC,s. Grafts were trimmed and implanted in a thoracoabdominal position as described previously, 8 with as1 end-to-end anastomosis between the graft and the proximal descending thoracic aorta between the second and third intercostal arteries and an end-to-side anastomosis to the infrarenal aorta. Graft removal. The grafts and adjacent arteries were removed for analysis either 4 or 20 weeks after implantation. To minimize F,latelet deposition on the graft material, 325 mg aspirin was administered to the dogs the evening before graft removal. Animals were anesthetized as described above. Heparin sodium (150 tmits/kg; Elkins.-Sinn Inc., Cherry Hill, N.J.) and papaverine (90 ng/kg; Eli Lilly & Co., Indianapolis, Ind.) were administered intravenously. Both common carotid arteries were isolated and pinned at anatomic length on Sylgard 184 silicone elastomer disks (Dow Coming Corp., Midland, Mich.) as described previously.6,7 Similarly, the graft, the entire aorta, and both iliac and femoral arteries were isolated and flushed with 1 L medium 199. Particular care was taken to remove all tissue from the outer surface of the graft and arteries. The vessels were then opened longitudinally, pinned onto Sylgard disks, excised, and placed in organ culture. Organ culture. The arterial and graft segments were rinsed thoroughly again 60 minutes after removal and placed in six-well Falcon tissue culture

860

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plates (Becton Dickinson Labware, Lincoln Park, N.J.) for organ culture by a modification of a method described by Pederson and Bowyer9 and Fingerle and Kraft) ° The tissue was incubated in medium 199 containing 15 units/ml heparin sodium, 0.15 mg/ml EC growth supplement isolated from bovine hypothalami (Rockland, Inc., Gilbertsville, Pa.), 0.05 mg/ml gentamicin (Sigma Chemical Co.), 2.2 mg/ml sodium bicarbonate (Sigma Chemical Co.), and 10% controlled processed serum replacement-2 (Sigma Chemical Co.), a serum substitute with low mitogenic activity. The tissue was incubated for 72 hours at 37 ° C in a 5% CO2 atmosphere. The conditioned medium was then removed and centrifuged for 5 minutes at 1000g and the supematant stored at 4 ° C for PDGF assay. Medium containing 2 ~Ci/ml tritiated leucine (Du Pont Diagnostic Imaging Division, N. Billerica, Mass.) was then added to the organ culture for an additional 24 hours for determination of secreted protein synthesis as a measure of cell viability and metabolic activity. PDGF assay. The PDGF was measured by radioreceptor assay as described by Fox and DiCorleto. n The radioreceptor assay measures competition with iodine 125-labeled PDGF for binding to PDGF receptors on human foreskin fibroblasts. This assay detects all three isoforms of the PDGF dimer (AA, AB, and BB) and has a sensitivity of 10 to 20 pg. The PDGF AB (Boehringer Mannheim Biochemicals) was radiolabeled according to the method of Heldin et al.12 Conditioned medium was concentrated approximately sevenfold with a Centriprep- 10 concentrator (Amicon Division/W. R. Grace & Co., Beverly, Mass.). Sparse, quiescent cultures of human foreskin fibroblasts were then incubated for 1 hour with 0.2 ml concentrated conditioned medium and 0.1 ml HEPES-buffered Dulbecco's modified Eagle medium/F12 mixture (Sigma Chemical Co.) with 2 mg/ml bovine serum albumin (Sigma Chemical Co.). After washing the cells, 0.3 ml medium containing 12SI-labeled PDGF was added for an additional hour. The medium was then aspirated, the cells were washed and solubilized with i% Triton X-100 (Sigma Chemical Co.), and cell-bound radioactivity was determined in a y-radiation counter. A standard curve was prepared with known amounts of purified PDGF, and results were fit by nonlinear regression with the logistic equation. The PDGF contributed by the complete organ culture medium was quantitated and subtracted from the PDGF measured for each sample. The PDGF production by graft and arterial segments was expressed as picograms per square centimeter of tissue.

JOURNAL OF VASCULARSURGEI~F May 1993

Protein synthesis. Secreted protein synthesis was determined by incorporation of tritiated leucine into trichloroacetic acid (TCA)-precipitable material as described previously by Fox and DiCorleto. n The conditioned medium from organ culture was centrifuged and the supernatant precipitated with 2 ml 5% TCA and 0.25% tannic acid solution. This was repeated three times and the final precipitate was solubilized with 0.25 mol/L NaOH. The radioactivity was determined in a liquid scintillation counter. Secreted protein synthesis was expressed as the percentage of added tritiated leucine incorpor~.7?d into TCA-precipitable material per square centimeter of tissue in organ culture × 1000. Graft analysis by microscopy. After organ culture, the arterial and graft segments were fixed in 2.5% glutaraldehyde (Sigma Chemical Co.) for 24 hours and processed for both light microscopy and scanning electron microscopy. Specimens for light microscopy were dehydrated in graded alcohols and embedded in Spurr low-viscosity resin (Polysciences, Inc., Warrington, Pa.). With a Reichert-Jung U1tracut E Microtome (Reichert-Iung, Vienna, Austria.), 0.6 ~m sections were cut and stained with toluidine blue. Intimal thickness at the anastomosis was measured at points 500 and 1000 I~m from the suture line along both the graft and arterial segments. The pseudointima (inner capsule) thickness of the midgraft was measured at five points on tl-=ee different graft segments. The specimens for scanning electron microscopy were dehydrated in graded alcohol solutions followed by a final dehydration in hexamethyldisilazine (Polysciences, Inc.) and coated with gold in an RMCEIKO IB-3 ion coater (Research Manufacturing Co., Inc., Tucson, Ariz.). Ten random fields were scanned at x 1000 magnification with a Ieol JSM-840-A electron microscope (Ieol USA, Peabody, Mass.). Percent endothelial coverage was determined by the presence or absence of ECs at 10 grid intersections on 10 fields per segment. Platelet deposition was estimated on the same 10 fields by comparison with standards rated 0 to 4, with 0 representing an absent/5 of platelets and 4 representing heavy platelet deposition. Erythrocytes and leukocytes were counted in the same fields. All studies were performed by an observer blinded with respect to graft treatment and implantation time. Statistical analysis. PDGF production by segments of the same animal were compared by the paired t test. Results between groups were analyzed with the unpaired t test. Linear regression analysis was used to study relationships between PDGF production and cell coverage or deposition.

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Table I. PDGF production by canine arterial and graft segments 4 wk (pg/cm2/72 hr) Seeded (n = 7) Proximal aorta Proximal anastomosis Midgraft Distal anastomosis Distal aorta

18 33 41 36 53

20 wk (pg/cm2/72 hr)

Control (n = 6)

_+ 5 _+ 6 -+ 6 + 6 -+ 131-

14 38 58 30 39

-+ -+ + + -+

6 7 21 7 11

Seeded (n = 7) 9 83 148 115 59

-+ 5 + 24 _+ 27* _+ 30* - 11t

Control (n = 6) 15 114 163 169 84

_+ 9 + 30* + 42* -+ 38* + 11"t

Data are means + SEM. *p < 0.(15 compared w i t h 4-week group. t ~ < 0.(12 compared w i t h proximal aorta. i

Table II. Protein synthesis by canine arterial and graft segments 4 wk (% applied tritiated leucine incorporated/cm2/24 hr × 1000) Seeded (n = 7) Proximal aorta Proximal anastomosis Midgraft Distal anastomosis Distal aorta

159 100 70 53 256

_+ 17 + 28 + 10 -+ 11 _+ 30*

Control (n = 6) 244 142 53 58 525

_+ 82 + 49 + 17 + 23 _+ 207

20 wk (% applied tritiated leucine incorporated/cm2/24 hr × 1000) Seeded (n = 7) 138 100 77 58 280

+ 16 + 6 + 9 + 5 _+ 4 1 "

Control (n = 6) 144 104 64 93 232

_+ 11 _+ 14 -+ 8 + 37 -+ 15"

Data are means + SEM. *p < 0.01 compared w i t h proximal aorta.

RESULTS All grafts were patent at removal and there was no e~'~lence of excessive thrombus formation or graft infection. Arterial and graft segments were placed in organ culture for 72 hours and PDGF in the conditioned medium was measured (Table I). The EC-seeded grafts removed at 20 weeks produced nearly four times as much PDGF as seeded grafts removed at 4 weeks. Interestingly, a similar increase was seen for control grafts, and in fact there was no significant difference in PDGF production between seeded and control grafts. Production of PDGF by anastomotic segments did n o t differ significantly from that by the midgraft in any group. Production of PDGF by the proximal and distal aorta did not change over time and was not influenced by the graft 8eatment. However, consistent with previous findings, 6,7 the distal aorta produced approximately four times more PDGF than the proximal aorta in all groups: 59 + 6 and 14 _+ 3 pg/cm2/72 b_r, respectively (p = 0.0001). The higher PDGF production by the distal aorta was also found in dogs not undergoing graft placement and represented a difference in PDGF production by the native aorta, not a change induced by graft placement. Secreted protein synthesis was used to assess tissue viability and general metabolic activity. As seen in Table II, all graft segments secreted similar

amounts of protein, documenting similarity of metabolic activity. There was no significant difference between the seeded and control grafts at any time point. Protein synthesis in the distal aorta was approximately twice that of the proximal aorta in both seeded and control groups, suggesting that an increase in metabolic activity or number of cells accounted for only a portion of the greater PDGF production by the distal aorta. Endothelial cell coverage as determined by scanning electron microscopy was significantly greater for the seeded grafts at both time points. At 4 weeks 57% _+ 7% of the luminal surface of the seeded grafts was covered with ECs compared with 21% +_ 7% for the control grafts (p < 0.004). Similarly, at 20 weeks the EC coverage of the seeded grafts was 80% _+ 4% compared with 48% _+ 10% for control grafts (p < 0.007). The increase in EC coverage between 4 and 20 weeks was statistically significant for both seeded (p < 0.01) and control grafts (p = 0.05). The EC coverage correlated positively with PDGF production in seeded grafts (r = 0.7; p < 0.006), suggesting that seeded ECs contributed to PDGF production. However, no significant correlation between EC coverage and PDGF production was found in the control grafts, suggesting that other cells, such as platelets or SMCs, were important sources of PDGF in grafts with lower EC coverage.

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862 Margolin et al.

Table III. Intimal thickness of canine arterial and graft segments 4 wk (pro) Seeded (n = 7) Proximal anastomosis A-200 A-100 G-100 G-200 Midgraft Distal anastomosis G-200 G-100 A-100 A-200

20 wk (i.tm) Control (n = 6)

18 42 147 100 79

+ 9 _+ 14 -+ 58 _+ 17 + 14"

41 34 72 118 131

62 74 84 66

-+ 37 _+ 30 -+- 33 + 41

64 69 69 37

Seeded (n = 7)

Control (n = 6)

+ 24 -+ 12 _+ 16 + 33 _+ 19

68 50 138 193 137

+ 31 -+ 9 -+ 33 _+ 351 -+ 2 2 t

80 47 181 183 115

-+ 26 -4- 11 -+ 3 3 t + 38 _+ 11

+ -+ + +-

232 221 126 67

-+ 58t _+ 67 -+ 29 + 41

196 274 213 178

+ 53t -+ 89~ _+ 95 -4- 81

17 20 29 28

Data are means +- SEM. A-200 and A-100 represent site of measurement of intimal thickness of the artery 1000 and 500 Ixm, respectively, from the suture line. G-100 and G-200 represent site of measurement of neointimal thickness on the graft 500 and 1000 ~m, respectively, from the suture line. *p < 0.05 compared with control. tp < 0.05 compared with 4 weeks.

Platelet and leukocyte deposition were evaluated by scanning electron microscopy because both cell types were a possible source for PDGF. Significantly fewer platelets were present on the seeded grafts at both time points. The platelet scores at 4 weeks averaged 1.0 _ 0.2 for seeded grafts and 2.4 - 0.3 for control grafts (p < 0.002). By 20 weeks platelet deposition had decreased on control grafts but was still significantly higher than on seeded grafts, the scores averaging 1.0 +_ 0.1 for the seeded group and 1.7 _ 0.3 for the control grafts (p < 0.04). No correlation between platelet deposition and PDGF production was found for either seeded (r = 0.2; p = 0.50) or control grafts (r = 0.14; p = 0.67). However, in the 4-week control grafts, platelet deposition did correlate with PDGF production (r = 0.8; p < 0.05), suggesting that PDGF from platelets may account for a portion of the measured PDGF in this subset of grafts with a thrombogenic surface relatively soon after implantation. White blood cell deposition on seeded grafts was not significantly different from that of control grafts at 4 weeks, with 3.9 _+ 1.0 and 5.6 + 1.1 white blood cells per field, respectively (p = 0.3). Similarly, at 20 weeks no significant difference was seen in leukocyte deposition on seeded and control grafts, with 1.6 _+ 0.5 and 4.4 + 2.2 white blood cells per field, respectively (p = 0.2). As with platelets there was no significant positive correlation between leukocyte deposition and PDGF production on seeded grafts (r = 0.51; p = 0.06) or control grafts (r = 0.27; p = 0.40), suggesting that leukocytes on these grafts were not a major source of PDGF. Intimal thickness was evaluated by light micros-

copy (Table III). At 4 weeks the midgraft pseudointimal thickness of seeded grafts was significantly thinner than that of control grafts, but by 20 weeks the thickness of the inner capsule of seeded and control grafts was similar. The pseudointima of midgraft segments of the seeded grafts increased significantly in thickness between 4 and 20 weeks (p < 0.05), whereas the fining of control grafts remained constant. At the proximal and distal arC~tomoses of seeded and control grafts, there was a trend toward increasing thickness with time, although this was not always statistically significant. Interestingly, increased PDGF production in seeded grafts correlated with increasing midgraft innercapsule thickness (r = 0.78; p < 0.001). The same correlation was not found in the control grafts. The measurements of inner-capsule thickness included all tissue, both cellular and acellular material, lining the graft. In seeded grafts at 4 weeks the majority of the inner capsule was composed of a monolayer of ECs with underlying mesenchymal cells (Fig. 1, A). However, there was substantial: variability in the histologic appearance of the i n n ~ capsule of control grafts at 4 weeks with regions of fresh and organizing thrombus, as well as regions with an EC surface and mesenchymal cells forming the subendothelial tissue (Fig. 1, B). By 20 weeks the pseudointima of seeded grafts was thicker and contained more cells having the appearance of SMCs (Fig. 2, A). Control grafts at 20 weeks had less surface thrombus and the pseudointima was more organized and more cellular than at 4 weeks (Fig. 2, B). The Dacron graft material was surrounded by inflammatory cells in both seeded and control grafts

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Fig[ 1. Histologic sections of midgraft segments 4 weeks after implantation. (Toluidine blue stain; original magnification x 400.) A, EC-seeded graft demonstrates monolayer of ECs with underlying mesenchymal cells. B, Control graft demonstrates thicker inner capsule with thrombus on luminal surface.

Fig. 2. Histologic sections ofmidgraft segments 20 weeks after implantation. (Toluidine blue stain; original magnification × 400.) A, EC-seeded graft demonstrates monolayer of ECs with thicker subendothelial tissue than was apparent at 4 weeks. B, Control graft demonstrates more organized and cellular pseudointima than was present at 4 weeks.

at 4 weeks. The number of inflammatory cells did not increase with time, but more multinucleated giant cells vcere present and organized fibrous tissue surrounded the graft at 20 weeks.

DISCUSSION The purpose o f this study was to explore changes in growth factor production during healing of a porous graft. The results of riffs study showed that

864 Margolin et aL

PDGF production by anastomotic and midgraft segments of Dacron grafts was significantly greater after a 20-week than a 4-week implantation period and surpassed that of the native aorta. This temporal increase in PDGF production was seen in previous studies of ePTFE grafts, z However, Golden et al.~ found that total mitogenic activity of ePTFE graft perfusates decreased with time during a 3-month study period. This difference may relate to differences in species, hemodynamic factors, or the characteristics of graft healing by ECs and SMCs. Organ culture was employed at the time of graft removal as in previous studies. Use of organ culture maintains the tissue architecture and does not disrupt cellular interactions that may contribute to control of functions such as mitogen production. In addition, direct measurement of PDGF secretion rather than mRNA levels is possible, an important advantage because gene expression does not necessarily correlate with protein production. Finally, organ culture allows PDGF measurements to be well localized to specific anatomic sites within the graft and arterial system, unlike perfusion studies. Unfortunately, organ culture does have the disadvantage of removing hemodynamic factors that may affect PDGF production in vivo. Furthermore, identification of the cell type primarily responsible for mitogen production and the contribution of the luminal cells, as opposed to cells in the interstices or on the outer wall of the graft, is difficult. Finally, the accuracy with which PDGF production by vessel segments in organ culture reflects that in vivo is not certain. However, very reproducible differences in PDGF production by different arteries and arteries and veins have been documented in organ culture, suggesting that it is a reflection of the tissue's capacity to produce PDGF in vivo. A dramatic difference was noted between PDGF production by Dacron grafts segments in this investigation and ePTFE graft segments studied earlier.7 After implantation for 20 weeks, EC-seeded Dacron graft segments produced twice as much PDGF as did seeded ePTFE grafts: 148 +_ 27 pg and 73 _ 22 pg/cm2/72 hr, respectively (p = 0.05). This difference occurred despite similar EC coverage for both groups: 80% __ 4% for Dacron and 70% __ 7% for ePTFE grafts, suggesting that ECs were not the primary source of PDGF at 20 weeks unless the ECs were more "activated" on Dacron than on ePTFE grafts. However, if all PDGF was produced by ECs, PDGF production would have correlated with EC coverage on both seeded and control Dacron grafts. A positive correlation existed between PDGF pro-

JOURNAL OF VASCULARSURGERi May 1993

duction and EC coverage on seeded Dacron grafts but not on control Dacron grafts. In fact, PDGF production by seeded and control grafts was similar, despite more rapid endothelialization of the seeded grafts. Although ECs may have produced a significant amount of PDGF, these data suggested that cells other than ECs also contributed to PDGF production. Although platelets and leukocytes may produce PDGF, neither cell type appeared to account for differences observed in PDGF production between 4- and 20-week graft segments or between Dacrc"~ and ePTFE grafts. No significant correlation was found between platelet or leukocyte deposition and PDGF production. However, platelets may have contributed to the PDGF measured at 4 weeks on control grafts because this group showed a positive correlation between platelet deposition and PDGF production. Aspirin was administered to the dogs the evening before sacrifice to lessen platelet deposition during manipulation of the graft during removal. This may have decreased the number of platelets on the grafts at the time ofplatelet assessment but should not have effected the correlation between platelet number and PDGF production, if the platelets were a major source of PDGF in the organ culture system. The role of platelets or antiplatelet agents in the development of pseudointimal hyperplasia was not assessed in this study. :~ Another possible source of PDGF was macrophages, which were shown by Shimokado et al.13 to produce PDGF. The contribution of tissue macrophages to PDGF was not addressed directly in this study. Histologic sections of Dacron and ePTFE grafts documented a larger number of macrophages adjacent to the Dacron graft material than the ePTFE prostheses, but the number of macrophages did not increase between 4 and 20 weeks, the time during which the difference in PDGF Production by the two types of prostheses became apparent. This suggested that macrophages were not the primary source of PDGF. The results of this study combined with those of the earlier study ofePTFE grafts suggested a role for SMCs in PDGF production, z The temporal increase in PDGF production was greater for Dacron grafts than for ePTFE grafts and was accompanied by the rapid appearance of SMCs in the pseudointima of the Dacron grafts. Twenty weeks after implantation, smooth muscle-like cells were abundant in the inner capsule of Dacron grafts but were virtually absent in the midportion of ePTFE grafts because of differences in healing of the Dacron and cPTFE grafts.

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PorotLs Dacron grafts heal by transmural ingrowth of capillary buds, which provide a source of ECs and S M C s , 14 whereas standard ePTFE grafts with a nominal 30 ~m internodal distance heal primarily by migration of ECs and SMCs from the cut end of the adjacent arteries, 1 resulting in few SMCs in the inner capsule of the midportion of ePTFE grafts. The SMCs did accumulate in the inner capsule of ePTFE grafts adjacent to the anastomoses, the areas with the most marked temporal increase in PDGF production. 7 In addition, SMCs were present in the midgraft ~eudointima of 52-week ePTFE grafts, which also had a marked increase in PDGF production. Thus in both ]Dacron and ePTFE grafts the presence of SMCs in the pseudointima was associated with a substantial increase in PDGF production. Whether this was cause or effect could not be determined from these data, but these findings suggested that SMCs might be a source of PDGF production in graft healing. The level of PDGF produced per cell was not determined in this study. Differences in PDGF production between Dacron and ePTFE graft segments could be a reflection of differences in SMC accumulation caused by porosity or differences in SMC stimulation as a result of the chemical or physical properties of the grafts. BAthough SMCs do not normally produce PDGF, other investigators have shown that under certain F-thologic conditions SMCs are capable of secreting PDGF. 15,16Birinyi et al) 7 found that SMCs cultured f r o m hyperplastic lesions obtained from clinical specimens released PDGF-like activity and expressed genes for the PDGF A chain. Thus the SMCs that grew onto the graft may have become modified to produce PDGF. The stimulus for the alteration of SMC function has not been defined but may have been a response to the foreign graft material. On the other hand, SMCs that grew onto prosthetic grafts may have represented a subset of normal SMCs that responded more readily to chemotactic stimuli and produced PDGF. Although the ability of SMCs to secrete PDGF under certain conditions is well documented, the ability of SMCs on grafts to respond to PDGF remains in question. The literature contains conflicting information on the response of modified SMCs to PDGF and other serum mitogens. The SMCs from restenosing lesions were stimulated significantly by PDGF, ~8 and SMCs from atherosclerotic plaques expressed more PDGF J3-type receptors than did SMCs of normal arteries, x9 In addition, SMCs from hyperplastic lesions of grafts expressed mRNA for PDGF receptor, 17 and neointimal SMCs in injured

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arteries expressed higher levels of PDGF J3-receptor mRNA than did SMCs of uninjured arteries. 2° On the other hand, Walker et al. 16 demonstrated that SMCs cultured from injured rat arteries secreted PDGF but had fewer PDGF receptors than normal SMCs and were not stimulated to proliferate by exogenous PDGF. Thus lesional SMCs may vary in their response to PDGF depending on degree of injury or pathologic state) 9 If SMCs on grafts are capable of both producing and responding to PDGF, they may regulate their proliferation in an autocrine fashion, as well as respond to continued PDGF production by ECs. This could lead to further SMC migration and proliferation, resulting in increased inner capsule thickness. The occurrence of this process at the anastomoses where ECs and SMCs migrate onto the graft from the native artery could account for the development of anastomotic intimal hyperplasia leading to graft failure. In summary, in a canine model PDGF production by Dacron graft segments was significantly greater than that by ePTFE grafts 20 weeks after implantation. The higher rate of PDGF production corresponded to the more rapid appearance of SMCs in the inner capsule of Dacron grafts, suggesting that SMCs play a role in PDGF production. EC production of PDGF may stimulate SMC ingrowth into the graft, but ECs may contribute a decreasing percentage of the total PDGF production over time. Clearly, PDGF is only one mitogen that may play a role in the development of anastomotic intimal hyperplasia. Further study of mitogen production by cells on prosthetic grafts and the responsiveness of graft SMCs to these mitogens will contribute to the understanding and potential control of anastomotic intimal hyperplasia. REFERENCES

1. Clowes AW, Gown AM, Hanson SR, Reidy MA. Mechanisms of arterial graft failure, 1: Role of cellular proliferation in early healing of PTFE prostheses. Am J Pathol 1985;118:4354. 2. Clowes AW, Kirkman TR, Reidy MA. Mechanisms of arterial graft heating: rapid transmural capillary ingrowth provides a source of intimal endothelium and smooth muscle in porous PTFE prostheses. Am J Pathol 1986;123:220-30. 3. Clowes AW, Kirkman TR, Clowes MM. Mechanisms of arterial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses. J VAse SURG 1986;3:877-84. 4. Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell 1986;46:155-69. 5. Golden MA, Au YPT, Kirlmaan TR, et al. Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. IFClin Invest 1991;87:406-14. 6. Kaufman BR, Fox PL, Graham LM. Platelet-derived growth

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14.

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factor production by canine aortic grafts seeded with endothelial cells. J VASCSURG 1992;15:699-707. Kaufman BR, DeLuca Dj', Folsom DL, et ai. Elevated platelet-derived growth factor production by aortic grafts implanted on a long-term basis in a canine model. J VASC SURG 1992;15:806-16. Graham LM, Vinter DW, Ford JW, Kahn RH, Burkel WE, Stanley JC. Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium. Arch Surg 1980;115:929-33. Pederson DC, Bowyer DE. Endothelial injury and healing in vitro: studies using an organ culture system. Am J Pathol 1985;119:264-72. Fingerle J, Kraft T. The induction of smooth muscle cell proliferation in vitro using an organ culture system. Int Angiol 1987;6:65-72. Fox PL, DiCorleto PE. Regulation of production of a platelet-derived growth factor-like protein by cultured bovine aortic endothelial cells. J Cell Physiol 1984;121:298-308. Heldin C-H, Westermark B, Wasteson A. Platelet-derived growth factor: purification and partial characterization. Proc Natl Acad Sci USA 1979;76:3722-6. Shimokado K, Raines EW, Madtes DK, Barrett TB, Benditt EP, Ross R. A significant part of macrophage-derived growth factor consists of at least two forms of PDGF. Cell 1985;43: 277-86. Zacharias RK, Kirkman TR, Clowes AW. Mechanisms of healing in synthetic grafts. J VASC SURG 1987;6:42936.

DISCUSSION Dr. Howard P. Greisler (Maywood, Ill.). The regulation of pseudointimal hyperplasia after the implantation of synthetic vascular grafts is very complex, and the continuing exploration of at least one possible modulator of this process (i.e., the production of PDGF by the tissues in response to the implanted material) presents interesting bits of data but also leads to a number of important questions. This study shows a temporal increase in PDGF production from 4 to 20 weeks after the implantation of knitted Dacron grafts. The PDGF production by the tissues incorporating these Dacron grafts was greater than that reported previously after the implantation of PTFE grafts. By contrast, and of great importance, no difference was seen comparing EC-seeded and unseeded grafts. The data presented also demonstrate no difference in PDGF production by explanted tissues in organ culture comparing the midgraft versus the anastomotic regions. This is of interest considering the clinical localization of the intimal hyperplastic activity to the perianastomotic regions. As is true of all good research, these data pose many questions. You demonstrate a significantly greater luminal surface coverage by ECs in the cell-seeded group at both 4 and 20 weeks. This being the case, one must then postulate

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15. Libby P, Warner SJC, Salomon RN, Birinyi LK. Production of platelet-derived growth factor-like mitogen by smoothmuscle cells from human atheroma. N Engl J Med 1988;318: 1493-8. 16. Walker LN, Bowen-Pope DF, Ross R, Reidy MA. Production of platelet-derived growth factor-like molecules by oalmred arterial smooth muscle cells accompanies proliferation after arterial injury. Proc Natl Acad Sci USA 1986;83: 7311-5. 17. Birinyi LK, Warner SJC, Salomon RN, Callow AD, Libby P. Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: production of a platelet-derived growth factor-like mitogen and expression of a gene for a platelet-derived growth factor receptory~ preliminary study. ~[VAsc SURG 1989;10:157-65. 18. Dartsch PC, Voisard R, Bauriedel G, H6fling B, Betz E. Growth characteristics and cytoskeletal organization of cultured smooth muscle cellsfrom human primary stenosing and restenosing lesions. Arteriosclerosis 1990;10:62-75. 19. Rubin K, Hansson GK, R6nnstrand L, et al. Induction of B-type receptors for platelet-derived growth factor in vascular inflammation: possible implications for development of vascular proliferative lesions. Lancet 1988;i: 1353-8. 20. MajeskyMW, geidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Bio11990; 111:2149-58. Submitted Sept. 14, 1992; accepted Dec. 19, 1992.

that either the ECs in the seeded group produce less PDGF per cell or PDGF in the unseeded group is produced by other cell types. You suggest the contribution of smooth cells and have provided indirect evidence against the role of either platelets or leukocytes. However, using ~-imaging techniques with indium 11 I-labeled platelets, other investigators have demonstrated a chronically elevated rate of platelet deposition onto the surface of both Dacron and ePTFE grafts. A differential increase in platelet deposition and activation caused by the Dacron could result in platelet degranulation and enhanced PDGF release. Similarly, macrophage interaction with Dacron is more extensive than that seen with ePTFE, and macrophages may wel~; produce at least a portion of the measured PDGF. Has immunohistochemistry or in situ hibridization techniques been used to identify the cell type from which the PDGF protein is secreted and in which the message is presumably located? Second, would you care to speculate as to the lack of difference in PDGF production by tissues explanted from the midgraft versus the perianastomotic zones? The measurement of inner capsule thickness shows a greater thickness in the perianastomotic zones, although these differences do not reach statistical significance. Are the

,rOURNAL OF VASCULAR SURGERY Volume 17, Number 5

thicker tissues in these zones a reflection of greater cell numbers or greater matrix deposition; if it is greater cell numbers, do they represent a PDGF-indnced alteration in smooth muscle cell migration, proliferation, or both? Do yo u have any data as to the rate of cell division in the inner capsules, and if so do these rates correlate with the measured PDGF production? Similarly, do )rou have data as to whether the PDGF is mitogenically active? Third, recent data from the Seattle group suggest that, at least in some models of injury, smooth muscle cell basic fibroblast growth factor (FGF) production may be more relevant to the issue of cell proliferation than is PDGF rodnction. Do you have data on FGF release? Fourth, these data strongly suggest significantly greater PDGF production after implantation of Dacron compared with ePTFE grafts; however, it is not at all clear that this is a function of the composition of the graft material. Other possibilities include differences in surface texture, graft construction, porosity, and the like. Of note, however, is that this study used 6 mm internal diameter Dacron grafts compared with 8 mm internal diameter ePTFE grafts that were evaluated previously. Thus one may postulate a difference in flow characteristics with greater flow velocity through these narrower grafts. Why did you choose a different diameter graft and do you have information as to the role of these different diameters and hemodynamic characteristics on PDGF production? Fifth, the explants were placed into organ culture for a 72-hour period before the measurement of PDGF in the culture medium. The results therefore may or may not reflect the PDGF productivity seen in vivo. Of particular "~ncern is the use of EC growth supplement in culture. This growth supplement contains acidic FGF, which may up-regulate the PDGF message and thus effect the results described. Furthermore, the acute change from in vivo pulsat~le hemodynamic characteristics to static conditions of culture may also affect PDGF expression. Dr. David A. Margolin. The ability of ECs to produce PDGF is well documented, but other cell types including leukocytes and SMCs are capable of producing PDGF. The cellular source of PDGF was not identified in dais study, although indirect evidence suggests that SMCs are an important source of PDGF after the acute phase of graft healing. Identification of the cellular source of the PDGF production with in situ hybridization histochemistry and quantitation of m R N A for PDGF with polymerase chain reaction is being pursued in our laboratory. Unfortunately gene expression does not necessarily correlate with protein production. In the present study no difference was found in PDGF production by the midgraft and at the anastomoses. In a previous study of ePTFE grafts, PDGF production by anastomotic regions was significantly greater than midgraft

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segments. This difference may have been due to PDGF production by SMCs. SMCs migrated onto the graft from the artery adjacent to the anastomoses but were virtually absent in the pseudointima of the midgraft. In contrast, in Dacron grafts SMCs were abundant in the pseudointima of both the midgraft and anastomotic segments. The composition of the inner capsule in the midgraft and at the anastomoses did not seem to be significantly different with respect to cellularity or matrix deposition. The rate of cell division in these areas was not quantitated. The PDGF measured in this study was biologically active in that it was able to bind to the PDGF receptor. The conditioned medium from organ culture stimulated SMC proliferation, although the contribution of PDGF to this mitogenic activity has not been evaluated. PDGF is only one of the SMC mitogens secreted by ECs. Other investigators have reported that PDGF comprises less than half of the total mitogenic activity in the perfusates of endothelialized prosthetic grafts. ~ Other mitogens, particularly basic FGF may be important in stimulating SMC proliferation. Determination of the level of basic FGF in the conditioned medium and in cell lysates is currently underway in our laboratory. The role ofhemodynamk: factors in stimulating PDGF production is unknown. The 6 mm internal diameter Dacron grafts and 8 mm internal diameter ePTFE grafts were selected for optimal match to the native thoracic aorta to minimize mitogen production caused by hemodynamic factors. In fact the internal diameter measurements, after the grafts were distended at physiologic pressure, were equivalent for Dacron and ePTFE grafts. The effect of increased flow and anastomotic configuration on PDGF production are the topics of current studies in our laboratory. Organ culture methods were selected for this study to allow measurement of PDGF production by specific arterial or graft segments while preserving the normal structural and intercellular relationships of the vessel wall. A 72-hour incubation period in organ culture is required for secretion of sufficient quantities of PDGF to be measured with the radioreceptor assay, so the accuracy with which PDGF production in organ culture reflects that in vivo is not certain. However, care is taken to eliminate known stimulators of PDGF production from the organ culture system. In preliminary experiments with and without ECGS in tissue culture and organ culture, no significant difference in PDGF production in the presence or absence of ECGS was found. One of the limitations of an organ culture system is that organ culture is a static model, and hemodynamic factors that may affect PDGF production in vivo are removed. The effect of flow on PDGF production is a subject for future studies.