Growth factors and myointimal hyperplasia in experimental aortic allografts

Eur J Vasc Endovasc Surg 13, 66-71 (1997)

Growth Factors and Myointimal Hyperplasia in Experimental Aortic AIIografts B. Randone 1, A. V. SterpettP*, F. Stipa 1, P. ProiettP, C. Aromatario 1, M. B. GuglielmP, M. PalestinP, L. Santoro-D'Angelo 2, A. Cavallaro ~ and A. Cucina 1 1Department of Surgery and 2Department of Histology and Embryology, University of Rome, "La Sapienza", Italy Objectives: To analyse the role of growth factors (platelet derived growth factor, PDGF; basic fibroblast growth factor, bFGF; interleukin 1, IL-1) in the genesis of myointimal hyperplasia in arterial allografts. Materials: Two groups of experiments were performed: isografts and allografls. The isograft group consisted of 15 inbred Lewis rats in which a i cm long segment of aorta was inserted as an abdominal aortic interposition graft. The aortic segments were obtained from syngenic Lewis rats. The allograft group consisted of 15 inbred Lewis rats, in which a I cm long segment of aorta was interposed at the abdominal aorta level. The aortic segments were obtained from alIogenic BrownNorway rats. Chief outcome measures: The animals were killed 4 weeks after surgery and were analysed by morphometric analysis (n = 3for each group). In addition, production of PDGF, bFGF and IL-1 by aortic segments (n = 12for each group) in organ culture was assessed. Main results: Allografts had more myointimal hyperplasia, than isografts (p < 0.05). PDGF and bFGF production, generally considered to be the cause of myointimal hyperplasia, was not increased in allografts. IL-1 production was higher in allografts (p < 0.001). Main conclusions: Myointimal hyperplasia in aortic allografts is dependent on growth factors produced by the graft itself. These growth factors are different from PDGF and bFGF that generally have been implicated in the genesis of naturally occurring myointimal hyperplasia and atherosclerosis. IL-1 may have a principal role in the genesis of myointimal hyperplasia in arterial allografts. Key Words: PDGF; bFGF; IL-1; myointimal hyperplasia; atherosclerosis; aortic allografts.

Introduction The autogenous saphenous vein is the conduit of choice for small artery bypass surgery. However, it is not always available. Theoretically, arterial allografts could represent a valid alternative. The use and complications that occur in allografts were first described by Carell I and Guthrie 2 and several studies have defined the response of transplanted arterial allografts. 3~s The pathologic changes are either graft dilatation and rupture caused by media destruction or a narrowing of the graft lumen caused by myointimal cell proliferation, which may lead to arterial occlusion. The factors leading to myointimal hyperplasia in *Please address all correspondence to: Antonio V. Sterpetti, Laboratorio Castro Laurenziano-I Clinica Chirurgica, University of Rome, Via A. Scarpa, 14, 00161 Rome, Italy.

arterial allografts are unknown. Growth factors induce and regulate numerous cell functions during the process of myointimal hyperplasia and atherosclerosis. They may act in cell recruitment and migration, cell proliferation and the control of protein synthesis, including extracellular matrix proteins. Platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are two of the best characterised mitogens for endothelial cells and smooth muscle cells.9-- 11 Several reports have demonstrated increased expression of PDGF and bFGF in association with naturally occurring atherosclerosis, experimentally induced atherosclerosis and stenoses associated with failure of autologous and synthetic vascular grafts. ~2-14 The aim of this study was to determine if PDGF and bFGF were also responsible for myointimal hyperplasia occurring in arterial allografts, and if other factors were also involved.

1078-5884/97/010066+ 06 $12.00/0 © 1997W. B. Saunders Company Ltd.

Aortic AIIografts

Material and Methods

Operative procedures Fifteen Brown-Norway rats and 45 Lewis rats (average weight 250 g) were used for the experiments. These inbred strains were chosen because of their immunologic histoincompatibility. Experiments were performed in two groups of 30 rats (15 donors, 15 recipients). In the autograft, Lewis rats were both donors and recipients. In the allograft group, the donors were Brown-Norway rats and the recipients were Lewis rats. All surgery was performed under general anaesthesia with intramuscular Xylazine (3 mg/kg) and Ketamine (50mg/kg) supplemented by intraperitoneal Ketamine for maintenance. Surgery was performed with the use of an operating microscope (Zeiss OPMI-7D). A midline laparotomy was performed on the donor and the aorta was exposed from the renal arteries to the bifurcation. The exposed aortic segment was then excised and placed in Dulbecco Modified Eagle Medium (DMEM) for approximately 20 min while the abdominal aorta of the recipient was exposed and proximal and distal controls obtained. One centimetre long segments of the previously harvested graft were anastomosed with end-to-end anastomoses to the native aorta, with continuous 10-0 nylon sutures (Ethicon Inc.). Four weeks after surger~ the rats were killed and the aortas, above and below the graft, and the aortic grafts were exposed and excised. No immunosuppression was used in these experiments.

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200 ~g/ml, Streptomycin 100 ~g/ml, Penicillin 100 IU/ ml). The vessels were opened longitudinally and placed in organ cultures. The specimens were placed in 48-well Costar tissue culture plates for organ culture. The tissue was incubated for 5 days at 37°C in a 5% CO2 atmosphere. Aliquots of conditioned media were collected at 72 h and centrifuged for 5 min at 15 000 rpm and the supernatant was stored at -80°C for assay of mitogenic activity and assay for PDGF, bFGF and interleukin 1 (IL-1) production.

Assay for mitogenic activity Swiss 3T3 cells were placed in 96 well plates (Falcon Plastics) at a density of 4 × 104 cells/ml in 200 ~1 of DMEM supplemented with 0.1% foetal calf serum (FCS). At 72 h, conditioned serum-free media from aortic allo- and isografts, and aortic segments were added to Swiss 3T3 cells (20~1). Positive controls received an equivalent volume of DMEM plus human recombinant bFGF (20 ng/ml) or human recombinant PDGF (50 ng/ml) (Genzyme Co, Boston, MA, U.S.A.); negative controls received only DMEM. After 2 days, trifiated thymidine (0.5 ~t Ci per well plate) was added and the cultures were incubated for 18 h and collected on Skatron Filters (Skatron Instruments; Sterling, VA, U.S.A.) for radioactivity determination in a LKB scintillation counter.

Analysis of reduction of mitogenic activity by anti-PDGF and anti-bFGF antibody Histology (n = 3 for each group) Grafts were perfusion fixed with Hanks' solution and 10% formaldehyde. Following standard procedures, the specimens were stained with haematoxylin and eosin, giemsa and carmallum. Transverse and sagittal sections were cut for each graft and from the native aorta at different levels. Slides were analysed in a semi-automatic image processor to measure intima~ media area.

Measurement of the Swiss 3T3 cells DNA synthesisstimulating activity of the conditioned media from aortic allo- and isografts was repeated in presence of an excess of monospecific antibody to PDGF (Genzyme) and in the presence of monoclonal antibody to bFGF (produced in our laboratory). Tritiated thymidine was again added and the cultures were incubated for 18 h. After further processing the radioactivity was measured.

Assay of PDGF, bFGF and IL-1 in the conditioned media Organ culture(n= 12) The aortic iso- and allografts and the control aortic segments were rinsed thoroughly for 10 min with DMEM supplemented with antibiotics (Gentamycin

The presence of bFGF, PDGF and IL-1 in the serumfree conditioned media from aortic allo- and isografts, and aortic segments was determined by inhibition antibody-binding assay. A dilution of anti-bFGF Eur J Vasc Endovasc Surg Vol 13, January 1997

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B. Randone e t al.

monoclonal antibody that showed 50% maximal reactivity against bFGF (4~g/ml) was incubated with various dilutions of conditioned media in 400 ~1 tubes precoated with phosphate buffered saline (PBS) gelatin 2%. After 20 h of incubation at 4°C, Staphylococcus aureus protein A was added, and the immunoaggregates were removed by centrifugation. The residual antibody-binding activity in the supernatant was measured by ELISA, according to a method already described. 15 In brief, plastic wells were coated with bFGF (10ng/well) for 8 h at 4°C. Plates were then washed twice with PBS and saturated with PBSgelatin 1% for 2 h at 37°C. Washed wells were then filled with 50 ~1 per well of supernatant obtained after immunoprecipitation. After 2 h of incubation at 37°C the wells were washed with PBS gelatin 0.1%. Peroxidase labeled goat anti-mouse immunoglobulin antibody was added. After 60 rain of incubation at 37°C, the plate was washed three times in PBS gelatin 0.1% and once in distilled water. Finally, O-phenylenediamine dihydrochloride (0.4mg/ml)(Sigma, St. Louis, MO, U.S.A.) was added as substrate for the enzyme. Bound specific antibody was quantitatively measured by optical density reading at 492nm with a spectrophotometer (Beckman Instruments, Inc., Palo Alto, CA, U.S.A.). The amount of bFGF in the conditioned media was determined by using a reference curve obtained by using known quantities of human recombinant bFGF. We used mouse anti-bFGF monoclonal antibody as a positive control and antibody without specificity as a negative control. Similar experiments were used to determine the presence and amount of PDGF and IL-1 in the conditioned media, with rabbit polyclonal antibody to PDGF (Genzyme) and mouse monoclonal antibody to IL-1 (Genzyme) respectively.

hyperplasia. There were signs of rejection in aortic allografts, with diffuse cellular infiltration in the adventitia, medial thinning and intimal proliferation. The intima-media area was statistically greater in the aortic allografts (p < 0.05) (Fig. 1) (Table 1).

Mitogenic activity of the conditioned media and the effect of anti-PDGF and anti-bFGF There was a statistically higher mitogenic activity in the conditioned media from aortic allografts than in the conditioned media from isografts (Fig. 2) (p < 0.001). The addition of serum-free conditioned media from allografts produced a three-fold mean increase of tritiated thymidine uptake of Swiss 3T3 cell cultures as compared with conditioned media from isografts. This increased production of mitogens could stimulate the growth of smooth muscle cells and lead to myointimal hyperplasia and atherosclerosis. Reduction of mitogenic activity by anti-PDGF and anti-bFGF antibodies means that the mitogenic activity is due to these particular growth factors. The addition of monospecific anti-PDGF antibody to the medium of 3T3 cell cultures exposed to conditioned medium from allografts had a negligible effect on the uptake of tritiated thymidine (Fig. 3). Similarly, the addition of monospecific anti-bFGF antibody to the medium of 3T3 cell cultures exposed to conditioned medium from allografts had a negligible effect on the uptake of tritiated thymidine (Fig. 4).

PDGF, bFGF and IL-1 assay by ELISA Statistical analysis Chi-squared test, analysis of variance and Student's t-test were used where appropriate. Data were expressed as mean + S.D. Differences were considered significant at the 5% level.

Results

All grafts were patent at the time of death.

Histology Aortic isografts showed minor degree of myointimal Eur J VascEndovascSurg Vo113,January 1997

Table 2 shows the release of PDGF, bFGF and IL-1 by aortic allo- and isografts. The release of PDGF and bFGF paradoxically was increased in aortic isografts (p <0.01). IL-1 production was higher in allografts than in isografts (p < 0.001).

Discussion

Accelerated graft myointimal hyperplasia and atherosclerosis are the main factors that limit the success of cardiac and renal transplantation. 16-2° In peripheral vascular surger)5 the use of arterial allografts has shown a sequence of pathologic events Consisting in thinning of the media, dislocation of elastic laminae, invasion of the adventitia by inflammatory cells and

Aortic AIIografts

intimal hyperplasia. 7's The factors leading to intimal hyperplasia are probably secondary to an immunologic process. In our study we tried to determine if the myointimal hyperplasia seen in aortic allografts was caused by an increased production of PDGF and bFGF. M a n y studies support a role for PDGF and bFGF in the genesis of myointimal hyperplasia and atherosclerosis, Birinyi et al. 14 cultured cells from myointimal

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hyperplasia obtained from patients at the time of reexploration for lower extremity graft failure. They found that cultured smooth muscle cells from myointimal hyperplasia express genes for PDGF and PDGF receptors. Several other reports have demonstrated increased expression of PDGF and bFGF with naturally occurring myointimal hyperplasia and atherosclerosis,

(a)

(b)

Fig. 1. Light microscopyof aortic isograft (a) and allograft (b). Aortic isografts appeared normal whereas aortic allografts showed signs of rejection, with diffuse cellular infiltration in the adventitia, medial thinning and intimal proliferation. The intima-media area was statistically greater in the aortic allografts. L = Lumen; MH = myointimal hyperplasia. Eur J Vasc Endovasc Surg Vol 13, January 1997

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B. Randone et al.

Table 1. Intima-media area.

Area infima + media (~tm2 x 10s) Normal aorta Isograf~ Allograft

2.74 _+0.2 3.11 _+0.3 4.00 + 0.2

Each value represents the mean + S.D, of three separate experiments.

experimentally induced atherosclerosis, and stenoses associated with failure of v a s c u l a r grafts. 13'14'16 The principal animal model that has been used to study myointimal hyperplasia is balloon angioplasty of the normal carotid artery. Jawien et aI. 21 found that PDGF infused into rats subjected to balloon carotid injury produced a two- to three-fold increase in medial smooth muscle cell proliferation, and a 20-fold increase in migration of smooth muscle cells from the media to the intima. Administration of antibodies to PDGF resulted in a 40% reduction in area of myointimal hyperplasia. 22 Similarly, it has been shown that 16 000

14 000

administration of antibodies to bFGF significantly reduced smooth muscle cell proliferation after balloon injury.2s In our study we found that aortic allografts release a greater quantity of mitogens than aortic isografts. This phenomenon could represent the cause for the occurrence of myointimal hyperplasia and atherosclerotic changes in arterial allografts. 4 The source of these growth factors can be only speculative. Results from our and other laboratories have shown that CD4 and CD8 lymphoid cells infiltrate the adventitia and the intima of aortic allografts (S. Lepidi, unpublished data). These cells could produce many growth factors including IL-1, IL-2 and GM-CSF. From our study it is evident that PDGF and bFGF are not implicated in the genesis of myointimal hyperplasia in aortic allografts and that other growth factors are involved including IL-1. The results of our experiments demonstrate that the mechanisms leading to myointimal hyperplasia and probably atherosclerosis in aortic aUografts are different from the pathogenesis of naturally occurring

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Allograft

Thoracic aorta

DMEM

Fig. 2. The addition of serum-free conditioned media from allografts produced a three-fold mean increase of tritiated thymidine uptake of Swiss 3T3 cell cultures as compared with conditioned media from isograffs. Eur J Vasc Endovasc Surg Vol 13, January 1997

_t_ Isograft

Allograft

hrPDGF

Fig. 3, The addition of monospecific anti-PDGF antibody to the medium of 3T3 cell cultures exposed to conditioned medium from aUografts had a negligible effect on the uptake of tritiated thymidine. (B) without anti-PDGF; (71) with anti-PDGF.

Aortic AIIografts

16 000

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'~

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Isograft

Allograft

hrbFGF

Fig. 4. The addition of monospecific anti-bFGF antibody to the medium of 3T3 cell cultures exposed to conditioned medium from allografts had a negligible effect on the uptake of tritiated thymidine. ( , ) without anti-bFGF; ([~) with anti-bFGF. Table 2. Release of bFGF, PDGF and IL-1 (ng/cm2/72 h).

Isograft Allograft Thoracic aorta

bFGF

PDGF

IL-1

79 _+6 25 _+3 46 + 8

39 ± 7 15 ± 5 32 ± 8

0.3 _+0.04 1.1 + 0.13 0.17 + 0.02

Each value represents the mean ± S.D. of 12 separate experiments.

atherosclerosis. Further research is needed to identify these growth factors.

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5. IsrK FE McDONALDTO, FERGUSONM, YAMANAKAE, GORDOND. Transplant arteriosclerosis in a rat aortic model. Am ] PathoI 1992; 141: 1139-1149. 6. ALLAIRE E, GUETTIER C, BRUNEVALP~ PLISSONIER D, MICHEL JP. Cell-free arterial grafts: Morphologic characteristics of aortic isografts, allografts, and xenografts in rats, J Vasc Surg 1994; 19: 446-456. 7. PETERSONMJ, ABBOTTWM, H'DoUBLERPB et aI. Hemodynamics and aneurysm development in vascular allografts. ] Vasc Surg 1993; 18: 955-964. 8. DUMONT CE, PLISSONIER D, GUETTIER C, MICHEL JP. Effects of glutaldehyden on experimental arterial iso- and allografts. J Surg Res 1993; 54: 61-69. 9. Ross R~ RAINES EW, BOWEN-POPEDE The biology of plateletderived growth factor. Ceil 1986; 46: 115-119. 10. RIFKIN DB, MOSCATELLID. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 1989; 109: 1-6. 11. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: 801-809. 12. BARRETTTB, BENDITTEP. Sis (platelet derived growth factor b chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery. Proc Natl Acad Sci USA 1987; 84: 1099-1103. 13. LIBBYP~ WARNERSJC, SALOMONRN, BIRINYILK. Production of platelet derived growth factor-like mitogen by smooth muscle cells from human atheroma. N Engl ] Med 1988; 318: 1493-1498. 14. BiRn,~YILK, WARNER SJC, SALOMONRN et al. Observation on human muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: Production of a platelet-derived growth factor receptor - - A preliminary study. J Vasc Surg 1989; 10: 157-164. 15. STERPETTI AM, CUCINA A, SANTORO-D'ANGELO L, CARDILLO B, CAVALLAROA. Shear stress modulates the proliferation rate, protein synthesis and mitogenic activity of arterial smooth muscle cells. Surgery 1993; 113: 691~99. 16. L~u G, BUTANYJ. Morphology of graft arteriosclerosis in cardiac transplant recipients. Hum Pathol 1992; 23: 768-773. 17. ONI AA, RAY J, HOSENPUD JD. Coronary venous intimal thickening in explanted cardiac allografts. Transplantation 1992; 53: 1247-1251. 18. IP JH, FUSTERV~BADIMONL, BADIMONJ, TAUBMANMB, CHESEBRO JH. Syndromes of accelerated atherosclerosis: Role of vascular injury and smooth muscle cell proliferation. ] Am Coll Cardiol 1990; 15: 1667-1687. 19. L1BByPt SALOMONRN, PAYNEDD, SCHOENFJr POBERJS. Functions of vascular wall cells related to the development of transplantation-associated coronary arteriosclerosis. TransplantationProceedings 1989; 21: 3677-3684. 20. HRUBAN RH, BESCHORNER WE, BAUMGARTNERWA et al. Accelerated arteriosclerosis in heart transplant recipients is associated with a T-lymphocye-mediated endothelialitis. Am J Pathol 1990; 137: 871-882. 21. JAWIENK, BOWEN-POPEDF, LINDNER V~ SCHWARTZSM, CLOWES AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest 1992; 89: 507-511. 22. FERN GAA, RAINES EW, SPRUGELKH, MONTANI AS, REIDY MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991; 253: 1129-1132. 23. LINDNERV, REIDyMA. Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc NatI Sci USA 1991; 88: 3739-3743.

Accepted 4 June 1996

Eur J Vasc Endovasc Surg Vol 13, January 1997