Smooth muscle cell proliferation in response to injury in an organ culture of human saphenous vein

Eur J VascSurg 5, 5-12 (199l)

W I N N E R OF THE ESVS PRIZE 1990

Smooth Muscle Cell Proliferation in Response to Injury in an Organ Culture of Human Saphenous Vein* G. D. Angelini, ~ A. A. S o y o m b o 2 and A. C. Newby 2

DepartmenZ of lCardiac Surgery, University of Shef/field and D~artment of 2Cardiology, University of Wales College of Med&ine

The principal cause of late vein graft occlusion is intimal smooth muscle cell proliferation, the underlying basis of which remains an enigma. Early theories implicating platelet activation now appear untenable since intimaI proliferation progresses after endothelial repair, and is little influenced by ant±thrombotic treatments. We developed an organ culture of human saphenous vein to investigate the basis of intimaI proliferation in a preparation which preserved the anatomical relationships of endothelium, smooth muscle and extracellular matrix. Tissue viability remained high during culture for up to 14 days and intimaI smooth muscle proliferation occurred. The removal of endothelium reduced intimal thickening in Cultured veins from 26 + 5 to 6 ± 3 tzm and also reduced the number of intimal cells/mm labelled with [3H]-thymidine from 12 ± 4 to 3 ± 1 (both p < O.01, n = 10). Surgical preparation of vein resulted in sign~cant injury to medial smooth muscle cells, which was only partially reversed during culturing. Surgical preparation did not affect intimal proliferation, but stimulated medial proliferationfrom 3 + 1 to 32 ± 9 [3H]thyroid±he-labelled cells/ram (p < O.01, n = 11). These experiments reveal evidence for proliferation enhancing factors derived from endothelium and injured smooth muscle cells, which probably participate in intimal proliferation in vein grafts. Inhibiting their action may therefore present new possibilitiesfor therapy. Key Words: Arteriovenous bypass grafting; Endothelium; Arteriosclerosis.

Introduction

Surveys of current practice L2 indicate c o n t i n u e d w i d e s p r e a d use of autologous s a p h e n o u s vein for c o r o n a r y and peripheral bypass grafting, despite longt e r m patency rates of approximately 50% after 10 years. 3 I m p r o v i n g these patency rates is therefore a w o r t h w h i l e goal. Early occlusions result from platelet activation leading to thrombosis, and can be minimised by ant±thrombotic treatments 4'5 and by avoiding endothelial injury d u r i n g preparation of vein for arterial implantation. 6 Late occlusions result from lumenal n a r r o w i n g caused by intimal s m o o t h muscle * Presented at the 4th Annual Meeting of the European Society f o r Vascular Surgery, Rome, September 1990.

Please address all correspondence to: Mr G. D. Angel±n±,Department of Cardiac Surgery, University of Sheffield, Northern General Hospital, Sheffield, $5 7AU, U.K. 0950-821X/91/010005+08$03.00/0© 1991Grune & Stratton Ltd.

proliferation with s u p e r i m p o s e d atheroma. Intimal proliferation does not appear to be related to platelet activation, since it progresses u n d e r a morphologically intact and n o n t h r o m b o g e n i c e n d o t h e l i u m 7 and is little influenced by ant±thrombotic treatments. 8"9 O t h e r inter-related mechanisms have therefore b e e n proposed, including the influence of surgical preparative d a m a g e to e n d o t h e l i u m and s m o o t h muscle cells, increased wall stress, 1° and the p r o d u c t i o n of end o g e n o u s g r o w t h factors. 11 Previous studies of intimal proliferation in m a n have relied on limited a m o u n t s of p o s t - m o r t e m material, and have not b e e n directed at mechanisms. 12-14 Culture of isolated s m o o t h muscle cells in vitro, by contrast, has identified g r o w t h factors derived from platelets, endothelial cells, leucocytes and s m o o t h muscle cells themselves. 1~ Such isolated cell preparations can not, however, take into account the normal anatomical relationships in the vessel wall or

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G.D. Angelini et aL

the negative influence of the extracellular matrix on smooth muscle proliferation. 15 We therefore sought first to establish whether intimal proliferation could be quantified in an organ culture of human saphenous vein. To investigate the effect of tissue injury on the extent and location of smooth muscle proliferation, we then investigated the influence of endothelial removal and surgical preparation.

bonate was substituted for the 20 mM-Hepes), supplemented with 2.5~g/ml of gentamicin (David Bull Laboratory, Warwick), 100 ~g/ml of penicillin: streptomycin (Flow Labs), 0.8mM-glutamine (Flow Labs) and 30% (v/v) of foetal bovine serum (Gibco). The culture medium was changed every 2 days. The medium was also supplemented with [3H]-thymidine (1 ~Ci/ml, 15.6 Ci/mmol; NEN) as indicated in the text. The concentration of ATP and the total activity of [3H]-thymidine incorporated into DNA were measured in perchloric acid extracts of veins as previously described. 16,18 Materials and Methods To study endothelial morphology by scanning Surgical procedures electron microscopy, vein segments were fixed in 0.1 M Sorensen's phosphate buffer (pH 7.3) containSegments of saphenous vein were obtained from 65 ing 2.5% glutaraldehyde (Agar Aids) for 24-36h at patients (49 male, mean age 60 years, range 41-79 room temperature, rinsed in distilled water and years) who were undergoing a coronary artery bypass dehydrated through 30% to absolute alcohol. Dehydgraft operation. Routine premedication, anaesthesia rated specimens were critical point dried, sputter and intra-operative heparinisation were performed as coated with gold and were then examined in a Jeol described previously. 16 A segment (ca. 2.5cm) of 840A scanning electron microscope. Endothelial freshly isolated vein was taken from the lower portion coverage was also scored visually under light microof the long saphenous vein using a no touch technique scopy after staining segments with silver nitrate. 19 as soon after the first incision as possible. Segments of These segments were not used for culturing or other surgically prepared vein were obtained from vein in analyses. excess on completion of the last proximal anastomosis. For measurement of intimal thickness, vein segSurgical preparation consisted of adventitial strip- ments were fixed in 0.1 M phosphate buffer (pH 7.2), ping, side branch ligation, gentle manually-controlled containing 10% formaldehyde and paraffin sections distension and storage in patients' heparinised blood (5 ~m), and were stained with Miller's elastic and van at room temperature (23°C). Gieson's stain. 2° Intimal thickness was measured with a calibrated graticule (Agar Aids) at three points on each of three serial sections per vein. For quantification of cell proliferation by autoraPreparation and tissue culture of vein segments diography, serial unstained sections were deparaffinised at 60°C for 30 rain, rehydrated through alcohol to Segments of vein were transported to the laboratory at water, coated with K2 nuclear emulsion (Ilford, Chesroom temperature (23°C) in sterile RPMI 1640 tissue hire) and exposed for 2 weeks at 4°C. Sections were culture medium, containing 20mM-Hepes buffer then developed and fixed, washed in distilled water, (Flow Labs), 4 IU/ml of sodium heparin (CP Pharma- post-stained with Harris' haematoxylin and eosin 2° ceuticals, Wrexham), 0.2 mg/ml of papaverine hydro- and dehydrated through alcohols to xylene. The total chloride (McCarthy Medical, Essex), and 5 ~g/ml of number of intimal and medial cells labelled with more amphotericin B (Flow Labs). Vein was processed and than ca. 20 silver grains was counted in three sections cultured by a modification of the method of Pederson from each vein and related to the length of intimal and Bowyer 17 as described in detail elsewhere. 18 surface, which was measured with a calibrated gratiBriefly, excess fat and adventitial tissue was removed cule, as described above. and the vein was opened along its upper aspect, pinned out, endothelial surface uppermost, and divided into I cm lengths. In one series of experiments one of a pair of replicate segments of freshly isolated Results vein from the same patient was subjected to removal of the endothelium by gentle abrasion of the intimal Culture offreshly-isolated vein surface with moist, sterile cotton wool. Vein segments were cultured at 37°C under 5% (v/v) CO2 in air in 6 ml Our experimental strategy was first to determine of RPMI 1640 medium (in which 2 g/1 of sodium bicar- whether the tissue remained viable during processing Eur J VascSurg Vol 5, February1991

Organ Culture of Human Saphenous Vein

and culture. Scanning electron microscopy showed t h a t e n d o t h e l i a l cells w e r e l a r g e l y u n d a m a g e d in t h e c e n t r a l p o r t i o n of f r e s h l y i s o l a t e d v e i n s p r e p a r e d for c u l t u r e (Fig. l a ) . N e a r t h e c u t e d g e s of s e g m e n t s , endothelial damage and loss did occur (not shown). L i g h t m i c r o s c o p y of s i l v e r - s t a i n e d s e g m e n t s s h o w e d t h a t 70% ( r a n g e 6 0 - 8 0 , n = 10) of t h e t o t a l s u r f a c e w a s c o v e r e d b y e n d o t h e l i a l cells. S c a n n i n g e l e c t r o n m i c r o g r a p h s of s e g m e n t s of f r e s h l y - i s o l a t e d v e i n t h a t h a d b e e n c u l t u r e d for 14 d a y s s h o w e d t h a t cells in t h e c e n t r a l r e g i o n w e r e s i m i l a r a l t h o u g h less f l a t t e n e d t h a n b e f o r e c u l t u r i n g (Fig. l b ) . C l o s e to t h e c u t e d g e s o f s e g m e n t s , cells a p p e a r e d l a r g e r , s o m e a p p e a r e d l e s s c u b o i d , a n d h a d f i l a m e n t o u s p r o j e c t i o n s (cf. L i n Fig. lf), w h i c h h a v e p r e v i o u s l Y b e e n r e f e r r e d to a s l a m e l l i p o d i a . 21 S t a i n i n g of t r a n s v e r s e s e c t i o n s w i t h U l e x e u r o p a e u s lectin c o n f i r m e d t h a t t h e m a j o r i t y o f t h e i n t i m a l s u r f a c e cells w e r e e n d o t h e l i u m ( r e s u l t s n o t shown). Endothelial coverage was estimated in silvers t a i n e d s e g m e n t s to b e 80% ( r a n g e 7 0 - 9 5 , n = 10) of t h e t o t a l s u r f a c e . M e a s u r e m e n t s of A T P c o n c e n t r a t i o n w e r e p e r f o r m e d to i n v e s t i g a t e t h e v i a b i l i t y of t h e p r e d o m i n a n t v a s c u l a r s m o o t h m u s c l e cells. 22 T h e A T P c o n c e n t r a t i o n i n s e g m e n t s of f r e s h l y - i s o l a t e d v e i n s h a d d e c l i n e d b y ca. 20% a f t e r 14 d a y s in c u l t u r e (Table 1). T h e s e m e a s u r e m e n t s e s t a b l i s h e d t h a t a h i g h d e g r e e of e n d o t h e l i a l a n d s m o o t h m u s c l e cell v i a b i l i t y was maintained during preparation and culturing. Table 1. Effect of culturing, endothelial removal and surgical preparation on medial integrity measured by ATP concentration

ATP concentration (nmol/g wet weight) Days in culture

0

14

Freshly-isolated vein

280 _+20 (20)

220 + 20 (20)*

Endothelial removal

210 ± 50 (10)

210 + 20 (10)*

Surgical preparation

130 + 20 (20)

150 + 20 (20)*f

* p < 0.05 vs. freshly-isolated vein on day 0. f p < 0.05 vs. freshly-isolated vein on day 14. Paired segments of freshly-isolated vein, one of which was subjected to endothelial removal, or freshly-isolated vein and surgically-prepared vein from the same patients were either frozen in liquid N2 immediately, or after culturing for 14 days. ATP concentration was measured in trichloroacetic acid extracts of pulverized frozen tissue. Values are means _+SEM for the number of experiments shown in parenthesis. Cell p r o l i f e r a t i o n in c u l t u r e d s e g m e n t s w a s d e m o n s t r a t e d b y i n c o r p o r a t i o n of [ 3 H ] - t h y m i d i n e (Table 2). T r a n s v e r s e s e c t i o n s of c u l t u r e d v e i n s s h o w e d t h e d e v e l o p m e n t of n e w i n t i m a (Fig. 2a, cf. Fig. 2b). I m m u n o c y t o c h e m i s t r y w i t h m o n o c l o n a l a n t i b o d i e s to s m o o t h m u s c l e a c t i n c o n f i r m e d t h a t t h e n e w i n t i m a c o m p r i s e d s e v e r a l l a y e r s of s m o o t h m u s c l e cells

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( r e s u l t s n o t s h o w n ) . T h e t h i c k n e s s of t h e i n t i m a a n d t h e n u m b e r of t h y m i d i n e - l a b e l l e d cells p e r m i l l i m e t r e of i n t i m a l l e n g t h a r e s h o w n for t w o d i f f e r e n t s e r i e s of e x p e r i m e n t s in T a b l e s 2 a n d 3. B o t h p a r a m e t e r s w e r e Table 2. Effect of endothelial removal on cell proliferation after 14 days in culture

Freshly-isolated

Minus endothelium

3500 + 400 (10)

2400 + 300 (10)*

Intimal thickness (~m)

26 + 5 (10)

6 + 3 (10)*

Intimal [3H]-thylabelled nuclei/ram of section length

12 + 4 (10)

3 _+1 (10)*

Medial [3H]-thylabelled nuclei/ram of section length

0.4 + 0.1 (10)

2 _+1 (9)

Total [3H]-thy incorporation (dpm/ mg wet weight)

* p K 0.01 vs. freshly-isolated vein. Paired segments of freshly-isolated vein from the same patient, one of which had been subjected to endothelial removal by gentle abrasion, were cultured for 14 days. The medium was supplemented with [3H]-thymidine for two periods of 48 hours on days 8 to 10 and 12 to 14. Total [3H]-thymidine incorporation was measured in trichloroacetic acid extracts of frozen pulverised tissue. Other parameters were measured in 5 btm paraffin sections as described in the Materials and Methods section. Values are means_+ SEM. Background levels of [3H]-thymidine incorporation were determined by culturing freshly-isolated veins for 24 h only. The values obtained were 270 + 60 dpm/mg wet weight (n = 12). Intimal thickness was less than 1 #,m, and there were no [3H]-thymidine-labelled cells in the intimal or medial layers of such cultured veins. Table 3. Effect of surgical preparation on cell proliferation after 14 days in culture

Total [3H]-thy incorporation (dpm/ mg wet weight)

Freshly-isolated

Surgically-prepared

3000 + 400 (10)

7900 + 1200 (10)*

intimal thickness (b~m)

22 _+5 (11)

23 + 4 (9)

Intimal [3H]-thylabelled nuclei/mm of section length

13 + 4 (11)

11 + 2 (9)

Medial [3H]-thylabelled nuclei/ram of section length

3 + 1 (11)

32 + 9 (9)*

* p ~ 0.01 vs. freshly-isolated. Segments of freshly-isolated and surgically prepared vein, from the same patients, were cultured for 14 days in medium supplemented throughout with [3H]-thymidine. The other experimental details are given in the legend to Table 2. Background levels of [3H]-thymidine incorporation were determined by culturing fleshly-isolated, and surgically-prepared veins for 24 h only. The values obtained were 270 + 60 dpm/mg wet weight (n = 12) and 110 _+10 (n = 11), respectively. Intimal thickness was less than 1 p,m, and there were no [ H]thymidine-labelled cells in the intimal or medial layers of such cultured veins. Eur J Vasc Surg Vol 5, February 1991

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G.D. Angelini et aL

Fig. 1. Scanning electron micrographs of the intimal surface of freshly-isolated, de-endothelialised and surgically-prepared veins before and after culturing. (a) Typical appearance of freshly-isolated vein before culture. (b) Part of the central portion of a freshly-isolated vein after 14 days in culture. Note the slight separation of some cells. (c) Part of the surface of a de-endotheliatised vein before culture. Note the smooth basement membrane (BM), which typically covered most of the intimal surface. Note also the filamentous internal elastic lamina (IEL), which was exposed in isolated areas such as the one s h o w n here. (d) Typical appearance of de-endothelialised vein cultured for 14 days. Note the absence of cuboid, endothelial cells, but the presence of a fibroblast-like (probably smooth muscle) cell on the intimal surface. (e) Frequently-observed morphology of surgically-prepared vein before culture. Note the rolled-up appearance of endothelial cells with exposure of smooth basement membrane and deeper filamentous structures. (f) Typical appearance of surgically-prepared vein after 14 days in culture. Note the presence of cuboid cells with fine projections, which have been referred to as lamellipodia (L). Initial magnification x 1000. Eur J Vasc Surg Vol 5, February 1991

Organ Culture of Human Saphenous Vein

highly reproducible. Proliferating cells in cultured, freshly-isolated vein were found almost exclusively in the new intima (Fig. 2b and Tables 2 and 3). Many fewer labelled cells were found in the medial layer, even close to the cut edges or near the adventitial surface of the segments (not shown). These observations established that intimal smooth muscle cell proliferation occurs and can be quantified in organ cultures of human saphenous vein.

Effect of endothelial removal Scanning electron microscopy showed that gentle abrasion of the intimal surface of freshly-isolated veins removed the endothelium. This exposed a smooth subendothelial basement membrane (BM) over most of the surface (right-hand side of Fig. lc), although the filamentous internal elastic lamina (IEL) was exposed in some isolated areas (left-hand side of Fig. lc). Endothelial coverage in silver-stained preparations was reduced to ca. 10% (range 0-20, n = 5). Scanning electron microscopy showed that endothelium was not present in cultured de-endothelialised vein, although there were occasional fibroblast-like cells on the intimal surface (Fig. ld). Silver-stained preparations (n = 5) also lacked endothelium. Removal of endothelium did not significantly affect ATP concentration either before or after culturing, showing that the medial smooth muscle cells were not grossly injured by intimal abrasion. Removal of endothelium greatly attenuated intimal proliferation, both in terms of total thymidine incorporation (Table 2) and, more dramatically, in terms of the number of intimal cells labelled with [3H]thymidine (Fig. 2c, Table 2). The low level of medial cell proliferation was not affected by endothelial removal (Table 2). These results suggest that the presence of endothelium normally promotes intimal proliferation.

Effect of surgical preparation Scanning electron microscopy showed that routine surgical preparation, involving gentle unlimited pressure distension, resulted in patchy damage to endothelium (Fig. le) distributed widely over the intimai surface. Endothelial coverage in silver-stained preparations was also patchy and appeared to be reduced to 50% (range 30-70, n = 10) by surgical preparation. After culturing for 14 days, the infimal surface

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appeared more uniform in scanning electron micrographs (not shown). Individual endothelial cells appeared slightly larger in cultured, surgicallyprepared vein than in cultured, freshly-isolated vein (Fig. lf, cf. Fig. lb), and cells with prominent lamellipodia (L) were more common. Endothelial coverage in silver-stained preparations appeared greater than 65% (range 50-80, n = 10) in surgically-prepared veins after rather than before culturing. ATP concentration was significantly reduced by surgical preparation (Table 1), and remained lower after culturing than in freshlyisolated or cultured, freshly-isolated veins (Table 1). Thus, surgical preparation led to endothelial and smooth muscle cell injury, which was not fully reversed during culture. Surgical preparation significantly increased cell proliferation in terms of total [3H]-thymidine incorporation (Table 3). This resulted from a stimulation of medial cell proliferation (Fig. 2d, Table 3). Neither intimal thickness nor the number of intimal cells labelled with [3H]-thymidine was significantly altered by surgical preparation (Table 3).

Discussion

Our experiments demonstrate that human saphenous vein maintained in organ culture" for up to 14 days retains a high degree of cell viability. Most interestingly, intimally-directed proliferation of vascular smooth muscle occurs, as it does in grafted vein. Several possible mechanisms might underlie this selective intimal proliferation. A concentration of growth factors or nutrients is an unlikely possibility because the cultures were totally immersed in a high concentration of serum-derived mitogens and remained viable. Moreover, such a gradient should have produced zones of proliferation at the cut edges and on the adventitial surface, neither of which occurred. An intrinsic difference in the responsiveness to the serum of the most intimal smooth muscle cell layers is another possibility, but this cannot explain the absence of proliferation in de-endothelialised vein. The same observation argues against the possibility that the suppressive effect of the extracellular matrix or cell to cell contacts was least in the most intimal cell layers. Our observations suggest instead that an additional endothelium-derived proliferation enhancing factor is required to initiate proliferation of smooth muscle cells in their normal anatomic location, even when exposed to a high concentration of serumderived mitogens. The intimal localisation of proliferation would then be readily explained by a concenEur J VascSurg Vol5, February1991

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G.D. Angelini et al. a

C

~

d

I

Fig. 2. Autoradiograms of transverse sections of cultured, freshly-isolated de-endothelialised and surgically-prepared veins. Transverse sections (5 p~m)were subjected to autoradiography and counterstained with haematoxylin and eosin. Photographs show the intima and the intimal third of the medial layer (initial magnification × i12). (a) Freshly-isolated vein cultured for 1 day only in the presence of [3H]thymidine. Note the presence of counterstained nuclei and the background of individual silver grains but the absence of heavily labelled nuclei. (b) Freshly-isolated vein cultured for 14 days in the presence of [3H]-thymidine. Note the presence of a new intima with many counterstained nuclei and some heavily [3H]-thymidineqabelled cells. (c) De-endothelialised vein cultured for 14 days with [3HJ-thymidine present from days 8 to 10 and 12 to 14. Note the much less prominent neointima, which nevertheless contains some heavily [3H]-thymidinelabelled cells (arrows). (d) Surgically-prepared vein cultured for 14 days in the presence of [3H]-thymidine. Note the prominent neointima with heavily [3H]-thymidine-labelled cells similar to (b). There are also [3H]-thymidine-labelled cells in the medial layer (arrows). Such cells were observed more frecluentlv in the middle- and outer-thirds of the medial laver (not shown).

Organ Culture of Human Saphenous Vein

tration gradient of this factor. Surgical preparation also resulted in some injury to endothelium but sufficient endothelium apparently remained to sustain intimal proliferation. Since the removal of endothelium did not stimulate medial proliferation, injury to the medial layer must have been the component of surgical preparative damage that was responsible for medial cell proliferation. This observation reveals the existence of a second proliferation enhancing factor derived from injured vascular smooth muscle ceils. Intimal proliferation has been previously detected in organ cultures of human, pig and rat aorta. 23-25 In the pig aorta, Koo and Gotlieb 26 demonstrated that intimal proliferation is also attenuated by removal of endothelium. They 26 showed, moreover, that conditioned medium from cultured, endothelium-intact aorta can restore intimal proliferation to de-endothelialised segments. This work and ours, provide strong evidence for an endothelium-derived proliferation enhancing factor, the nature of which must now be elucidated. Culture of rabbit aorta 17"27 and human internal mammary artery (Holt C, Soyombo AA, N e w b y AC, Angelini GD, unpublished observations), under the same experimental conditions used here, produces a lower rate of intimal thickening. This difference in behaviour does not correlate with the presence or absence of pre-existing neointimal cells and so must result from other unknown differences in structure or regulatory properties. This difference is clearly worth further study because it may explain the much lower incidence of intimal proliferation in internal mammary artery rather than saphenous vein grafts. 3 Interestingly, Fingerle and Kraft 27 observed that deliberate mechanical injury to the medial cell layer of rabbit aorta stimulated smooth muscle cell proliferation, consistent with our data on incidental surgical preparative damage. There is also evidence from in vivo studies of mechanically-induced endothelial removal, 28 that simultaneous injury to the medial layer is necessary to initiate intimal proliferation, even in the presence of persistent platelet adhesion and therefore release of platelet-derived mitogens. These data and our own, therefore, provide circumstantial evidence for the existence of a second proliferation enhancing factor derived from injured smooth muscle cells. It is unlikely that these factors are conventional growth factors related to those already known to be released by endothelium and smooth muscle cells 11 because they are active over and above a high concentration of serum. Our current working hypothesis is that the factors may be chemoattractants, which draw smooth muscle cells into the subendothelium or into areas of cell loss. Alternatively, they may free cells from the

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growth suppression exerted by an intact extracellular matrix. Altered endothelial function, rather than denudation, is probably responsible for progressive intimal proliferation in vein grafts. 7 Given the evidence that mitogens alone are insufficient to initiate intimal proliferation, a role for endothelium-derived proliferation enhancing factors, such as that revealed by our experiments is likely. Vein graft medial and intimal proliferation are also associated with increased wall stress, 1° and excessive stretch is known to be the cause of medial injury during surgical preparation. 22 Exposure to arterial pressure may therefore result in medial injury, in addition to that caused by loss of vasa vasora. There is indeed ample evidence for medial cell necrosis in grafted v e i n , 7"12-14 and the conditions for the production of the smooth muscle cell-derived proliferation enhancing factor are therefore met. Direct confirmation of the role of the two factors must nevertheless await their chemical characterisation. Most studies concur that intra-operative factors such as distension pressure or cardioplegia have little effect on subsequent intimal proliferation, 7'29-31 although they may influence lipoprotein accumulation. 3°'3l Thus, hopes of improving long-term patency rates of bypass grafts must rely largely on postoperative treatments. Since none of the regimes in current use reduces intimal proliferation, 7 new approaches are required, such as preventing the action of the proliferation enhancing factors described here.

Acknowledgements This work was supported by grants from the British Heart Foundation and the Heart Research Fund for Wales. We thank Mrs A. Williams for excellent technical assistance.

References 1 LEFRAKEA. The internal mammary artery bypass graft: praise versus practice. Texas H lnst J 1987; 14: 139-143. 2 ANGELINIGD, BRYANAI, WESTRR, NEWBYAC,BRECKENRIDGE1M. Coronary artery bypass surgery: current practice in the United Kingdom. Thorax 1989; 44: 721-724. 3 GRONDINCM. Late results of coronary artery grafting. Is there a flag on the field? J Thorac Cardiovasc Surg 1984; 87: 161-166. 4 VERSTRAETEM, BROWN BG, CHESEBROJH, et al. Evaluation of antiplatelet agents in the prevention of aorto-coronary bypass occlusion. Eur Heart [ 1986; 7: 4-13. 5 GOLDMANS, COPELANDl, MORITZT, et al. Improvement in early saphenous vein graft patency after coronary artery bypass surgery with antiplatelet therapy: Results of a veterans administration cooperative study. Circulation 1988; 77: 1324-1332. Eur J Vasc Surg Vol 5, February 1991

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