Eur J Vasc Surg 6, 354-361 (1992)
Endothelial Cell Injury Secondary to Surgical Preparation of Reversed and In situ Saphenous Vein Bypass Grafts R. D. Sayers 1, P. A. C. Watt 2, S. Muller 3, P. R. F. BelP and H. Thurston 2 Departments of ~Surgery, 2Medicine and 3pathology, Clinical Sciences Building, Leicester Royal Infirmary, P. 0. Box 65, Leicester, LE2 7LX, U.K. Failure of infra-inguinal vein grafts appears to be due to the development of intrinsic lesions (intimal hyperplasia, fibrous stenoses) within the graft which lead to narrowing of the h~men, poor blood flow and thrombosis. The cause of Nese lesions remains unknown but recently it has been suggested that endothelh~l injury might be an aetiologicalfactor. The damage that can occur after preparation of reversed vein grafts includes loss of endothelial cells and functional impairment of those cells that remain, in that the ability to produce prostacyclin and endothelium-derived relaxing factor (EDRF) is reduced. The preparation of in situ grafts is different to that for reversed grafts in that a valvulotome is passed along the lumen of the vein to destroy the valves. However, little is known about the degree of endothelial injury that this technique causes. Vein samples were obtained from patients undergoing infra-inguinal and coronary artery bypass grafting. The veins were mounted in an organ bath system to measure isometric tension and exposed to the endothelium-dependent agents acetylcholine, bradykinin, adenosine, histamine and the endothelium-independent agent sodium nitroprusside. The results indicate that preparation of reversed vein grafts leads to some loss of endothdiaI cells with functionaI impairment of the cells that remain. However, preparation of in situ grafts leads to severe or total loss of endotheliaI cells which resulted in an absence of detectable EDRF release. Thesefindings were confirmed by histological examination of the vein samples. Key Words: Endothelium; Injury; Saphenous vein; Reversed vein graft; In-situ vein graft; Endothelium-derived relaxing factor; Valvulotome.
Introduction In infra-inguinal bypass procedures, the patient's o w n long s a p h e n o u s vein is the conduit of choice, with patency rates that are superior to prosthetic alternatives. 1 For an above-knee femoro-popliteal bypass, the vein is reversed so that the valves do not obstruct the flow of blood, whereas, in femoro-distal bypass to the below knee popliteal artery or single calf vessels, the in situ m e t h o d is often preferred. 2 This technique uses the vein non-reversed, to allow a better size match b e t w e e n artery and vein graft at the distal anastomosis, and involves passage of a valvulotome along the vein to r e n d e r the valves incompetent. 3 Failure of infra-inguinal vein grafts remains a serious problem which m a y result in loss of the limb. 4 Please address all correspondence to: R. D. Sayers, Department of Surgery, Clinical Sciences Building, Leicester Royal Infirmary, P.O. Box 65, Leicester, LE2 7LX, U.K. 0950-821X/92/040354+08$03.00/0© 1992Grune & Stratton Ltd.
One of the major causes of failure is the d e v e l o p m e n t of intrinsic lesions within the graft, particularly d u r i n g the first postoperative year, which lead to narr o w i n g of the lumen, poor blood flow and graft thrombosis. 5 Recent interest has focused on graft surveillance p r o g r a m m e s to detect these lesions 6'7 and allow their correction, by p e r c u t a n e o u s transluminal angioplasty or revisional surgery, before graft failure occurs. The two major types of intrinsic lesions that m a y develop in vein grafts are short isolated graft stenoses 5'8 or longer areas of intimal hyperplasia. 5 Their aetiology remains u n k n o w n and it is not clear w h e t h e r they r e p r e s e n t similar or different pathological processes. The changes that occur in reversed vein grafts d u r i n g and after surgical preparation have been well d o c u m e n t e d . 9-11 It has b e e n s h o w n that surgical preparation causes both structural and functional endothelial cell damage. There is a considerable loss of endothelial cells 9-11 and the remaining ones are func-
Endothelial Cell Injury
355
tionally impaired with reduced production of prosta- of side-branches and uncontrolled distension with cyclin12 and endothelium-derived relaxing factor saline. The third group (in situ) were obtained after (EDRF). 13 preparation for in situ vein grafting which included Endothelium-derived relaxing factor is released exposure and mobilisation of the vein, ligation of side from endothelial cells and acts on the underlying branches and the passage of a valvulotome (9 vessels, smooth muscle cells to produce relaxation via the cyc- 7 patients). A 2.5-ram Hall valvulotome was used for lic GMP pathway. 14 Functional injury or loss of endo- each in situ graft and it was passed as atraumatically thelial cells impairs this response so that it may be as possible. All samples of vein for each group were used as an assessment of endothelial damage. 13 taken from the calf region to ensure that they were Endothelium-independent vasodilators act directly matched for site and size. In the in situ group the vein on the smooth muscle and are used to confirm that was obtained from the distal end of the graft. the smooth muscle cells are able to relax despite enThe veins were transported from theatre to the laboratory in cold (4°C) calcium-free physiological salt dothelial damage. Recently a link has been suggested between en- solution in a sealed container. In the laboratory the dothelial injury caused by surgical preparation of vein was carefully cleaned of fat and adventitia and a reversed vein grafts and the development of intimal ring of vein 5 mm in length was cut open to form a hyperplasia using an in vitro organ culture technique rectangular strip. All dissection was performed under with human saphenous vein. is a dissecting microscope with the vein immersed in However, little is known about endothelial calcium-free physiological salt solution. One end of damage after preparation of in situ vein grafts. We the vein was attached to a stainless steel plate and the have therefore studied veins prepared for both in situ other to the force transducer to measure isometric and reversed bypass grafting in order to determine tension. The stainless steel plate and vein were the degree of structural and functional endothelial placed in an organ bath (Fig. 1) containing physiocell damage that occurs with each technique. logical salt solution (composition: NaC1 118mM; NaHCO3 25mM; KC1 4.5mM; KH2PO4 lmM; CaCI2 2.5mM; MgSO4.7H20 lmM; glucose 6mM) at 37°C and gassed with a 5% carbon dioxide/95% oxygen mixture to achieve a pH of 7.45.16 Methods The initial study was on 28 samples of saphenous vein which were obtained from 23 patients. These patients underwent either infra-inguinal or coronary artery bypass grafting. The patients were neither diabetic nor hypertensive and their mean ages and blood pressures are shown in Table 1. Table 1. A g e and m e a n b l o o d p r e s s u r e of p a t i e n t s in the control, reversed and in situ g r o u p s of p a t i e n t s e x p r e s s e d as m e a n + S.E.
Age (years)
Blood pressure (mmHg)
Control
73 + 4
109 + 3
Reversed
63 + 3
107 + 5
In situ
71 + 2
109 + 4
~ ] ~
Organ c h a m b e r ~ o o o ,
Strip of vessel/
l
Y
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i °
I
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Stainlesssteel plate
Fig. 1. Diagrammatic illustration of a vein mounted on a stainless steel plate in a heated gassed organ bath. Three groups of veins were studied which had been surgically handled in different ways. The first group (control) were obtained after minimal surgical dissection using a minimal-touch technique (10 vessels, 8 patients). The second group (reversed) were samples taken after the veins had been fully prepared for reversed bypass grafting (9 vessels, 8 patients). This included full exposure and mobilisation, ligation
After an equilibrium period of i h each vein was stretched and the optimum passive tension that would produce the largest contraction to noradrenaline (10 -7M) was determined. A further equilibrium period of I h was allowed to elapse before a cumulative dose response curve to noradrenaline (10-8M to 10-5M) was performed in the presence of cocaine Eur J VascSurgVol6, July1992
356
R.D. Sayers et al.
(10-6M). Following this, each vein was sub-maximally contracted with noradrenaline and relaxation studies were performed using the endotheliumdependent vasodilators acetylcholine (10-SM to 10-5M), bradykinin ( 1 0 - 9 M to 1 0 - 6 M ) , adenosine diphosphate ( 1 0 - 8 M to 10 -5 M), h i s t a m i n e ( 1 0 - 9 M to 10-5M) and the endothelium-independent vasodilator sodium nitroprusside (10-9 M to 10 - 5 m). Finally, the veins were fixed with 2% buffered formaldehyde and processed for histological assessment.
to noradrenaline, but the in situ group of veins failed to re-contract to a second exposure. This prevented the study of relaxation in the in situ group because the veins are required to be sub-maximally contracted before exposure to a relaxing agent. However, endothelium-dependent and independent relaxation responses were studied in the control and reversed groups. The relaxation produced by acetylcholine, bradykinin, adenosine, histamine and sodium nitroprusside are shown in Figures 2-6. The -10
Preparation of tissue for histological assessment Paraffin sections (4 txm) of veins were prepared and stained with haematoxylin and eosin (H&E) and elastin Van Gieson (EVG). In addition, the presence of endothelial cells was assessed with a novel monoclonal antibody to human endothelium, Q Bend 10 (Unipath, Norse Road, Bedford, MK41 0QG, U.K.) using the labelled avidin biotin immunoperoxidase technique with the primary antibody being used at a dilution of 1:50. The degree of endothelial cell loss and smooth muscle damage was assessed by a pathologist who had no prior knowledge of the group origin of the vein samples. Seven vein samples from each group were examined. A simple semi-quantitative scoring system was employed with the degree of endothelial damage assessed as negligible (0), minimal/patchy cell loss (1), and severe/total cell loss (2).
O
~
n,-
25
50
I 10-8
I 10-7
I i0 -6
I 10-5
Acefylcholine (M)
Fig. 2. Relaxation response of the control (O), reversed (A) and in situ (rq) groups of veins to acetylcholine.
Statistical analysis The relaxation responses are reported as a percentage of the initial sub-maximal contractile response to noradrenaline. Significant differences were determined by non-parametric analysis with the Mann-Whitney U-test accepting p < 0.05 as being significant.
"-7-
25
O
Results
The optimum passive tension that produced the maximum contractile response to noradrenaline was the same for the control and reversed groups but reduced in the in situ group (2.50 + 0.27g, 2.50 + 0.27g and 1.5 + 0.55g, respectively). It was found that the control and reversed groups of veins produced repeated contractions of the same magnitude Eur J Vasc Surg Vol 6, July 1992
50
J i0 -9
10-8
I 10-7
iO-S
Brodykinin (M)
Fig. 3. Relaxation response of the control (O) and reversed (A) groups of veins to bradykinin.
Endothelial
Cell
Injury
357
\\\
25
,I
04
P, 25
o
5C
g~
n-
75
50
I iO -8
I iO-7
I iO-S
I iO-5
I00
I i0-9
I (o-e
I
iO-7
\ 1(3-6
iO-5
Sodium nitroprusside(M)
Adenosine (M)
Fig. 4. Relaxation response of the control (O) and reversed (A) groups of veins to adenosine.
Fig. 6. Relaxation response of the control (O) and reversed (A) groups of veins to sodium nitroprusside.
-I0 0 x\\
25
Table 2. Maximum relaxation responses produced by the control and reversed groups of veins to acetylcholine (ACH), bradykinin (BK), adenosine (ADEN), histamine (HIST) and sodium nitroprusside (SNP)
\
\\\
\\
5(?
\
\
\
\
75
{00
i
10-9
1
10-8
I
10-7
I
i0-6
Control
Reversed
ACH (%)
10 _+6
6 _+5
BK (%)
29 _+9
17 _+9
ADEN (%)
17 _+8
4 _+3
HIST (%)
38 + 8
62 + 15
SNP (%)
100 + 0
100 _+0
I
10-5
Histamine (M) Fig. 5. Relaxation response of the control (O) and reversed (A)
groups of veins to histamine. m a x i m u m relaxation r e s p o n s e s to these agents are s h o w n in Table 2. There w a s no significant difference b e t w e e n the control a n d r e v e r s e d g r o u p s in their responses to a n y of the relaxing agents u s e d (p > 0.05, M a n n - W h i t n e y U-test). In order to p e r m i t relaxation studies to be perf o r m e d in the in situ group, a second series of veins which h a d b e e n p r e p a r e d for use as in situ grafts w e r e obtained f r o m patients u n d e r g o i n g femoro-distal b y p a s s p r o c e d u r e s (5 patients, 8 vessels). T h e s e
patients w e r e n o r m o t e n s i v e ( m e a n blood p r e s s u r e 106 _+ 2 m m H g ) a n d not diabetic w i t h a m e a n age of 67 + 4 years. The,veins w e r e collected, p r e p a r e d a n d m o u n t e d in the o r g a n b a t h as described previously. The passive tension w h i c h w o u l d give the best contractile r e s p o n s e w a s not d e t e r m i n e d for these veins as this w o u l d involve r e p e a t e d e x p o s u r e s to noradrenaline. Instead, the passive tension w a s preset to the p r e v i o u s l y o b s e r v e d m e a n resting tension of 1.5 g in the first in situ g r o u p of veins. The veins w e r e t h e n s u b - m a x i m a l l y contracted w i t h a single b o l u s injection of n o r a d r e n a l i n e a n d a c u m u l a t i v e relaxation curve p e r f o r m e d w i t h acetylcholine. T h e s e vessels did not p r o d u c e a n y e n d o t h e l i u m - d e p e n d e n t relaxation r e s p o n s e to acetylcholine (Fig. 2). Eur J Vasc Surg Vol 6, July 1992
358
R.D. Sayers et al.
Table 3. Histological scoring assessment of endothelial injury
Total
Control (n = 7)
Reversed(n = 7)
0
1
2
1
1
1
1
1
2
0
1
2
0
2
2
0
!
1
0
1
2
2
8
12
In situ
(n = 7)
Histological assessment
The injury scores for each of the three groups of veins are shown in Table 3. The control veins showed minimal endothelial cell loss and therefore produced low "injury scores" (Fig. 7). Assessment of veins from the reversed group showed patchy endothelial cell loss (Fig. 8) and the veins from the in s i t u group showed severe or complete endothelial cell loss (Fig. 9). This was reflected in the higher injury scores for the reversed and in s i t u groups. The anti-endothelial monoclonal Q Bend 10 confirmed the presence of endothelial cells in the control (Fig. 10) and reversed groups with absence or near total loss in the in s i t u group (Fig. 11).
Discussion
It has been well documented that surgical preparation of reversed vein grafts leads to loss of endothelial cells 9-11 and that the remaining cells are functionally impaired with a reduction of their ability to produce prostacyclin 12 and EDRF. 13 Endothelial injury associated with the in s i t u technique of vein grafting has not been extensively investigated, although one study has suggested that this technique produces superior endothelial cell preservation. 17 Recently a link has been suggested between endothelial and smooth muscle cell injury during preparation of reversed vein grafts and the subsequent development of intimal hyperplasia, is It is possible that a similar process may be important in the development of lesions after in s i t u grafting and therefore the degree of endothelial cell injury associated with this technique requires further study. Our results support the view that surgical preparation of reversed grafts leads to endothelial cell damage.9-11,13 These veins exhibited reduced relaxation responses to all of the endothelium-dependent agents when compared with controls although these differences did not reach statistical significance. The ability of the smooth muscle cells of these veins to fully relax was confirmed by their response to the endothelium-independent agent sodium nitroprusside, indicating that the final pathway was intact. In addition histological examination of veins from the reversed group showed a degree of loss of the endothelial cell layer when compared to controls.
Fig. 7. Histologyof a vein from the control group showing intact endothelium. Eur J VascSurg Vol 6, July1992
Endothelial Cell Injury
359
Fig. 8, Histology of a vein from the reversed group showing patchy loss of endothelium.
Fig. 9. Histology of a vein from the in situ group showing complete loss of endothelium,
Fig. 10 Histology of a vein from the control group showing endothelial cells stained with Q Bend 10. Eur J Vasc Surg Vol 6, July 1992
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R.D. Sayers et al.
Fig. 11. Histology of a vein from the in situ group stained with Q Bend 10 showing absence of endothelial cells.
The in situ group of veins all showed severe endothelial cell injury. These veins did not produce any endothelial-dependent relaxation when exposed to acetylcholine and there was severe or complete endothelial cell loss on histological examination. This study shows the traumatic effects of the in situ technique of vein grafting. However the precise relationship between the severity of endothelial injury and the development of vein graft lesions has still to be defined. In addition, the exact role of smooth muscle cell injury which also occurs after surgical preparation of both reversed and in situ vein grafts requires further investigation. Interestingly, it has been reported that there is no difference in the incidence of intrinsic lesions in reversed or in situ vein grafts, is However this study only involved belowknee femoro-popliteal procedures and not distal grafts to single calf vessels. In addition the mere presence of endothelial 'injury and not its severity may be the most important factor in the development of these lesions. Therefore further studies are required to define the exact relationship between endothelial injury and intrinsic vein graft lesions before attempts can be made to prevent them.
Acknowledgements This work was funded by the Ivy Walsh and Edith Powell research grants awarded by the British Medical Association. We would like to thank Ethicon Ltd for supplying sutures. Eur J Vasc Surg Vol 6, July 1992
References 1 BERGANJJ, YAO JST, FLINN WR, GRAHAMLM. Prosthetic grafts for the treatment of lower limb ischaemia: present status. Br ] Surg 1982; 69: $34-$37. 2 LEATHERRP, POWERSSR, KARMODYAM. A reappraisal of the in situ saphenous vein bypass: its use in limb salvage. Surgery 1979; 85: 453-461. 3 HALL KV. The great saphenous vein used in situ as an arterial shunt after extirpation of vein valves. Surgery 1962; 51: 492-495. 4 WOLFEJHN, MCPHERSONGAD. The failing femoro-distal graft. Eur J Vasc Surg 1987; 1: 295-296. 5 SZIEAGYIDE, ELLIOTTJP, HAGEMANJH, SMITH RE, DALL'OLMo CA. Biological fate of autogenous vein implants as arterial substitutes. Ann Surg 1973; 78: 232-246. 6 BRENNANJA, WALSHAKM, BEARDJD, BOLIAAA, BELLPRE. The role of simple non-invasive testing in infra-inguinal vein graft surveillance. Fur J Vasc Surg 1991; 5: 13-17. 7 BARTLETTST, KILLEWICHLA, FISHERC, WARDRE. Duplex imaging of in-sitt~ saphenous vein bypass grafts and late failure reduction. Am J Surg 1988; 156: 484-487. 8 MOODYP, DE COSSARTLM, DOUGLASHM, HARRISPL. Asymptomatic strictures in femoro-popliteal vein grafts. Eur J Vasc Surg 1989; 3: 389-392. 9 FUCHSJCA, MITCHENERJS, PER-OTTo H. Postoperative changes in autologous vein grafts. Ann Surg 1978; 188: 1-15. 10 DILLEY RJ, McGEACHIE JK, PRENDERGASTFJ. A review of the histological changes in vein-to-artery grafts, with particular reference to intimal hyperplasia. Arch Surg 1988; 123: 691-696. 11 RAMOSJR, BERGERK, MANSFIELDPB, SAUVAGELR. Histological fate and endothelial changes of distended and nondistended yein grafts. Ann Surg 1976; 183: 205-228. 12 ANGELINI GD, BRECKENRIDGEIM, PSAILA JV, WILLIAMS HM, HENDERSONAH, NEWBYAC. Preparation of human saphenous vein for coronary artery bypass grafting impairs its capacity to produce prostacyclin. Cardiovasc Res 1987; 21: 28-33. 13 ANGELINIGD, CHRISTIEMI, BRYANAJ, LEWISMJ. Surgical preparation impairs release of endothelium derived relaxing factor from human saphenous vein. Ann Thorac Surg 1989; 48: 417420. 14 FURCHGOTTRE, ZAWADZKIJV. The obligatory role of endothelial
Endothelial Cell Injury
cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: 373-376. 15 ANGELINI GD, SOYOMBO AA, NEWBY AC. Smooth muscle cell proliferation in response to injury in an organ culture of h u m a n saphenous vein. Eur J Vasc Surg 1991; 5: 5-12. 16 RUBANYGM, VANHOUTTE PM. Hypoxia releases a vasoconstrictor substance from the canine vascular endothelium. J Physiol 1985; 364: 45-56.
361
17 CAMBRIA RP, MEGERMAN J, ABBOTT WM. Endothelial preservation in reversed and in-situ autogenous vein grafts. Ann Surg 1985; 202: 50-55. 18 HARRIS PL, TOW TV, JONES DR. Prospective randomised clinical trial to compare in situ and reversed saphenous vein grafts for femoro-popliteal bypass. Br J Surg 1987; 74: 252-255.
Accepted 11 March 1992
Eur J Vasc Surg Vol 6, July 1992
Endothelial Cell Injury Secondary to Surgical Preparation of Reversed and In situ Saphenous Vein Bypass Grafts R. D. Sayers 1, P. A. C. Watt 2, S. Muller 3, P. R. F. BelP and H. Thurston 2 Departments of ~Surgery, 2Medicine and 3pathology, Clinical Sciences Building, Leicester Royal Infirmary, P. 0. Box 65, Leicester, LE2 7LX, U.K. Failure of infra-inguinal vein grafts appears to be due to the development of intrinsic lesions (intimal hyperplasia, fibrous stenoses) within the graft which lead to narrowing of the h~men, poor blood flow and thrombosis. The cause of Nese lesions remains unknown but recently it has been suggested that endothelh~l injury might be an aetiologicalfactor. The damage that can occur after preparation of reversed vein grafts includes loss of endothelial cells and functional impairment of those cells that remain, in that the ability to produce prostacyclin and endothelium-derived relaxing factor (EDRF) is reduced. The preparation of in situ grafts is different to that for reversed grafts in that a valvulotome is passed along the lumen of the vein to destroy the valves. However, little is known about the degree of endothelial injury that this technique causes. Vein samples were obtained from patients undergoing infra-inguinal and coronary artery bypass grafting. The veins were mounted in an organ bath system to measure isometric tension and exposed to the endothelium-dependent agents acetylcholine, bradykinin, adenosine, histamine and the endothelium-independent agent sodium nitroprusside. The results indicate that preparation of reversed vein grafts leads to some loss of endothdiaI cells with functionaI impairment of the cells that remain. However, preparation of in situ grafts leads to severe or total loss of endotheliaI cells which resulted in an absence of detectable EDRF release. Thesefindings were confirmed by histological examination of the vein samples. Key Words: Endothelium; Injury; Saphenous vein; Reversed vein graft; In-situ vein graft; Endothelium-derived relaxing factor; Valvulotome.
Introduction In infra-inguinal bypass procedures, the patient's o w n long s a p h e n o u s vein is the conduit of choice, with patency rates that are superior to prosthetic alternatives. 1 For an above-knee femoro-popliteal bypass, the vein is reversed so that the valves do not obstruct the flow of blood, whereas, in femoro-distal bypass to the below knee popliteal artery or single calf vessels, the in situ m e t h o d is often preferred. 2 This technique uses the vein non-reversed, to allow a better size match b e t w e e n artery and vein graft at the distal anastomosis, and involves passage of a valvulotome along the vein to r e n d e r the valves incompetent. 3 Failure of infra-inguinal vein grafts remains a serious problem which m a y result in loss of the limb. 4 Please address all correspondence to: R. D. Sayers, Department of Surgery, Clinical Sciences Building, Leicester Royal Infirmary, P.O. Box 65, Leicester, LE2 7LX, U.K. 0950-821X/92/040354+08$03.00/0© 1992Grune & Stratton Ltd.
One of the major causes of failure is the d e v e l o p m e n t of intrinsic lesions within the graft, particularly d u r i n g the first postoperative year, which lead to narr o w i n g of the lumen, poor blood flow and graft thrombosis. 5 Recent interest has focused on graft surveillance p r o g r a m m e s to detect these lesions 6'7 and allow their correction, by p e r c u t a n e o u s transluminal angioplasty or revisional surgery, before graft failure occurs. The two major types of intrinsic lesions that m a y develop in vein grafts are short isolated graft stenoses 5'8 or longer areas of intimal hyperplasia. 5 Their aetiology remains u n k n o w n and it is not clear w h e t h e r they r e p r e s e n t similar or different pathological processes. The changes that occur in reversed vein grafts d u r i n g and after surgical preparation have been well d o c u m e n t e d . 9-11 It has b e e n s h o w n that surgical preparation causes both structural and functional endothelial cell damage. There is a considerable loss of endothelial cells 9-11 and the remaining ones are func-
Endothelial Cell Injury
355
tionally impaired with reduced production of prosta- of side-branches and uncontrolled distension with cyclin12 and endothelium-derived relaxing factor saline. The third group (in situ) were obtained after (EDRF). 13 preparation for in situ vein grafting which included Endothelium-derived relaxing factor is released exposure and mobilisation of the vein, ligation of side from endothelial cells and acts on the underlying branches and the passage of a valvulotome (9 vessels, smooth muscle cells to produce relaxation via the cyc- 7 patients). A 2.5-ram Hall valvulotome was used for lic GMP pathway. 14 Functional injury or loss of endo- each in situ graft and it was passed as atraumatically thelial cells impairs this response so that it may be as possible. All samples of vein for each group were used as an assessment of endothelial damage. 13 taken from the calf region to ensure that they were Endothelium-independent vasodilators act directly matched for site and size. In the in situ group the vein on the smooth muscle and are used to confirm that was obtained from the distal end of the graft. the smooth muscle cells are able to relax despite enThe veins were transported from theatre to the laboratory in cold (4°C) calcium-free physiological salt dothelial damage. Recently a link has been suggested between en- solution in a sealed container. In the laboratory the dothelial injury caused by surgical preparation of vein was carefully cleaned of fat and adventitia and a reversed vein grafts and the development of intimal ring of vein 5 mm in length was cut open to form a hyperplasia using an in vitro organ culture technique rectangular strip. All dissection was performed under with human saphenous vein. is a dissecting microscope with the vein immersed in However, little is known about endothelial calcium-free physiological salt solution. One end of damage after preparation of in situ vein grafts. We the vein was attached to a stainless steel plate and the have therefore studied veins prepared for both in situ other to the force transducer to measure isometric and reversed bypass grafting in order to determine tension. The stainless steel plate and vein were the degree of structural and functional endothelial placed in an organ bath (Fig. 1) containing physiocell damage that occurs with each technique. logical salt solution (composition: NaC1 118mM; NaHCO3 25mM; KC1 4.5mM; KH2PO4 lmM; CaCI2 2.5mM; MgSO4.7H20 lmM; glucose 6mM) at 37°C and gassed with a 5% carbon dioxide/95% oxygen mixture to achieve a pH of 7.45.16 Methods The initial study was on 28 samples of saphenous vein which were obtained from 23 patients. These patients underwent either infra-inguinal or coronary artery bypass grafting. The patients were neither diabetic nor hypertensive and their mean ages and blood pressures are shown in Table 1. Table 1. A g e and m e a n b l o o d p r e s s u r e of p a t i e n t s in the control, reversed and in situ g r o u p s of p a t i e n t s e x p r e s s e d as m e a n + S.E.
Age (years)
Blood pressure (mmHg)
Control
73 + 4
109 + 3
Reversed
63 + 3
107 + 5
In situ
71 + 2
109 + 4
~ ] ~
Organ c h a m b e r ~ o o o ,
Strip of vessel/
l
Y
Forcetransducer
i °
I
n o - ~7-
Stainlesssteel plate
Fig. 1. Diagrammatic illustration of a vein mounted on a stainless steel plate in a heated gassed organ bath. Three groups of veins were studied which had been surgically handled in different ways. The first group (control) were obtained after minimal surgical dissection using a minimal-touch technique (10 vessels, 8 patients). The second group (reversed) were samples taken after the veins had been fully prepared for reversed bypass grafting (9 vessels, 8 patients). This included full exposure and mobilisation, ligation
After an equilibrium period of i h each vein was stretched and the optimum passive tension that would produce the largest contraction to noradrenaline (10 -7M) was determined. A further equilibrium period of I h was allowed to elapse before a cumulative dose response curve to noradrenaline (10-8M to 10-5M) was performed in the presence of cocaine Eur J VascSurgVol6, July1992
356
R.D. Sayers et al.
(10-6M). Following this, each vein was sub-maximally contracted with noradrenaline and relaxation studies were performed using the endotheliumdependent vasodilators acetylcholine (10-SM to 10-5M), bradykinin ( 1 0 - 9 M to 1 0 - 6 M ) , adenosine diphosphate ( 1 0 - 8 M to 10 -5 M), h i s t a m i n e ( 1 0 - 9 M to 10-5M) and the endothelium-independent vasodilator sodium nitroprusside (10-9 M to 10 - 5 m). Finally, the veins were fixed with 2% buffered formaldehyde and processed for histological assessment.
to noradrenaline, but the in situ group of veins failed to re-contract to a second exposure. This prevented the study of relaxation in the in situ group because the veins are required to be sub-maximally contracted before exposure to a relaxing agent. However, endothelium-dependent and independent relaxation responses were studied in the control and reversed groups. The relaxation produced by acetylcholine, bradykinin, adenosine, histamine and sodium nitroprusside are shown in Figures 2-6. The -10
Preparation of tissue for histological assessment Paraffin sections (4 txm) of veins were prepared and stained with haematoxylin and eosin (H&E) and elastin Van Gieson (EVG). In addition, the presence of endothelial cells was assessed with a novel monoclonal antibody to human endothelium, Q Bend 10 (Unipath, Norse Road, Bedford, MK41 0QG, U.K.) using the labelled avidin biotin immunoperoxidase technique with the primary antibody being used at a dilution of 1:50. The degree of endothelial cell loss and smooth muscle damage was assessed by a pathologist who had no prior knowledge of the group origin of the vein samples. Seven vein samples from each group were examined. A simple semi-quantitative scoring system was employed with the degree of endothelial damage assessed as negligible (0), minimal/patchy cell loss (1), and severe/total cell loss (2).
O
~
n,-
25
50
I 10-8
I 10-7
I i0 -6
I 10-5
Acefylcholine (M)
Fig. 2. Relaxation response of the control (O), reversed (A) and in situ (rq) groups of veins to acetylcholine.
Statistical analysis The relaxation responses are reported as a percentage of the initial sub-maximal contractile response to noradrenaline. Significant differences were determined by non-parametric analysis with the Mann-Whitney U-test accepting p < 0.05 as being significant.
"-7-
25
O
Results
The optimum passive tension that produced the maximum contractile response to noradrenaline was the same for the control and reversed groups but reduced in the in situ group (2.50 + 0.27g, 2.50 + 0.27g and 1.5 + 0.55g, respectively). It was found that the control and reversed groups of veins produced repeated contractions of the same magnitude Eur J Vasc Surg Vol 6, July 1992
50
J i0 -9
10-8
I 10-7
iO-S
Brodykinin (M)
Fig. 3. Relaxation response of the control (O) and reversed (A) groups of veins to bradykinin.
Endothelial
Cell
Injury
357
\\\
25
,I
04
P, 25
o
5C
g~
n-
75
50
I iO -8
I iO-7
I iO-S
I iO-5
I00
I i0-9
I (o-e
I
iO-7
\ 1(3-6
iO-5
Sodium nitroprusside(M)
Adenosine (M)
Fig. 4. Relaxation response of the control (O) and reversed (A) groups of veins to adenosine.
Fig. 6. Relaxation response of the control (O) and reversed (A) groups of veins to sodium nitroprusside.
-I0 0 x\\
25
Table 2. Maximum relaxation responses produced by the control and reversed groups of veins to acetylcholine (ACH), bradykinin (BK), adenosine (ADEN), histamine (HIST) and sodium nitroprusside (SNP)
\
\\\
\\
5(?
\
\
\
\
75
{00
i
10-9
1
10-8
I
10-7
I
i0-6
Control
Reversed
ACH (%)
10 _+6
6 _+5
BK (%)
29 _+9
17 _+9
ADEN (%)
17 _+8
4 _+3
HIST (%)
38 + 8
62 + 15
SNP (%)
100 + 0
100 _+0
I
10-5
Histamine (M) Fig. 5. Relaxation response of the control (O) and reversed (A)
groups of veins to histamine. m a x i m u m relaxation r e s p o n s e s to these agents are s h o w n in Table 2. There w a s no significant difference b e t w e e n the control a n d r e v e r s e d g r o u p s in their responses to a n y of the relaxing agents u s e d (p > 0.05, M a n n - W h i t n e y U-test). In order to p e r m i t relaxation studies to be perf o r m e d in the in situ group, a second series of veins which h a d b e e n p r e p a r e d for use as in situ grafts w e r e obtained f r o m patients u n d e r g o i n g femoro-distal b y p a s s p r o c e d u r e s (5 patients, 8 vessels). T h e s e
patients w e r e n o r m o t e n s i v e ( m e a n blood p r e s s u r e 106 _+ 2 m m H g ) a n d not diabetic w i t h a m e a n age of 67 + 4 years. The,veins w e r e collected, p r e p a r e d a n d m o u n t e d in the o r g a n b a t h as described previously. The passive tension w h i c h w o u l d give the best contractile r e s p o n s e w a s not d e t e r m i n e d for these veins as this w o u l d involve r e p e a t e d e x p o s u r e s to noradrenaline. Instead, the passive tension w a s preset to the p r e v i o u s l y o b s e r v e d m e a n resting tension of 1.5 g in the first in situ g r o u p of veins. The veins w e r e t h e n s u b - m a x i m a l l y contracted w i t h a single b o l u s injection of n o r a d r e n a l i n e a n d a c u m u l a t i v e relaxation curve p e r f o r m e d w i t h acetylcholine. T h e s e vessels did not p r o d u c e a n y e n d o t h e l i u m - d e p e n d e n t relaxation r e s p o n s e to acetylcholine (Fig. 2). Eur J Vasc Surg Vol 6, July 1992
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R.D. Sayers et al.
Table 3. Histological scoring assessment of endothelial injury
Total
Control (n = 7)
Reversed(n = 7)
0
1
2
1
1
1
1
1
2
0
1
2
0
2
2
0
!
1
0
1
2
2
8
12
In situ
(n = 7)
Histological assessment
The injury scores for each of the three groups of veins are shown in Table 3. The control veins showed minimal endothelial cell loss and therefore produced low "injury scores" (Fig. 7). Assessment of veins from the reversed group showed patchy endothelial cell loss (Fig. 8) and the veins from the in s i t u group showed severe or complete endothelial cell loss (Fig. 9). This was reflected in the higher injury scores for the reversed and in s i t u groups. The anti-endothelial monoclonal Q Bend 10 confirmed the presence of endothelial cells in the control (Fig. 10) and reversed groups with absence or near total loss in the in s i t u group (Fig. 11).
Discussion
It has been well documented that surgical preparation of reversed vein grafts leads to loss of endothelial cells 9-11 and that the remaining cells are functionally impaired with a reduction of their ability to produce prostacyclin 12 and EDRF. 13 Endothelial injury associated with the in s i t u technique of vein grafting has not been extensively investigated, although one study has suggested that this technique produces superior endothelial cell preservation. 17 Recently a link has been suggested between endothelial and smooth muscle cell injury during preparation of reversed vein grafts and the subsequent development of intimal hyperplasia, is It is possible that a similar process may be important in the development of lesions after in s i t u grafting and therefore the degree of endothelial cell injury associated with this technique requires further study. Our results support the view that surgical preparation of reversed grafts leads to endothelial cell damage.9-11,13 These veins exhibited reduced relaxation responses to all of the endothelium-dependent agents when compared with controls although these differences did not reach statistical significance. The ability of the smooth muscle cells of these veins to fully relax was confirmed by their response to the endothelium-independent agent sodium nitroprusside, indicating that the final pathway was intact. In addition histological examination of veins from the reversed group showed a degree of loss of the endothelial cell layer when compared to controls.
Fig. 7. Histologyof a vein from the control group showing intact endothelium. Eur J VascSurg Vol 6, July1992
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359
Fig. 8, Histology of a vein from the reversed group showing patchy loss of endothelium.
Fig. 9. Histology of a vein from the in situ group showing complete loss of endothelium,
Fig. 10 Histology of a vein from the control group showing endothelial cells stained with Q Bend 10. Eur J Vasc Surg Vol 6, July 1992
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Fig. 11. Histology of a vein from the in situ group stained with Q Bend 10 showing absence of endothelial cells.
The in situ group of veins all showed severe endothelial cell injury. These veins did not produce any endothelial-dependent relaxation when exposed to acetylcholine and there was severe or complete endothelial cell loss on histological examination. This study shows the traumatic effects of the in situ technique of vein grafting. However the precise relationship between the severity of endothelial injury and the development of vein graft lesions has still to be defined. In addition, the exact role of smooth muscle cell injury which also occurs after surgical preparation of both reversed and in situ vein grafts requires further investigation. Interestingly, it has been reported that there is no difference in the incidence of intrinsic lesions in reversed or in situ vein grafts, is However this study only involved belowknee femoro-popliteal procedures and not distal grafts to single calf vessels. In addition the mere presence of endothelial 'injury and not its severity may be the most important factor in the development of these lesions. Therefore further studies are required to define the exact relationship between endothelial injury and intrinsic vein graft lesions before attempts can be made to prevent them.
Acknowledgements This work was funded by the Ivy Walsh and Edith Powell research grants awarded by the British Medical Association. We would like to thank Ethicon Ltd for supplying sutures. Eur J Vasc Surg Vol 6, July 1992
References 1 BERGANJJ, YAO JST, FLINN WR, GRAHAMLM. Prosthetic grafts for the treatment of lower limb ischaemia: present status. Br ] Surg 1982; 69: $34-$37. 2 LEATHERRP, POWERSSR, KARMODYAM. A reappraisal of the in situ saphenous vein bypass: its use in limb salvage. Surgery 1979; 85: 453-461. 3 HALL KV. The great saphenous vein used in situ as an arterial shunt after extirpation of vein valves. Surgery 1962; 51: 492-495. 4 WOLFEJHN, MCPHERSONGAD. The failing femoro-distal graft. Eur J Vasc Surg 1987; 1: 295-296. 5 SZIEAGYIDE, ELLIOTTJP, HAGEMANJH, SMITH RE, DALL'OLMo CA. Biological fate of autogenous vein implants as arterial substitutes. Ann Surg 1973; 78: 232-246. 6 BRENNANJA, WALSHAKM, BEARDJD, BOLIAAA, BELLPRE. The role of simple non-invasive testing in infra-inguinal vein graft surveillance. Fur J Vasc Surg 1991; 5: 13-17. 7 BARTLETTST, KILLEWICHLA, FISHERC, WARDRE. Duplex imaging of in-sitt~ saphenous vein bypass grafts and late failure reduction. Am J Surg 1988; 156: 484-487. 8 MOODYP, DE COSSARTLM, DOUGLASHM, HARRISPL. Asymptomatic strictures in femoro-popliteal vein grafts. Eur J Vasc Surg 1989; 3: 389-392. 9 FUCHSJCA, MITCHENERJS, PER-OTTo H. Postoperative changes in autologous vein grafts. Ann Surg 1978; 188: 1-15. 10 DILLEY RJ, McGEACHIE JK, PRENDERGASTFJ. A review of the histological changes in vein-to-artery grafts, with particular reference to intimal hyperplasia. Arch Surg 1988; 123: 691-696. 11 RAMOSJR, BERGERK, MANSFIELDPB, SAUVAGELR. Histological fate and endothelial changes of distended and nondistended yein grafts. Ann Surg 1976; 183: 205-228. 12 ANGELINI GD, BRECKENRIDGEIM, PSAILA JV, WILLIAMS HM, HENDERSONAH, NEWBYAC. Preparation of human saphenous vein for coronary artery bypass grafting impairs its capacity to produce prostacyclin. Cardiovasc Res 1987; 21: 28-33. 13 ANGELINIGD, CHRISTIEMI, BRYANAJ, LEWISMJ. Surgical preparation impairs release of endothelium derived relaxing factor from human saphenous vein. Ann Thorac Surg 1989; 48: 417420. 14 FURCHGOTTRE, ZAWADZKIJV. The obligatory role of endothelial
Endothelial Cell Injury
cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: 373-376. 15 ANGELINI GD, SOYOMBO AA, NEWBY AC. Smooth muscle cell proliferation in response to injury in an organ culture of h u m a n saphenous vein. Eur J Vasc Surg 1991; 5: 5-12. 16 RUBANYGM, VANHOUTTE PM. Hypoxia releases a vasoconstrictor substance from the canine vascular endothelium. J Physiol 1985; 364: 45-56.
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17 CAMBRIA RP, MEGERMAN J, ABBOTT WM. Endothelial preservation in reversed and in-situ autogenous vein grafts. Ann Surg 1985; 202: 50-55. 18 HARRIS PL, TOW TV, JONES DR. Prospective randomised clinical trial to compare in situ and reversed saphenous vein grafts for femoro-popliteal bypass. Br J Surg 1987; 74: 252-255.
Accepted 11 March 1992
Eur J Vasc Surg Vol 6, July 1992
