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Ann Thorac Surg 2003;76:1533-1538
© 2003 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, Onze Lieve Vrouwe Gasthuis, Amsterdam Netherlands
b Laboratory of Physiology, Amsterdam, Netherlands
c Institute of Pathology, Amsterdam, Netherlands
d Institute of Cardiovascular Research, University Hospital VU University Medical Center, Amsterdam, Netherlands
e Department of Experimental Thoracic Surgery, University of Groningen, Groningen, Netherlands
Accepted for publication May 14, 2003.
* Address reprint requests to Dr Stooker, Onze Lieve Vrouwe Gasthuis, Department of Cardiothoracic Surgery, PO Box 95500, 1090 HM Amsterdam, Netherlands.
e-mail: w.stooker{at}olvg.nl
Abstract
BACKGROUND: Compliance of artificial and autologous vascular grafts is related to future patency. We investigated whether differences in compliance exist between saphenous vein grafts derived from the upper or lower leg, which might indicate upper or lower leg saphenous vein preference in coronary artery bypass surgery. Furthermore, the effect of perivenous application of fibrin glue on mechanical vein wall properties was studied to evaluate its possible use as perivenous graft support.
METHODS: Vein segments (N = 10) from upper or lower leg saphenous vein grafts were collected for histopathologic examination and smooth muscle cell/extracellular matrix (SMC/ECM) ratio was calculated. This ratio is suggested to be related with vascular elastic compliance. In a second group vein graft segments (N = 6) from upper and lower leg were placed in an in vitro model generating stepwise increasing static pressure up to 150 cm H2O. Outer diameter was measured continuously with a video micrometer system. Distensibility was calculated from the pressure-diameter curves. A third group of vein graft segments (N = 7) was pressurized after fibrin glue application to prevent overdistension, and studied in the same setup.
RESULTS: Vein segments from the lower leg demonstrated a consistent higher relative response compared with the upper leg saphenous vein graft (0.9176 ± 0.03993 vs 0.5245 ± 0.02512). Both reach a plateau in the high-pressure range (> 100 cm H2O). A significant difference in in vitro distensibility between upper and lower leg saphenous vein was only found at a pressure of 50 cm H2O (p < 0.05). With fibrin glue, support overdistension is prevented as revealed by the maximum relative response between fibrin glue supported upper and lower leg saphenous vein segments (0.4080 ± 0.02464 vs 0.582 ± 0.051), and no plateau is reached in the pressure range up to 150 cm H2O.
CONCLUSIONS: No upper or lower leg saphenous vein preference could be deduced from the differences in pressure-diameter response due to loss of distensibility (and thus of compliance) in the high-pressure range. Fibrin glue effectively prevents overdistension and preserves some distensibility in the high-pressure range in both the upper and lower leg saphenous vein. This might provide a basis for clinical application of perivenous support.
When a vein is placed in the arterial circulation in coronary artery bypass graft (CABG) procedures, the vein graft wall is subjected to two mechanical factors: increased circumferential deformation and changed flow velocity, which have serious implications on future vein graft patency [1, 2]. The degree of the vein graft distension is related to vein properties like compliance. In vivo compliance is related to future patency according to Davies and coworkers [3, 4], who reported lower patency rates of in vivo less compliant vein grafts in peripheral bypass surgery. In vivo vein compliance measurements are limited to the venous pressure range with maximum pressures of about 75 mm Hg (100 cm H2O) [5], and are influenced by the elastic properties of surrounding tissues, whereas vein grafts in CABG procedures are dissected free of supporting perivenous tissue. Therefore, we evaluated whether in vitro pressure-diameter relationship differences exist between upper and lower leg saphenous vein grafts and whether morphologic differences can be demonstrated, which might give clues to upper or lower leg preference in the choice of venous bypass grafts.
Due to the sudden exposure to arterial blood pressure the vein graft will be overdistended with subsequent increase of wall tension. In the arterial pressure range the vein graft will be a rigid tube. Prevention of overdistention of venous bypass grafts by using nonrestrictive, microporous, elastic, and biodegradable extravascular graft support is reported to have a beneficial effect on the vein graft wall adaptation with more favorable patency in animal models [6–9], and is known to attenuate the injury pattern found in human saphenous vein segments in experimental models [10, 11]. In previous ex vivo experiments we demonstrated that human vein grafts were completely de-endothelialized and revealed disrupted media structures within 1 hour of perfusion at 60 mm Hg, which did not occur when overdistention was prevented by perivenous support [11]. However, easy to apply specific perivenous support is not available clinically. Every external application in the form of a graft or a stent implicates handling and instrumentation of the vein graft with subsequent risk of injury to the vein graft wall, technical limitations for side-to-side anastomoses, and sizing problems. A favorable alternative may be an external graft support in the form of a spray, which can be applied immediately after completion of the bypass graft anastomoses and before exposure to high arterial blood pressure [12]. Therefore we investigated whether prevention of overdistention with perivenous fibrin glue is able to change vein graft wall dynamics toward a more suitable conduit in coronary artery bypass graft procedures.
Material and methods
Patients were included in the study after informed consent. Anesthesia and cardiopulmonary bypass were performed according to the routine protocol.
After harvest of the saphenous vein for the CABG procedure in 10 patients, segments of a few millimeters from both the saphenous vein from the upper leg region and the lower leg region were stored in Ringers' lactate with papaverin (0.1 mmol/L) to prevent spasm, and were prepared for histopathologic investigation.
In a second series of experiments in 6 patients two paired segments of study vein graft, one from the above-knee region and one from the below-knee region, with a length of about 2 cm were collected in chilled MOPS buffer (composition see below) for in vitro study. Within 1-hour after harvest the vein graft segments were connected to a pressure myograph in a setup that was described before [13]. The vein segments were mounted on an inflow and outflow cannula and secured with ligatures. The cannula at the inflow site was connected to a micrometer to stretch the vein segment to its in vivo length. The cannula at the outflow site was connected to a column filled with MOPS buffer (see below) for stepwise pressurization of the vein segment from 0 to 150 cm H2O. The vessel chamber was placed under a microscope that was connected to a video camera and a monitor. The diameter of the vein graft was measured continuously with an electronic system and stored on a computer. The electronic system triggers on dark-light transitions in the video signal, corresponding with the left outer wall, and calculates the distance to the next light-dark transition corresponding with the right outer wall. Biologic materials are known to show creep, ie, gradually alter their dimensions with time, and hysteresis. Based on the studies of Wesly and colleagues [14], each pressure increase was followed by a 2-minute delay before taking measurements to allow for the creep to occur.
In the third series of experiments in 7 patients, segments of about 2 cm in length from both the upper leg and lower leg were studied for the effect of the application of fibrin glue on distensibility. After application of a fibrin glue layer of about the vein graft wall thickness, solidification was awaited, and the vein grafts were distended as mentioned in the second series of experiments. Distension was measured following the same protocol.
To illustrate the presence of functionally intact endothelium after pressure distension with fibrin glue support, in a fourth experiment the vessel wall vasoreactivity was tested in 2-cm vein graft segments of both the upper and lower leg saphenous vein in 9 patients after peeling off the fibrin glue. The fibrin glue was removed to allow for the registration of even small changes in vessel wall diameter. After preconstriction with norepinephrine (10-7 mol/L), the endothelium dependent dilator acetylcholine was added in increasing concentration (10-7 to 10-5 mol/L) to the medium and endothelium mediated dilatation was measured and expressed as percentage of the precontraction to norepinephrine, which was considered 100%. After washout and equilibration, sodium nitroprusside, also in increasing concentration (10-7 to 10-5 mol/L), was added and dilatation was measured again.
Calculations
Vein graft distensibility was calculated using the video record of vessel outer diameter and intraluminal pressure. Because distension characteristics were obtained from the slope of the pressure-diameter relation, a least-squares fit of the data to a sigmoid curve was performed:
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D/D)max is the maximal change in (D/D), p50 is the pressure with half the maximal response, and n is the Hill coefficient. This particular curve was selected because pressure-diameter curves of isolated vein segments demonstrate a sigmoid shape.
Distensibility is defined as the change in normalized volume brought about by a unit of change in pressure. Because the volume is difficult to ascertain we measured diameter, which is related to volume. Distensibility can be thus expressed as: Dist. = 2D/DP, where Dist. = distensibility, D = diameter, and P is pressure. From the above it follows that distensibility is the same as two times the differential of the pressure-normalized diameter curve.
Statistical analysis
The data were expressed as mean ± standard error of the mean. The curves were analyzed by using a computer program (Graph Pad Prism version 3.0 for Windows, GraphPad Software, San Diego, CA).
Differences between upper and lower leg were analyzed by means of two-way analysis of variance (ANOVA) with Bonferroni posthoc test for differences between groups. Significance was accepted at p less than 0.05.
Chemicals
The MOPS buffer consisted of (in mM) the following: 145 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 5 dextrose, 2 pyruvate, 0.02 EDTA and 3,3-(N-morpholino) propanesulfonic acid.
Histopathology
Ten paired upper and lower leg samples of leftover vein grafts were stored in Ringers' lactate with papaverin and collected for morphologic evaluation. The specimens were routinely fixed in 4% formalin and subsequently embedded in paraffin. Paraffin-embedded vascular tissue sections (4 µm) were mounted on microscope slides, deparaffinized for 10 minutes in xylene at room temperature, and rehydrated through descending concentrations of ethanol.
Immunohistochemistry
Subsequent to deparaffinization and rehydration, sections were treated with 0.3% H2O2 in methanol for 30 minutes to block endogenous peroxidase activity. Sections were then preincubated with normal rabbit serum (1:50, Dakopatts A/S, Stockholm, Sweden) for 10 minutes at room temperature and incubated for 60 minutes with anti-
-smooth muscle cell actin antibody (SMA; 1:200 Dakopatts A/S). After a wash in phosphate buffered saline solution (PBS), sections were incubated for 30 minutes with rabbit antimouse biotin-labeled antibody (1:500) at room temperature and subsequently washed in PBS. After incubation with biotin-labeled streptavidin-horseradish peroxidase (1:200, Dakopatts A/S) for 60 minutes at room temperature, horseradish peroxidase was visualized with 3,3-diaminobenzidine tetrahydrochloride/H2O2 (Sigma Chemical Company, St. Louis, MO) for 3 to 5 minutes.
Immunoquantification
Smooth muscle cell (SMC) ratios of the medial layer and measurement of the medial thickness was quantified by using a video overlay system (QPRODIT 5.2, Leica), which is described in more detail in Vermeulen and coworkers [16].
Results
Morphologic evaluation
No morphologic difference was found between upper and lower leg saphenous vein segments. Wall thickness, SMC, and ECM content in both the circular and longitudinal layer exhibited no significant difference between upper and lower leg saphenous vein graft segments as expressed in the SMC/ECM ratio (Fig 1).
Although a significant difference was found between the thickness of the circular SMC layer of the upper leg saphenous vein and the longitudinal muscle layer of the lower leg saphenous vein (not shown), this difference did not correspond with an all over difference in muscle layer thickness or ECM.
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40 cm H2O). The maximum distension response in the fibrin glue supported group never reaches the unsupported plateau of distension, which suggests that fibrin glue support is able to prevent overdistension of the vein graft in the arterial pressure range. The difference in distension range between supported upper and lower saphenous vein grafts is only significant in the high-pressure range (125 [p < 0.01] and 150 cm H2O [p < 0.001]).
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The extracellular matrix of the tunica media of the saphenous vein, which consists predominantly of collagen highly aligned in a circumferential direction, forms the greatest contribution to the stiffness of the wall at higher pressure [17]. Although we expected to find a difference in SMA/ECM ratio to explain the consistent finding of a higher distension range of the lower leg saphenous vein graft compared with the upper leg saphenous vein, no significant difference is found in histopathologic investigation in this study as expressed in SMC/ECM ratio. Perhaps a difference in collagen fiber arrangement could underlie this phenomenon.
In preoperative in vivo studies Davies and colleagues [4] report a more favorable patency of saphenous vein bypass graft in peripheral bypass surgery of more compliant vein grafts. Saphenous vein compliance was found to be inversely related with the degree of focal hyperplasia and circular muscle hypertrophy present in the vein preoperatively. Especially the distal (lower leg) saphenous vein revealed more focal hyperplasia and circular muscle hypertrophy compared with the proximal (upper leg) saphenous vein and, consequently, lower compliance was found in the lower leg saphenous vein. This could favor the upper leg saphenous vein concerning future patency. In our study we could not confirm the higher degree of focal hyperplasia and circular muscle hypertrophy.
Compliance of the in situ vein grafts is influenced by the surrounding tissues, which could explain the different findings in our study on explanted saphenous vein segments. In this study we demonstrated a consistent difference in pressure-diameter relationship between vein grafts from the upper leg compared with vein grafts from the lower leg. Lower leg saphenous vein grafts can be overdistended to a higher range compared with the upper leg saphenous vein grafts. However, both the upper leg and lower leg saphenous vein grafts appear to be rather rigid tubes in arterial pressure conditions and no significant difference in distensibility exists between upper and lower leg saphenous vein grafts in the arterial pressure range.
Although a significant difference in distensibility between upper and lower leg saphenous vein grafts at lower pressure is suggested in our investigation, this difference does not exist in the high-pressure range. An interesting finding was that the upper leg saphenous vein graft in the same pressure range (above 100 cm H2O) characterizes significant preserved compliance if surrounded by fibrin glue, which supports the hypothesis that the graft will preserve some pulsatility, and overdistension is prevented with fibrin glue support. Although the same pattern is found in the supported lower leg saphenous vein graft versus the unsupported graft, the difference in distensibility between supported and unsupported lower leg saphenous vein grafts did not reach significance at any pressure.
Compliance is proven to be an important factor in the etiology of graft failure, and mismatch in the elastic properties of native artery and anastomosed graft is suggested to promote intimal hyperplasia at anastomotic sites [17]. An apparent difference exists between the compliance of vein and artery. Future patency of saphenous vein bypass grafts might be favored if the wall properties could be modulated to approach the properties of an artery. Arteries are less distensible than the veins at low pressures, but remain relatively distensible in high-pressure range. This is explained from the arrangement of elastin lamellae with wavy collagen inbetween, which permit further extension of the artery wall [18]. Perivenous support is known to promote vein wall adaptation toward a more arterylike conduit in animal experiments if nonrestrictive, elastic, biodegradable and permeable.
In this study the perivenous application of fibrin glue is demonstrated to give adequate support to both the saphenous vein graft from both the upper leg and from the lower leg. Maximum distension is limited to a level somewhere on the slope of the pressure-diameter relation curve of the unsupported graft, which implies that overdistension of the vein graft is prevented. This corresponds with our earlier studies [11, 12] in which perfusion pressures of 60 mm Hg during 1 hour in the unsupported vein grafts complete de-endothelialization and media disruption was observed, although in the supported vein grafts this injury was prevented. In the supported vein grafts, wall stress will be reduced and the graft will remain pulsatile in the high-pressure range. From this it is to be expected that the basis for well-formed remodeling of the vein graft wall is provided.
In conclusion, a difference in distension characteristics is found between upper and lower leg saphenous vein graft segments, which might have implications for future patency. Distension studies report stiffening in the arterial pressure range of both the upper and lower leg saphenous vein graft. No morphologic substrate can be indicated in our study and no difference in distensibility in the arterial pressure range to favor the use of upper over lower leg saphenous vein or vice versa.
Application of fibrin glue prevents overdistension and preserves some distensibility even in the high-pressure range, which supports the hypothesis that fibrin glue application can provide adequate perivenous support.
Acknowledgments
Doctor Niessen is a recipient of the Dr E. Dekker program of the Netherlands Heart Foundation (D99025).
References
This article has been cited by other articles:
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  M. R. Dashwood, K. Savage, J. C.S. Tsui, A. Dooley, S. G. Shaw, M. S. Fernandez Alfonso, L. Bodin, and D. S.R. Souza Retaining perivascular tissue of human saphenous vein grafts protects against surgical and distension-induced damage and preserves endothelial nitric oxide synthase and nitric oxide synthase activity. J. Thorac. Cardiovasc. Surg., August 1, 2009; 138(2): 334 - 340. [Abstract] [Full Text] [PDF]
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P. Human, T. Franz, J. Scherman, L. Moodley, and P. Zilla Dimensional analysis of human saphenous vein grafts: Implications for external mesh support. J. Thorac. Cardiovasc. Surg., May 1, 2009; 137(5): 1101 - 1108. [Abstract] [Full Text] [PDF]
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 M. R. Dashwood, K. Savage, A. Dooley, X. Shi-Wen, D. J. Abraham, and D. S.R. Souza Effect of Vein Graft Harvesting on Endothelial Nitric Oxide Synthase and Nitric Oxide Production Ann. Thorac. Surg., September 1, 2005; 80(3): 939 - 944. [Abstract] [Full Text] [PDF]
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= lower leg; 
= upper leg.
= upper leg with fibrin glue; 
= lower leg with fibrin glue.)