Ann Thorac Surg 1997;63:429-433
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Surgical Preparation Abolishes Endothelium-Derived Hyperpolarizing Factor-Mediated Hyperpolarization in the Human Saphenous Vein
Jian-An Yang, MD,
Guo-Wei He, MD, PhD
Cardiovascular Research Laboratory, Department of Surgery, University of Hong Kong, Grantham Hospital, Aberdeen, Hong Kong
Accepted for publication August 21, 1996.
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Abstract
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Background. The impairment of the synthesis and release of endothelium-derived relaxing factors may be related to the high incidence of atherosclerosis and occlusion in saphenous vein grafts. This study focused on the effect of surgical preparation on one of the endothelium-derived relaxing factors, endothelium-derived hyperpolarizing factor, in the human saphenous vein.
Methods. Human saphenous vein segments taken from patients undergoing coronary bypass were placed in an organ bath. A glass microelectrode was inserted into a smooth muscle cell. The membrane potential in response to acetylcholine (-9 to -5 log M) was measured in normal or surgically prepared saphenous vein with presence or absence of NG-nitro-L-arginine (300 µmol/L) and indomethacin (7 µmol/L).
Results. The resting membrane potential was -71.28 ± 1.91 mV (n = 7) with intact endothelium and -65.5 ± 2.92 mV (n = 6, p > 0.05) without endothelium. Acetylcholine hyperpolarized membrane potential with intact endothelium (-90.57 ± 1.48 mV, n = 7, p < 0.001), but not without endothelium (-69.67 ± 2.93 mV, n = 6, p > 0.05). In the surgically prepared saphenous vein, acetylcholine did not hyperpolarize membrane potential (-71.83 ± 3.84 mV versus the resting membrane potential of -69.50 ± 3.53 mV, n = 6, p > 0.05).
Conclusions. The endothelium-derived hyperpolarizing factor plays a role in the human saphenous vein. The surgical preparation abolishes the endothelium-derived hyperpolarizing factor-mediated hyperpolarization in the saphenous vein. This study provides evidence of functional changes of endothelium by traditional surgical preparation from another point of view, and it may be related to the high incidence of occlusion in saphenous vein grafts.
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Introduction
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The vascular endothelial cell plays an important role, not only on modulating homeostasis, proliferation, inflammatory, and immune mechanisms in the vascular wall, but also on regulating the tone of the underlying vascular smooth muscle [1–4]. The vascular endothelium releases a number of endothelium-derived relaxing factors (EDRFs) such as endothelium-derived nitric oxide (EDNO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF) [1, 2]. The EDRFs may be major determinants of vascular tone and circulation resistance. It has been suggested that the endothelium and the EDRFs may protect vessels from pathologic changes [3–6]. With endothelial dysfunction or denudation, the synthesis and release of EDRFs are reduced and vascular spasm, thrombosis, and atherosclerosis may occur [4]. Recently, the effect of EDHF on regulating vascular tone [1, 2, 7] and its importance in pathologic changes in the coronary circulation [8, 9] have been reported. In particular, under conditions when the synthesis of EDNO is impaired (ie, in atherosclerosis, hypertension, and ischemia), EDHF is considered as an important factor in the aforementioned function [6, 10].
As reported, the incidence of atherosclerosis and occlusion is high in venous grafts implanted into the aorta-to-coronary arterial system [11, 12]. Therefore, studies have focused on the method to prepare saphenous vein (SV) grafts and improve long-term patency [13, 14]. The preparation of the veins is believed to be an important factor in influencing long-term results. Although the method to prepare venous grafts has been evolutive over the past years, the rate of long-term patency does not seem to be significantly enhanced.
Many factors such as the distention pressure during preparation, the application of solutions for storing and flushing, and the discontinuation of the vaso vasorum of blood supply to the venous wall have been suggested to be related to the patency [13–16]. Studies have demonstrated that surgical preparation reduces the production of EDNO in the SV graft [17, 18]. However, the role of EDHF in the human SV and whether it is affected by the surgical preparation has not been reported. Therefore, we designed this study to investigate the influence of surgical preparation on the effect of EDHF in the human SV.
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Material and Methods
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Preparation of Vessels
Human SV segments were harvested from patients undergoing coronary artery bypass grafting. Approval to use discarded SV tissue was given by the Ethics Committee of the Grantham Hospital. Segments of veins before surgical preparation were taken as control. The procedure for surgical preparation follows traditional method. The SV was dissected carefully and branches were ligated. The veins were then distended by injecting heparinized normal saline to check diameter and leakage. The required length was measured and the redundant vein segments were immediately placed in 4°C Krebs' solution (see below) and transferred to the laboratory. The connective tissue surrounding the SV was gently removed. Segments were placed in an organ bath (2.5 mL) and continuously superfused with Krebs' solution (3 to 5 mL/min) at 37.0°C bubbled with 95% O2 and 5% CO2. In six segments, endothelial cells were removed mechanically by gently rubbing the intimal surface of the veins with a blade.
Membrane Potential Measurement
Strips were cut from human SV along the longitudinal axis and mounted on the bottom of the organ bath with the intimal side upward. The SV was allowed to equilibrate for 60 minutes and a glass microelectrode filled with 3 mol/L KCl (tip resistance 40 to 80 M
) was then inserted into a smooth muscle cell from the endothelial side [2, 19–21]. The electrical signals were amplified by a microelectrode amplifier (Axoprobe-1A, H.V. Electrometer model 400B; Axon Instruments, Inc, Foster City, CA). The membrane potential was displayed continuously on a storage oscilloscope (COS5020-ST; Gould, Cleveland, OH) and simultaneously recorded (Recorder PCM-2 (A/D) VCR; Medical System Corp, Greenvale, NY) (see Fig 1
for the experimental set-up). The criteria of impaling cells are a sudden negative change in voltage and a stable negative voltage for more than 1 minute as well as an instantaneous return to the previous voltage level on dislodgment of the microelectrode [7, 22].
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Fig 1. . Diagram of measuring and recording set-up for membrane potential of human saphenous vein in the organ bath.
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Protocol
All SV segments were equilibrated again for 60 minutes in the organ bath. The following protocol for the measurement of the membrane potential was used.
CONTROL GROUP.
(1) In the endothelium-intact SV segments, the resting membrane potential was measured as the baseline. KCl (20 mmol/L) was added to test the viability and the depolarization of the SV. The vessel was washed for 30 minutes to restore the baseline and cumulative concentration–response curves of the membrane potential in response to acetylcholine chloride (ACh; -9 to -5 log M) were established (n = 7). The SV was then washed repeatedly to restore the baseline. NG-nitro-L-arginine (L-NNA; 300 µmol/L) and indomethacin (7 µmol/L) were added to the bath for 30 minutes. The concentration–response curve to ACh was established again. (2) The protocol as in (1) was used on endothelium-denuded SV segments (n = 6).
SURGICAL PREPARATION GROUP.
The same protocol was followed. However, in this group, the membrane was not hyperpolarized by ACh (-9 to -5 log M). Therefore, no further experiments with inhibitors were required.
Drugs and Solution
The Krebs' solution had the following composition (in mmol/L): 144 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.0 glucose. Drugs used were ACh chloride, indomethacin, and L-NNA (all from Sigma, St. Louis, MO). The drugs were prepared in distilled water. Indomethacin was dissolved in ethyl alcohol (100%) at a concentration of -2 log M.
Data Analysis
The results were expressed as means ± standard error. Statistical significance was tested with Student's two-tailed paired and unpaired t tests. Values of p less than 0.05 were considered statistically significant.
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Results
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Control Group
The resting membrane potential of the smooth muscle of human SVs was -71.28 ± 1.91 mV (n = 7) with intact endothelium. The membrane potential was not significantly changed after incubation with L-NNA and indomethacin (-68.72 ± 2.28 mV, p = 0.4). The membrane potential was depolarized to -33.29 ± 3.67 mV (n = 7, p < 0.001) by 20 mmol/L KCl. After the repeated wash for 30 minutes, the membrane potential recovered to -70.08 ± 2.22 mV (n = 7, p > 0.05 compared with the previous baseline). At the concentration of -7.5 log M, ACh caused membrane hyperpolarization (-77.00 ± 1.93 mV, n = 7, p = 0.039). ACh at -5 log M induced the most significant hyperpolarization (-90.57 ± 1.48 mV, n = 7, p < 0.001) (Figs 2, 3). The hyperpolarization decreased with time (2 to 3 minutes) (see Fig 2). Indomethacin (7 µmol/L) and L-NNA (300 µmol/L) did not inhibit the ACh-caused hyperpolarization (maximal, -84.43 ± 3.74 mV, n = 7, p > 0.05).
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Fig 2. . An actual trace of the membrane potential measurement in a single smooth muscle cell of the human saphenous vein. Concentration–response (membrane potential) curves were established for acetylcholine (ACh) in the presence of indomethacin (7 µmol/L) and NG-nitro-L-arginine (300 µmol/L) in the endothelium-intact (a), endothelium-denuded (b), and surgically prepared (c) human saphenous vein. The numbers refer to -log M concentrations.
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Fig 3. . Concentration-dependent curves of membrane potential by acetylcholine (-9 to -5 log M) in normal human saphenous vein (control group). Vertical bars are 1 standard error of the mean of the response at each concentration. (Solid circle = control; open circles = in the presence of NG-nitro-L-arginine [LNNA, 300 µmol/L] and indomethacin [INDO, 7 µmol/L]; triangles = endothelium-denuded preparation [E-].)
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In endothelium-denuded SV, the resting membrane potential was -65.50 ± 2.92 mV (n = 6, p > 0.05 compared with the control rings with intact endothelium). In these veins, ACh (-9 to -5 log M) did not cause significant membrane hyperpolarization (-69.33 ± 3.49 mV, n = 6, p > 0.05) (see Figs 2, 3
).
Surgical Preparation Group
The resting membrane potential of smooth muscle cells was -69.50 ± 3.52 mV (n = 6, p > 0.05 compared with the control rings with intact endothelium). ACh (-9 to -5 log M) did not significantly hyperpolarize the membrane potential (-71.83 ± 3.84 mV, n = 6, p > 0.05) (Fig 4).
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Fig 4. . Concentration-dependent curves of membrane potential by acetylcholine (-9 to -5 log M) in the human saphenous vein undergoing surgical preparation (surgical preparation group). Vertical bars are 1 standard error of the mean of the response at each concentration. (Closed circles = control; open circles = human saphenous vein undergoing surgical preparation; triangles = endothelium-denuded preparation [E-]; *p < 0.05 compared with the control.)
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Comment
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This study demonstrates that surgical preparation abolishes the EDHF-mediated, endothelium-dependent membrane hyperpolarization in the human SV. This is most likely attributable to the damage of surgical preparation on the endothelium of the grafts.
The effect of vascular endothelium on regulation of the vascular tone is mainly attributable to the biosynthesis and release of at least three EDRFs. They are PGI2, EDNO, and EDHF. Most studies have focused on the effect of EDNO and PGI2. More recently, studies on EDHF have revealed that the cytochrome P450-monooxygenase metabolite of arachidonic acid is possibly a candidate for EDHF [5], although the nature of EDHF has not been finally identified. EDHF induces vascular smooth muscle relaxation by hyperpolarization of the smooth muscle cells [1–5, 7–9], which may involve potassium channels [5, 8, 9]. In addition to the vascular relaxing effect, recent studies have suggested that EDHF may also play a role in the prevention of pathologic changes such as atherosclerosis [3, 4] in blood vessels.
There are many factors that contribute to the high occlusion rate of saphenous vein grafts for coronary artery bypass grafting [13–16]. Such factors may be categorized as unavoidable and avoidable. Unavoidable factors are the exposure of the vein to arterial pressure and the interruption of the nutritive blood supply (vaso vasorum) to the wall of the vein. However, some factors such as high-pressure distention and use of detrimental solutions are avoidable. Therefore, the method of surgical preparation is important with regard to preservation of the vein.
Previous studies have focused on the influence of surgical preparation on the production of EDNO and PGI2. One study [18] suggested that the synthesis and release of EDNO is significantly reduced in surgically prepared vein grafts. In contrast, PGI2 plays a small role in the SV as the production of PGI2 in the normal SV is low and it is unaffected by surgical preparation [23]. Logically, a question arises regarding the influence of surgical preparation on the third component of the EDRFs-EDHF, the newly discovered factor. However, such influence is unknown. If EDHF plays a role in normal vessels and the decrease of its biosynthesis and release is related to pathologic changes such as atherosclerosis as aforementioned, it may also play a role in the saphenous vein. In addition, the change of its biosynthesis or release, if there is any, may also correlate with the long-term patency of the venous graft. Therefore, we designed the present study to examine the role of EDHF in normal and surgically prepared SV grafts.
The present study demonstrates that in the normal human SV, EDHF may play a role in regulating the balance between contraction and relaxation. This is shown by the observation that ACh significantly hyperpolarized the membrane potential of the smooth muscle cell in the SV (-70.08 ± 2.22 versus -84.43 ± 3.74 mV, p < 0.05) in the presence of inhibitors for EDNO (L-NNA) and PGI2 (indomethacin). This hyperpolarization is abolished by the mechanical denudation of the endothelium. As demonstrated previously, ACh may stimulate the release of various EDRFs [1–3, 24]. However, because of the experimental design the biosynthesis and release of the other two EDRFs were inhibited, the hyperpolarization is the response to the release of EDHF. Therefore, our experiments have demonstrated that endothelium-derived hyperpolarization exists in the human SV. Previously we have demonstrated that EDHF plays a role in another human vessel-the internal mammary artery [26]. Although the exact role of EDHF in the human SV is still to be determined, our study opened a new area for investigators to search for the physiologic role of EDHF in the human SV and pathologic significance in the formation of atherosclerosis and late occlusion from another point of view, compared with previous studies.
In our experiments we have also demonstrated that the traditional method of preparing the human SV almost abolished the membrane hyperpolarization. This is attributable to the elimination of EDHF-related function, either its release or its effect on the smooth muscle, or both. Surgical preparation has been shown to alter the endothelial function in the human SV, as mentioned above [18, 23, 25]. From our experiments, the response to ACh in the distended SV was similar to the SV in which the endothelium was mechanically rubbed off (Fig 4). The ACh-mediated hyperpolarization in both veins was totally abolished. This implies that the eliminated hyperpolarization in the surgically prepared SV was attributable to the damage to the endothelium.
If it is true that EDHF, together with other EDRFs, plays a role in regulating vascular tone and in protecting vessels in some nonphysiologic situations such as ischemia and atherosclerosis as proposed [4], the present study suggests that the elimination of the endothelium-dependent hyperpolarization of the membrane potential of the smooth muscle in the human SV may be one of the causes for the development of atherosclerosis and late occlusion. Together with the reduced or eliminated production of other EDRFs, particularly EDNO, the pathologic changes such as platelet aggregation, thrombus formation, and atherosclerosis occur and these pathologic changes ultimately lead to the luminal occlusion of the graft. It is logical, therefore, to propose that an ideal method to preserve the SV for coronary artery bypass grafting should be able to preserve the EDHF-related membrane potential hyperpolarization in accordance with the preservation of other functions of the endothelium and the smooth muscle.
In conclusion, our study demonstrates that endothelium-dependent hyperpolarization exists in the human SV and that the traditional method to harvest the SV abolishes the EDHF-related hyperpolarization. Our study suggests that in addition to the alteration of the effect of other EDRFs (EDNO and PGI2), the elimination of the EDHF-related function may be also related to the development of atherosclerosis and late occlusion of the SV graft. We propose that an ideal method to preserve the SV during operation should include preservation of the EDHF-related function to improve long-term patency of the SV graft.
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Acknowledgments
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This study was supported by Committee of Research and Conference Grant (337/048/0018) and Vice-Chancellor Grant (SN/mp/350/172/019), University of Hong Kong. Doctor Jian-An Yang was a Visiting Fellow from the Department of Cardiovascular Surgery, The Great Wall Hospital, Beijing, China, and was supported by a fellowship provided by Department of Surgery, University of Hong Kong. We sincerely thank Drs Tak-Ming Wong and Jian-Zhong Sheng at the Department of Physiology, The University of Hong Kong for their kind assistance in the experiments to measure the membrane potential. We also thank Drs Shui-Wah Chiu and David Cheung for their support and assistance in providing some of the saphenous vein tissue.
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Footnotes
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Address reprint requests to Prof He, Division of Cardiothoracic Surgery, Department of Surgery, University of Hong Kong, Grantham Hospital, 125 Wong Chuk Hang Rd, Aberdeen, Hong Kong.
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Abstract
Introduction
