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Ann Thorac Surg 2003;76:2023-2028
© 2003 The Society of Thoracic Surgeons
a National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, United Kingdom
Accepted for publication June 6, 2003.
* Address reprint requests to Dr Amrani, Consultant Surgeon, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK.
e-mail: mr.amrani{at}rbh.nthames.nhs.uk
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Abstract |
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METHODS: RA and LITA specimens were divided into vascular rings, which were incubated in the absence or presence of 10-6 mol/L Cerivastatin for 2 or 24 hours. In terms of preoperative statin treatment, four groups included: group 1 [preop statin(-)/in vitro cerivastatin(-)]; group 2 [preop(-)/in vitro(+)]; group 3 [preop(+)/in vitro(-)]; and group 4 [preop(+)/in vitro(+)]. Endothelial function was assessed with acetylcholine (10-9 to 10-5 mol/L) following contraction by 3 x 10-8 mol/L endothelin-1.
RESULTS: Although endothelium-dependent vasodilatation was higher in RA (57.7% ± 3.5%) than in LITA (46.5% ± 3.8%, p = 0.046), there was no significant evidence that it depended on the preoperative use of statins or incubation period. In vitro incubation with cerivastatin significantly increased endothelium-dependent vasodilatation by 14.2% ± 2.4% (p < 0.0001) independent of artery types (RA/LITA). There was no significant evidence that endothelium-dependent vasodilatation depended on the preoperative use of statins or incubation period.
CONCLUSIONS: In vitro incubation with cerivastatin preserved endothelial function more effectively than preoperative use of statins. This could have implications to perioperative use of statins for patients undergoing coronary surgery.
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Introduction |
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Endothelial function could be ameliorated by pharmacologic interventions. The reductase inhibitors 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), or statins, are well known as lipid-lowering drugs that have recently been demonstrated to provide many other benefits [6–12]. Statins produce endothelium-dependent vasodilatation [6, 7] and inhibition of the smooth muscle cell proliferation [8], in addition to their antioxidant [9] and antiinflammatory [10, 11] effects. Furthermore, they provide an antisclerotic effect that had a major impact in reducing graft failure [12]. However, differential in vitro response of the human arterial grafts to cerivastatin have not yet been investigated.
Therefore, the aim of this study was to assess the effects of preoperative and in vitro exposure to statins on the endothelial function of the human RA and left internal thoracic artery (LITA) grafts used for CABG. It is hoped that this study would provide a rationale for the use of statins to augment endothelial function following CABG.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Plan of investigation
On processing the specimens, excess connective tissue was removed using a dissecting microscope followed by dividing each specimen into two to four pieces, approximately 3-mm each. In both types of artery specimens were divided into the following four groups from the point of view of preoperative treatment with statins and in vitro exposure to cerivastatin (BAY w6228; Bayer AG, Wuppertal, Germany):
Because groups 1 and 2, and groups 3 and 4 were necessarily paired, sources of the rings were exactly the same in the those groups. Patient preoperative characteristics including age, sex, smoking, hypertension, diabetes mellitus, hyperlipidemia, serum lipid data, and medications were compared.
For the 2-hour incubation, the specimens were immediately mounted on two L-shaped metal hooks in isolated organ baths without stretching. Vascular rings in groups 2 and 4 were incubated in organ baths with 10-6 mol/L cerivastatin, whereas those in the groups 1 and 3 were incubated without cerivastatin. The organ baths contained modified Tyrode's solution that is composed of (in mmol/L): NaCl, 136.9; NaHCO3, 11.9; KCl, 2.7; NaH2PO4, 0.4; MgCl2, 2.5; CaCl2, 2.5; glucose, 11.1; and disodiumethylenediaminetetraacetic acid, 0.04. The solution was continuously gassed with 95% O2 and 5% CO2 at the temperature of 37°C. In each organ bath, one hook was attached to a force-displacement transducer and this was fixed to a Grass 7D polygraph (Grass Instruments, Quincy, MA), which monitored and recorded changes in vessel-wall tension. The other hook was fixed to a screw gauge, which was used to stretch the vessel segments.
For the 24-hour incubation, the specimens were incubated in Dulbecco's Modified Eagles Medium (DMEM D6046; Sigma) containing penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mmol/L), and 15% heat-inactivated fetal bovine serum. Vascular rings in groups 2 and 4 were incubated with 10-6 mol/L cerivastatin, whereas those in groups 1 and 3 were incubated without cerivastatin. The rings were left in an incubator for 24 hours at 37°C. These incubation conditions have no significant effect on the viability of vessel segments.
Vascular function studies
After the incubation period, vascular function studies were started and contained 10-6 mol/L cerivastatin in groups 2 and 4 as previously reported [13, 14]. Initial pretensions of 80 mN and 50 mN were applied to each vascular ring of RA and LITA, respectively. Then they were relaxed out and were allowed to equilibrate for 30 minutes. Following that the rings were challenged with 90 mmol/L potassium chloride solution (KCl). The bath was washed out when the response reached a plateau followed by a return to the baseline. After the washout, 10-6 mol/L cerivastatin was supplemented in the bath for groups 2 and 4, and this allowed the study to keep the same concentration of cerivastatin in the organ baths throughout the experiment. This series of procedures was repeated again and the response to KCl at the second time was recorded as a result (pretensions of 80 mN for RA and 50 mN for LITA were applied on two separate occasions). When re-equilibration was obtained, tension was induced in each ring by the addition of 3x10-8 mol/L endothelin-1 (ET-1; Calbiochem, Nottingham, UK). This concentration of ET-1 was determined by a pilot study that was aimed to find out the minimum dose to achieve a stable plateau (data not shown). After a stable plateau of vasoconstriction, acetylcholine (ACH; 10-9 to 10-5 mol/L) was added to the bath in a cumulative fashion in 1/2 log10 units. The response to each concentration was allowed to reach a plateau before addition of the next concentration of ACH. Finally, 10-5 mol/L sodium nitroprusside (SNP) was added to the bath to ensure maximal relaxation.
Analysis of the data
All data were expressed as mean ± standard error of the mean (SEM). Changes in tension in response to ACH were normalized to the magnitude of the maximal contraction induced by ET-1. For analysis of the responses to ACH, median effective concentration (EC50) was calculated. The values of EC50 were transformed into geometric means (pEC50 = -log10EC50). The results of vascular function test was analyzed with ANOVA when normal distribution was confirmed, otherwise, nonparametric analysis was performed. To analyze the data of patients preoperative characteristics, X2 test was applied, except to patients'age and serum lipid data, which were analyzed by either Student's t test or Mann Whitney's U test. Results were considered significant if p values less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Vascular function studies
Vascular contraction by KCl was significantly higher in RA (113.2 ± 10.3 mN) than in LITA (36.4 ± 3.8 mN, p < 0.001). Similarly, vascular contraction by ET-1 was significantly higher in RA (78.4 ± 7.1 mN) than in LITA (32.0 ± 3.4 mN, p < 0.001). There was no significant evidence that contraction by KCl and ET-1 depended on incubation period or preoperative use of statins. Cerivastatin significantly decreased vascular contraction by KCl by 12.1 ± 5.1 mN independent of artery types, incubation periods, or preoperative use of statins. However, cerivastatin did not significantly change vascular contraction by ET-1 (decrease by 3.6 ± 4.0 mN, p = 0.37).
Although endothelium-dependent vasodilatation was higher in RA (57.7% ± 3.5%) than in LITA (46.5% ± 3.8%, p = 0.046), there was no significant evidence that it depended on the preoperative use of statins or incubation period. In vitro incubation with cerivastatin significantly increased endothelium-dependent vasodilatation by 14.2% ± 2.4% (p < 0.0001) independent of artery types (RA/LITA). After a 2-hour incubation preoperative use of statins tend to lead to a lower increase in endothelium-dependent vasodilatation with cerivastatin (6.0% ± 3.9% vs 18.6% ± 5.2%), although after a 24-hour incubation preoperative use of statins tend to lead to a higher increase with cerivastatin (23.4% ± 4.6% vs 8.2% ± 3.7%, p = 0.004; Fig 1A–1D; Tables 2 and 3).
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Vasodilatation by SNP was significantly higher in RA (116.9% ± 3.5%) than in LITA (106.2% ± 3.7%, p = 0.007). Preoperative use of statins decreased vasodilatation by SNP (103.1% ± 2.0% vs 119.6% ± 4.5%, p = 0.007). There was no significant evidence that vasodilatation by SNP depended on incubation period. In vitro incubation with cerivastatin did not significantly change vasodilatation by SNP (1.5% ± 3.6%, p = 0.67; Tables 2 and 3).
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Comment |
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The main use of statins is for their lipid lowering effect, which could reduce graft failure after CABG by an antisclerotic effect [12]. In addition, statins also have other beneficial effects, which include endothelium-dependent vasodilatation [6, 7], inhibition of the smooth muscle cell proliferation [8], and antioxidant [9] and antiinflammatory [10, 11] effects. We believe that these nonlipid-lowering actions of statins are largely responsible for the enhanced endothelial function seen in this study. Indeed patients may benefit from continued exposure to statins throughout the perioperative period (for example, by giving statins from a naso-gastric tube in early period).
Currently there are six different types of statins: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and cerivastatin [15]. Only pravastatin is hydrophilic and the others are lipophilic [15]. Inoue and colleagues [10] reported that lipophilic statins (simvastatin, fluvastatin, and cerivastatin) had an antiinflammatory effect on endothelial cells through reduction of mRNA levels for interleukin-1ß, interleukin-6, cyclooxygenase-2, and p22phox, however, pravastatin did not exhibit these kinds of effects. The usual dosage is 10 to 80 mg for lovastatin, 5 to 80 mg for simvastatin, 5 to 40 mg for pravastatin, 20 to 80 mg for fluvastatin, 10 to 80 mg for atorvastatin, and 0.1 to 0.8 mg for cerivastatin [15]. Only lovastatin and simvastatin are prodrugs, and bioavailability is less than 5% in lovastatin and simvastatin, 17% in pravastatin, 10% to 35% in fluvastatin, 12% in atorvastatin, and 60% in cerivastatin [15]. Therefore, in the present study, cerivastatin was selected for in vitro incubation because of nonprodrug, lipophilicity, and low dosage; although cerivastatin was withdrawn from the United States market in 2001 (http://www.fda.gov/cder/drug/infopage/baycol/default.htm). Cerivastatin is an entirely synthetic and enantiomerically pure HMG-CoA reductase inhibitor [16–18]. It was reported that 1-µM cerivastatin revealed antiinflammatory (no effect under this concentration) and antioxidant effect to protect endothelial nitric oxide synthase (eNOS) activity [9, 11]. Therefore, 1-µM cerivastatin was used in this study, although it was reported that maximum plasma concentration of cerivastatin was 2.27 to 2.88 µg/L (0.0047 to 0.0060 µM) after 200 µg of cerivastatin administration in healthy male volunteers [16].
Our data suggest that the in vitro administration of statin is more essential than the preoperative one. It is unclear from the present study if this relates to the inhibition of HMG-CoA reductase, antioxidant and antiinflammatory effects, stabilization of nitric oxide synthase (NOS) mRNA, or a yet undefined mechanism of action. However, it was reported that a 2-week treatment with Cerivastatin improved endothelial-dependent vasodilatation of human forearm vasculature, while this improvement was reversed by NOS inhibitor N(G)-monomethyl-L-arginine [7]. This could suggest that one of the important effects of cerivastatin on endothelium is related to NOS enhancement/preservation.
The RA and the LITA have been demonstrated to have different biological characteristics [13, 14, 19]. The RA is a thick-walled muscular artery with a mean width of the media reported to be approximately 500 µm, compared with 300 µm for the ITA [19, 20]. Chardigny and coworkers [21] reported that the prostacyclin (PGI2) basal production was greater in the ITA than in the RA, concluding that antispastic drugs were more indicated in case of using the RA as a conduit. We speculate that the regulation of NOS may also differ between LITA and RA. In addition, preexisting vascular disease might affect the results in our study. Kaufer and colleagues [22] investigated the incidence and the degree of arteriosclerosis in RA and ITA by classifying them to grade 0 to 4. Pathologically no atherosclerotic change (grade 0) was found in 77.4% of LITA and 46.2% of RA. In addition, grade 3 and 4 were 0% in LITA and 8.5% in RA. Kane-Toddhall and associates [23] also histologically examined the 177 RA, 168 IMA, and 86 long saphenous veins (SV) from the same patients undergoing CABG. Minimal atherosclerotic change (< 5% stenosis) were seen in 91% of ITA, 42% of RA, and 70% of SV. Thus the RA appears to be at risk of endothelial damage, thereby making it more amenable to the protective action of statins compared to the LITA.
Although it was initially difficult to determine the optimal incubation period, we decided on the periods of 2 and 24 hours in order to look for immediate effects mediated by cerivastatin and those associated with changes in gene expression and protein synthesis [8, 9, 11]. In vitro cerivastatin treatment displayed a better endothelial function, although the data of pEC50 exhibited deterioration of endothelial function between 2 and 24 hours. In addition, contraction by ET-1 did not decrease with time. Therefore, we speculate that the beneficial effect of cerivastatin on endothelium-dependent vasodilatation might be due to preserving the endothelium rather than inducing additional effect on the NOS. Possible mechanisms of endothelial deterioration could be oxidative stress, inflammatory change, or reduction of eNOS protein and mRNA [9–11, 24]. A limitation of this study is that we did not perform histologic studies, such as an immunohistochemistry and scanning with an electron microscope, which might be useful to examine the endothelial cells in detail.
In conclusion, in vitro incubation with cerivastatin preserved endothelial function more effectively than preoperative use of statins. This suggests that the postoperative (may include intraoperative) administration of statins could improve the endothelial function of the arterial grafts in patients undergoing CABG.
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Acknowledgments |
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= group 2; 
= group 3; 
= group 4. (ET-1 = endothelin-1; LITA = left internal thoracic artery; RA = radial artery.)