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Eur J Cardiothorac Surg 2004;26:1149-1155
© 2004 Elsevier Science NL
a Cardiothoracic Research Lab, Division of Cardiothoracic Surgery, Emory University School of Medicine, 550 Peachtree Street, NE, Atlanta, GA 30308-2225, USA
b Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA
Received 7 May 2004; received in revised form 21 August 2004; accepted 24 August 2004.
* Corresponding author. Tel.: +1 404 686 2511; fax: +1 404 686 4888. (E-mail: jvinten{at}emory.edu).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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A number of agents have been used for prophylaxis against arterial conduit vasospasm, including phosphodiesterase inhibitors [16], class I antiarrhythmic agents [5], alpha adrenergic receptor antagonists [15,18], calcium channel blockers, and nitric oxide donors. While these agents have been somewhat successful in alleviating intraoperative vasospasm, most are short-acting and provide for inhibition of vasospasm in the intraoperative period only. Furthermore, most of these antispasmodic therapies, which target specific stimulators of contraction, do not address the redundant extracellular stimuli and intracellular signals that regulate vascular smooth muscle cell contraction and arterial vasospasm. Therefore, although each of the above agents may inhibit one stimulus of vasospasm, other mechanisms remain operative that may cause vasospasm in the postoperative period.
It is well-known that phosphorylation of the 20kD subunit of myosin light chain (MLC) by myosin light chain kinase (MLCK) is a key regulatory step in the development of force in the vascular smooth muscle cell. Phosphorylation of MLC by MLCK allows binding of actin to myosin, followed by activation of the myosin-ATPase, and the subsequent contraction of the smooth muscle cell [13]. Inhibiting MLC phosphorylation, one of the final steps in the mechanism of vascular smooth muscle contraction, would address the multiplicity of causes of vasospasm. In theory, by inhibiting MLCK activity, smooth muscle cell contraction and vasospasm could potentially be abolished, independent of the source of stimulation. In addition, the ideal treatment agent would be long-acting, so that a brief treatment of the conduit vessel prior to implantation would be effective for several postoperative days.
Wortmannin (WT), a product of the fungus Talaromyces wortmannin, is an irreversible inhibitor of MLCK [8]. Although no previous studies have been conducted on the long-term effect of WT on vasospasm in arterial grafts, it has been shown to be an effective inhibitor of vasoconstriction in the rat thoracic aorta [8], the swine carotid artery [19], and the rabbit basilar artery [23] in the acute setting. In the present study, we tested the hypotheses that (1) blockade of MLCK activity with WT would attenuate vasoconstriction of canine brachial arteries in response to exogenous and endogenous stimuli in an ex vivo organ chamber model, and (2) this inhibition would be long-acting because of the irreversible nature of the interaction between WT and MLCK.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.2. Animal preparation and artery harvest
All animal care was conducted under the approval of the institutional animal care and use committee, and in compliance with the European Convention on Animal Care.
Canine brachial arteries, equivalents of radial arteries in humans, were used in this study since they were more readily available and we could obtain longer segments than human radial arteries. Based on our previous publications, we have determined that the contractile and relaxation properties of these canine vessels are similar to the properties of human radial arteries in organ chamber studies [3,18]. The arteries were harvested as a pedicle graft and stored in 4°C KH buffer (in mM/l: glucose 11; magnesium sulfate, 1.2; potassium phosphate 1.2; KCl, 4.7; sodium chloride 118; calcium chloride, 2.5) at pH 7.4 until ready for use.
2.3. Vessel preparation, treatment and testing of contractile function
The vessels were carefully skeletonized, cut into 4–5mm segments, and soaked in a 1mM solution of WT in KH buffer and 10% DMSO (used to increase solubility of WT), pH 7.4 at 37°C for 60min. Control vessels were soaked in KH buffer with 10% DMSO only. The vessels were then rinsed well with KH to remove all unbound drug. For testing of contractile responses, the arterial segments were mounted on steel hooks in glass organ chambers (Radnoti Glass, Monrovia, CA) in KH buffer at pH 7.4 and 37°C, and continuously bubbled with a gas mixture of 95% O2 and 5% CO2. One steel hook was held stationary while the other was attached to a force transducer. Force generated by the vessels in response to various agonists was recorded using an analog-to-digital converter and SPECTRUM Cardiovascular Acquisition and Analysis software (Wake Forest University, Winston–Salem, NC).
The vessel segments were allowed to stabilize at a baseline tension of 3g for 60min prior to testing the effect of various vasoconstrictor agents. During this time, the buffer was changed every 20min. The total time from the end of WT treatment until contractile responses were tested was approximately 2h. The contractile responses to 1µM NE (
1 receptor-dependent, calcium-dependent), 1µM 5-HT (serotonin receptor-dependent, calcium-independent), 3µM U46 (thromboxane A2 receptor-dependent, calcium-independent), and 60mM KCl (receptor-independent depolarizing agent) were then quantified. These drug concentrations were chosen after preliminary studies indicated that they would provide the maximal level of contraction that was still detectable by our system. The order in which the constricting agents were administered was varied randomly in order to avoid the effects of one drug on another. However, KCl was always administered last since high concentrations of KCl have been shown to have damaging effects on vascular endothelium. The vessels were thoroughly washed and allowed to stabilize for 20min between vasoconstrictor agents. The resting tension was adjusted to 3g during each stabilization period.
2.4. Contractile response at 48h
To test the duration of MLCK inhibition by the 60min pre-treatment with 1mM WT, a subset of vessels were harvested and treated with WT as above, then incubated in drug-free sterile culture medium [Dulbecco's modified eagle medium (Sigma, St Louis MI), with 10% newborn calf serum (Gibco, Inc.) and 1% penicillin–streptomycin] under sterile conditions for 48h in an incubator aerated with 95% O2 and 5% CO2. The culture medium was changed every 12h during this time. At the end of the 48h period, the vessels were removed, rinsed with KH buffer, and contractile responses were tested as described above.
2.5. Testing of smooth muscle relaxing capacity
Vascular smooth muscle integrity has been shown to be impaired after some treatment strategies [6,17]. Therefore, to test the vasorelaxation capacity of the vessels after treatment with WT, the vessels were pre-constricted with 3µM U46, and then exposed to incrementally increasing concentrations (50nM to 5mM) of SNP, a direct smooth muscle dilator.
Endothelial-dependent vasorelaxation (i.e. vasodilatation in response to acetylcholine or bradykinin) was not tested in this study since WT is a known inhibitor of phosphoinositide-3 (PI-3) kinase, which mediates acetylcholine- and bradykinin-induced activation of endothelial nitric oxide synthase (eNOS) [7].
2.6. Evaluation of vessels for morphologic injury and apoptosis
Prolonged storage in cell culture environments has the potential to cause cellular injury which might impair smooth muscle function. To confirm that the lack of contraction we observed in WT-treated vessels was not a function of cellular damage, a subset of vessels was tested for histological signs of injury and apoptosis after treatment with WT and incubation for 48h. For histology, formalin-fixed vessels were embedded in paraffin blocks, cut into sections for slide preparation, and stained with hematoxylin and eosin (H and E) prior to examination for signs of morphologic damage.
Endothelial and smooth muscle cell apoptosis was evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. In preparation for the TUNEL assay, the vessels were embedded in optimal cutting temperature compound (OCT, Sakura Finetek, Torrance, CA). Cryosections from frozen tissue were obtained using a Hacker-Bright cryostat and thaw-mounted onto Fisher-Plus slides (Fisher Scientific). Determination of apoptosis was performed using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN). DNA strand breaks were labeled with fluorescein-dUTP, followed by the addition of a secondary, antifluorescein antibody conjugated with alkaline phosphatase, a converter which generates a red color from Vector Red substrate. Following counterstaining with hematoxylin and dehydration in graded alcohols, the number of apoptotic cells (indicated by red-stained nuclei) per high power field (HPF) was counted under light microscopy and is expressed as a percentage of the total number of nuclei per HPF.
2.7. Statistical analysis
All data are expressed as mean±standard error of the mean (SEM). Differences in groups were determined by a one way analysis of variance (ANOVA) with Student–Newman–Keuls post-hoc analysis corrected for multiple comparisons. If data failed tests of normality, a Kruskal–Wallis ANOVA on ranks or a Mann–Whitney Rank Sum test was performed. A P-value of less than 0.05 was considered to be statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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At 48h, with lower concentrations of SNP (
0.5µM), the WT-treated group had a diminished relaxation response compared to controls (76.01±2.60% for WT-treated vs. 87.49±2.91% for controls, P=0.01, Fig. 2). However, maximal relaxation in response to SNP at higher concentrations (>0.5µM) did not differ between the two groups (115.15±3.90% for WT-treated vs. 107.90±2.21% for controls, P=0.085, Fig. 3).
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3.4. Evaluation of endothelial cell and smooth muscle cell apoptosis
Forty-eight hours after treatment with WT and incubation in culture medium, the vessels were examined for apoptosis. In the smooth muscle of WT-treated vessels, there was a greater percentage of TUNEL-positive cells (4.63±1.04%) than in control vessels (0.35±0.16%, P=0.003) (Fig. 3). In the endothelium, there were relatively more TUNEL-positive cells in the WT group (16.88%±4.90%) than in the control group (4.47±2.90%). This difference, however, did not reach statistical significance (P=0.058) (Fig. 4).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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In this study, we compared the effects of wortmannin-treated vs. vehicle-treated vessels. However, using a similar protocol, we have previously published the effects of phenoxybenzamine, an irreversible –1 receptor blocker [3,4,18] and papaverine on radial artery contraction both at 2 and 48h after treatment [18] In our previous study, phenoxybenzamine was effective in preventing alpha-agonist-induced contractions at 48h following treatment; however, while phenoxybenzamine provides long-lasting inhibition of alpha-adrenergic vasoconstrictors, it does not prevent vasospasm induced by other potentially important stimuli [2]. We have also previously demonstrated that papaverine, the current gold standard prophylactic treatment for intraoperative vasospasm, had a brief duration of action, with virtually no effect after the drug has been washed away [18]. In contrast, our results from the current study indicate that wortmannin significantly attenuates contraction induced by both calcium-dependent and independent mechanisms at 48h following treatment and washout.
Nitric oxide donors and calcium channel blockers are commonly used agents to treat postoperative vasospasm. Like papaverine, these vasodilators are short-acting and are usually administered as an intravenous infusion during the first 24 postoperative hours, followed by oral administration. As a result of this systemic administration, they may have undesirable side effects such as hypotension, bradycardia, and negative inotropy [1,12]. In addition, prolonged use of nitroglycerin has been associated with endothelial dysfunction and nitrate tolerance which may cause reflex vasospasm upon withdrawal [11]. Therefore, treatment of postoperative vasospasm with these presently administered agents may be of limited effectiveness.
Although direct MLCK inhibition prevents long-term vasospasm in the current study, the use of wortmannin as the specific inhibitor has some limitations, particularly the observation of smooth muscle and endothelial cell apoptosis. This finding can be explained by the fact that wortmannin, in addition to inhibiting MLCK, is a potent inhibitor of phosphoinositide-3 (PI-3) kinase [20], which has been shown to have antiapoptotic activity in some cell culture lines. Therefore, inhibition of the antiapoptotic effects of PI-3 kinase may allow initiation of apoptosis triggered by other signals. Accordingly, blockade of PI-3 kinase activity with wortmannin has been associated with the development of apoptosis in these cells in vitro [22]. It is therefore likely that the smooth muscle and endothelial cell apoptosis seen in our study is a result of PI-3 kinase inhibition by wortmannin.
While apoptosis of the smooth muscle and endothelium is undesirable, its implications for long-term graft function and patency are unclear. Other antispasmodic treatments, including papaverine, have been shown to have similar pro-apoptotic effects on vessels. Several studies have shown that papaverine causes both impairment of endothelial function and endothelial cell apoptosis in radial and internal mammary arteries [4,6,10]. Nevertheless, topical application of papaverine has not proven to have any long-term detrimental effects. With regard to the current study, inhibition of PI-3 kinase and its physiological consequences could potentially be averted by using a more selective MLCK inhibitor which is devoid of PI-3 kinase inhibitory activity. While such agents have been described in the literature, they are not currently commercially available [21].
In many ex vivo organ chamber studies, such as we have carried out, vascular endothelial function is tested by measuring relaxation responses to an endothelial-dependent vasodilating agent, such as acetylcholine or bradykinin. However, vasorelaxation induced by these agonists depends on the phosphorylation and activation of eNOS by Akt, a PI-3 kinase-dependent protein kinase [7]. Therefore, since wortmannin inhibits PI-3 kinase, and consequently phosphorylation of Akt into its active form, it follows that acetylcholine-dependent vasodilation would also be inhibited by wortmannin. Thus, in this study, we were unable to utilize endothelial-dependent relaxation as an assessment of endothelial function. In addition, since inhibition of MLCK with wortmannin limits vasoconstriction almost completely in the early period and by 50% at 48h, relaxation responses could not be accurately compared to control groups, which contracted to a greater extent. Therefore, we used histology and TUNEL staining to evaluate potential cellular damage caused by treatment with wortmannin.
Despite the potential detrimental effects of PI-3 kinase inhibition outlined above, a recent study by Su et al. [14] suggests that the inhibition of PI-3 kinase may indeed be an important mechanism in the antispasmodic action of wortmannin. While the study by Su did not examine the effects of wortmannin specifically, their results using a selective PI-3 kinase inhibitor suggest that the activation of PI-3 kinase may be involved in MLC-dependent and independent pathways of vascular smooth muscle contraction. Therefore, while the inhibitory effect of wortmannin on PI-3 kinase may have some undesirable consequences, it may also be partly responsible for the potent antispasmodic effect presented in this study.
Finally, wortmannin has been described in previous literature as an irreversible inhibitor of MLCK [20]. However, our results indicate that 48h after treatment with wortmannin, the vessels regained about 50% of their contractile activity. Why this occurs is not entirely clear, but may be explained in part by the turnover and new synthesis of MLCK. Alternatively, there may be unidentified MLCK-independent mechanisms of smooth muscle cell contraction which are not active initially, but become activated at the 48h time point. Furthermore, it is possible that in a biological setting, physiologically circulating factors might produce vascular changes that reverse the inhibitory properties of the drug. Although we tried to mimic the physiologic milieu by storing the treated vessels in culture medium at physiologic pH and temperature, the long-term inhibitory properties of wortmannin would ideally be assessed in an in vivo setting.
In summary, irreversible inhibition of MLCK by wortmannin nearly abolishes vasoconstriction stimulated by an array of vasopressor agents in the early period after pre-treatment. Furthermore, this treatment strategy provides sustained attenuation of vasoconstriction in response to all potentially vasoconstrictor stimuli for up to 48h. While we do not currently encourage the clinical use of WT for this purpose, we feel that further laboratory studies should be conducted in vivo to validate the concept of MLCK inhibition, evaluate graft patency, and identify MLCK inhibitors with fewer side effects. Inhibition of MLCK could be an effective technique to prevent conduit vessel vasospasm in the postoperative period.
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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-adrenegic stimuli. J Thorac Cardiovasc Surg 2003;126:1549-1554.
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