![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR
Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota (S.G., R.R.T., S.T.O.) and Department of Internal Medicine, University of North Dakota, Grand Forks, North Dakota (S.K., M.R.)
Received October 9, 2006; accepted December 11, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
,9-epoxymethano-prostaglandin F2; 10-8 M) [-log (M) EC50 (pD2) = 7.28 ± 0.1; Emax = 57 ± 6%] or phenylephrine (10-6 M) (pD2 = 7.16 ± 0.4; Emax = 45 ± 9%). Sirolimus-induced relaxation was unaffected by treatment with indomethacin (10-5 M) but was nearly abolished in tissues contracted by depolarization with elevated K+ (60 mM). In U46619
[GenBank]
-contracted rings, the response to sirolimus was markedly inhibited in the presence of the specific ATP-sensitive potassium (KATP) channel blocker, glyburide (10-6 M), but was unaffected by treatment with blockers of large conductance, calcium-activated potassium channel (iberiotoxin, 10-7 M), small conductance, calcium-activated potassium channel (apamin, 10-6 M), or voltage-gated potassium channel (4-aminopyridine, 10-3 M). The KATP channel opener, aprikalim (10-7 to 10-5 M), caused concentration-dependent relaxations that were inhibited by glyburide (10-6 M) and abolished in tissues contracted with elevated K+ (60 mM), thus confirming that KATP channel opening causes relaxation of these arteries. These data suggest that sirolimus, at concentrations attained in vivo, causes relaxation of human arteries, and this effect is mediated by opening of KATP channels in vascular smooth muscle. Reduced vasomotor tone is a heretofore unrecognized action of sirolimus that could potentially contribute to its efficacy in drug-eluting stents.
). In particular, stents coated with polymers that gradually release sirolimus have generated considerable interest. Sirolimus is a potent immunosuppressant and antiproliferative agent, and restenosis rates are almost negligible with sirolimus-eluting stents (Morice et al., 2002; Sousa et al., 2003).
The cellular effects of sirolimus are mediated by binding of the drug to its cytosolic receptor, the FK506-binding protein, which results in blockade of cell cycle progression at the G1/S transition (Sehgal, 2003). The inhibitory effects of sirolimus on cell growth and migration in blood vessels are widely recognized and are believed to play a pivotal role in the remarkable efficacy of sirolimus in maintaining the patency of implanted stents (Marks, 2003; Asnaghi et al., 2004). Surprisingly, little is known about the effects of sirolimus on blood vessel function (i.e., vasodilation and vasoconstriction). This is particularly notable since sirolimus is released directly into the circulation (Hiatt et al., 2001; Suzuki et al., 2001), where it could potentially alter blood flow and tissue perfusion in regions distal to the site of stent placement. A limited number of preliminary studies have attempted to address this issue by using animal models, but the results are conflicting (Corbin et al., 1994; Milliard et al., 1998; Jeanmart et al., 2002; Gardiner et al., 2004). Hence, the present study was designed to determine the vasomotor effects of sirolimus in human blood vessels.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Organ Chamber Studies. Arterial rings were suspended in water-jacketed organ chambers filled with 25 ml of physiological salt solution, which were aerated with a mixture of 95% O2 and 5% CO2 and maintained at 37°C (O'Rourke, 1996a,b). The rings were suspended by means of two fine stainless steel wire clips passed through their lumen; one clip was anchored to the bottom of the organ chamber, whereas the other was connected to a strain-gauge force transducer for the purpose of measuring isometric force. The rings were placed individually at the optimal point of their length-tension relationship by progressively stretching them until the contractile response to KCl (20 mM) was maximal. The blood vessels were then allowed to equilibrate at their optimal length for 1 h before exposure to vasoactive substances.
Vasodilator responses were determined in rings contracted with phenylephrine (10-6 M), U46619 [GenBank] (10-8 M), or KCl (60 mM). After the contractions reached a stable plateau, increasing concentrations of sirolimus (10-10 to 10-6 M), diltiazem (10-8 to 10-4 M), or aprikalim (10-7 to 10-5 M) were added to the organ chambers, and relaxations were recorded. In some experiments, indomethacin (10-5 M) was added to the organ chamber 30 min before addition of the contractile agent to inhibit the production of vasoactive prostanoids. In other experiments, glyburide (10-6 M), iberiotoxin (10-7 M), apamin (10-6 M), or 4-aminopyridine (10-3 M) were added to the organ chamber 30 min before addition of the contractile agent to inhibit ATP-sensitive potassium (KATP) channels, large conductance, calcium-activated potassium channels (BKCa), small conductance, calcium-activated potassium channels (SKCa), and voltage-gated potassium channels (Kv), respectively. Control and treated rings prepared from the same artery were studied in parallel.
Data Analysis. Relaxations are expressed as a percentage of the initial vasoconstrictor-induced tone. The maximal decrease in tension (Emax) and the concentration necessary to produce 50% of its own maximal response (EC50) were determined. The EC50 values were converted to the negative logarithms and expressed as -log (M) EC50 (pD2). Results are expressed as mean ± S.E.M., and n refers to the number of patients from whom blood vessels were taken. Mean values were compared by Student's t test or analysis of variance. Values were considered to be significantly different when P < 0.05.
Drugs and Solutions. The following drugs were used: acetylcholine, 4-aminopyridine, diltiazem, glyburide, indomethacin, and phenylephrine (Sigma-Aldrich, St. Louis, MO); apamin and iberiotoxin (Tocris, Ellisville, MO); aprikalim (Rhone Poulenc Rorer, Alfortville, France); sirolimus (LC Laboratories, Woburn, MA); and U46619 [GenBank] (Pharmacia and Upjohn, Kalamazoo, MI). Drug solutions were prepared daily, kept on ice, and protected from light until used. All drugs were dissolved initially in distilled water with the exception of aprikalim, which was dissolved in ethanol; glyburide, which was dissolved in 0.1 N NaOH; indomethacin, which was dissolved in an aqueous solution of sodium bicarbonate; and sirolimus, which was dissolved in dimethylsulfoxide, before further dilution in distilled water. Drugs were added to the organ chambers in volumes not greater than 0.2 ml. Drug concentrations are reported as final molar concentration in the organ chamber.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
1-adrenoceptor agonist, phenylephrine (10-6 M) (Fig. 1, bottom). These concentrations of sirolimus had no direct contractile or relaxant effects on unstimulated radial artery rings. Treatment of the rings with indomethacin (10-5 M) had no effect on the concentration-response curve to sirolimus (Fig. 2), suggesting that cyclooxygenase metabolites of arachidonic acid play no role in the relaxations elicited by sirolimus.
|
|
), completely inhibited the contractile response to KCl in a concentration-dependent manner (Fig. 3, bottom). The pD2 was 5.33 ± 0.1, and the maximal relaxation was 97 ± 3% for diltiazem-induced relaxation of rings contracted with KCl (60 mM).
|
Because the lack of effect of sirolimus in tissues contracted by depolarization with KCl suggested that sirolimus-induced relaxation is sensitive to changes in membrane potential, we tested whether or not the response to sirolimus is mediated via potassium channel activation. In rings contracted with U46619
[GenBank]
(10-8 M), the selective KATP channel blocker, glyburide (10-6 M) (Ashcroft and Ashcroft, 1990), significantly inhibited sirolimus-induced relaxation (Fig. 4, top), whereas selective blockers of other potassium channel subtypes, including iberiotoxin (10-7 M), apamin (10-6 M), and 4-aminopyridine (10-3 M) were without effect on the response to sirolimus (n = 5; data not shown). Aprikalim (10-7 to 10-5 M), a selective KATP channel opener (Atwal, 1992), caused concentration-dependent relaxations of human radial artery rings contracted with U46619
[GenBank]
, and this response was also inhibited significantly in the presence of glyburide (10-6 M) (Fig. 4, bottom). Like sirolimus, aprikalim (10-8 to 10-4 M) failed to cause relaxation in rings contracted by depolarization with KCl (60 mM) (n = 4; data not shown).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The vasodilator effect of sirolimus in human radial arteries is not likely due to the release of an endothelium-derived relaxing factor, such as nitric oxide or endothelium-derived hyperpolarizing factor, because the experiments were performed on arterial rings in which the endothelium had been removed. Moreover, the cyclooxygenase inhibitor, indomethacin, had no effect on the response to sirolimus, thus ruling out a role for prostacyclin or other vasodilator prostaglandins in the observed response. These findings are consistent with a direct action of sirolimus on vascular smooth muscle cells to cause relaxation of human arteries.
The smooth muscle relaxant effect of sirolimus was not limited to contractile responses evoked by a single vasoconstrictor. Sirolimus was effective in relaxing arteries contracted with the thromboxane A2-analog, U46619
[GenBank]
, and the 1-adrenoceptor agonist, phenylephrine, suggesting that sirolimus may interfere with multiple receptor-mediated vasoconstrictor mechanisms. In contrast, sirolimus had little effect on contractions induced by elevated extracellular potassium. Potassium-induced contractions are the result of membrane depolarization, which causes increased calcium entry via voltage-operated calcium channels in vascular smooth muscle cells (O'Rourke et al., 2005). This mechanism was verified in human radial arteries by the experiments with diltiazem, a selective inhibitor of voltage-operated calcium channels that caused complete relaxation of potassium-induced contractions in a concentration-dependent manner. The lack of effect of sirolimus under these same conditions suggests that the mechanism of action of sirolimus is dependent on membrane potential but, unlike diltiazem, does not involve direct blockade of extracellular calcium entry via voltage-operated calcium channels.
One mechanism by which sirolimus could elicit vascular smooth muscle relaxation is via activation of potassium channels, which results in smooth muscle relaxation that is dependent on membrane potential and is abolished in the presence of high concentrations of extracellular potassium (Hamilton et al., 1986; Cook et al., 1988). Several potassium channel subtypes are expressed in vascular smooth muscle cells (Brayden, 1996), including KATP,BKCa,SKCa, and Kv. Because glyburide, a potent and selective KATP channel blocker (Ashcroft and Ashcroft, 1990), markedly inhibited sirolimus-induced relaxations, it is likely that the mechanism of this response to sirolimus involves opening of KATP channels. Moreover, the effect of sirolimus seems to be selective for KATP channels because blockers of BKCa (i.e., iberiotoxin) (Galvez et al., 1990), SKCa (i.e., apamin) (Banks et al., 1979), and Kv (i.e., 4-aminopyridine) (Hille, 2001) had no effect on the concentration-response curve to sirolimus. That opening of KATP channels causes relaxation of human radial arteries is confirmed by the results with aprikalim (Atwal, 1992), a selective KATP channel opener that caused concentration-dependent relaxations that were, like those to sirolimus, inhibited by glyburide and abolished in the presence of high concentrations of extracellular potassium. These results are in agreement with previous studies indicating a role for KATP channels in regulating vasomotor tone in human arteries (Miura et al., 2003; Wareing et al., 2006).
The molecular events underlying sirolimus-induced relaxation of human vascular smooth muscle cells remain to be elucidated. The pharmacologic data presented in the current study provide strong evidence for a role for KATP channels in the smooth muscle-relaxing effect of sirolimus; however, this interpretation requires confirmation by electrophysiological studies of the effects of sirolimus on potassium currents in human vascular smooth muscle. Nevertheless, the feasibility of this mechanism is supported by recent studies in neurons and cardiac myocytes, where sirolimus modulates potassium channel activity through binding to FK506-binding protein, which may interact directly with potassium channels or associated proteins (Terashima et al., 1998; DuBell et al., 2000).
There are few reports at present of studies designed to investigate the effects of sirolimus on vasomotor tone. A direct effect of sirolimus on vasomotion was initially demonstrated in rat isolated aortic rings (Corbin et al., 1994), where acute in vitro administration of sirolimus reduced certain vasoconstrictor responses in an endothelium-dependent manner. Under in vivo conditions, however, i.v. injection of sirolimus into conscious rats caused a modest pressor response and decrease in regional blood flow (Gardiner et al., 2004), consistent with a vasoconstrictor effect. In light of the present results demonstrating that sirolimus causes endothelium-independent relaxation of human arteries, the importance of species differences and the need to assess the pharmacologic activity of vasoactive drugs in human tissues are readily apparent.
Chronic exposure to sirolimus may indirectly alter arterial diameter by causing endothelial dysfunction. In an in vitro porcine model, incubation of isolated coronary arteries with sirolimus for 48 h caused impairment of endothelium-dependent relaxations evoked by serotonin and bradykinin (Jeanmart et al., 2002). In humans, implantation of sirolimus-eluting stents, but not bare metal stents, was associated with impaired endothelium-dependent vasodilation in response to exercise or acetylcholine in coronary arteries distal to the stent (Togni et al., 2005; Hofma et al., 2006). Coronary artery dilation by the endothelium-independent vasodilator, nitroglycerin, was not altered. These data, although not directly comparable with the present results due to differences in experimental conditions, emphasize that the vasomotor effects of sirolimus are complex and may differ with regard to acute effects (i.e., vasodilation) versus long-term effects (i.e., endothelial dysfunction).
In summary, sirolimus causes relaxation of human arterial smooth muscle in a manner consistent with the opening of KATP channels. The smooth muscle-relaxing effect of sirolimus is observed at physiologically relevant concentrations, inasmuch as systemic whole-blood levels of sirolimus on the order of 
10 nM may be achieved following stent implantation in coronary arteries (Suzuki et al., 2001; Yu et al., 2004) and oral administration of sirolimus to prevent restenosis (Guarda et al., 2004; Rodriguez et al., 2005). Although sirolimus concentrations of this magnitude are on the low end of the concentration-response relationship reported in the present study, it is likely that significantly higher local drug concentrations are attained in arteries located in close proximity to a drug-eluting stent because the lipophilicity of sirolimus considerably enhances its uptake, distribution, and retention in the arterial wall (Schreiber, 1991; Suzuki et al., 2001). Thus, relaxation of vascular smooth muscle is a heretofore unrecognized action of sirolimus that could potentially contribute to its efficacy in drug-eluting stents.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: FK506, tacrolimus; U46619
[GenBank]
, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2; KATP, ATP-sensitive potassium; BKCa, large conductance, calcium-activated potassium channel; SKCa, small conductance, calcium-activated potassium channel; KV, voltage-gated potassium channel; pD2, -log (M) EC50.
1 Current affiliation: Respiratory and Inflammation Center of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, Pennsylvania. 
Address correspondence to: Dr. Stephen T. O'Rourke, Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105-5055. E-mail: stephen.orourke{at}ndsu.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ashcroft SJ and Ashcroft FM (1990) Properties and functions of ATP-sensitive K-channels. Cell Signal 2: 197-214.[CrossRef][Medline]
Asnaghi L, Bruno P, Priulla M, and Nicolin A (2004) mTOR: a protein kinase switching between life and death. Pharmacol Res 50: 545-549.[CrossRef][Medline]
Atwal KS (1992) Modulation of potassium channels by organic molecules. Med Res Rev 12: 569-591.[CrossRef][Medline]
Banks BE, Brown C, Burgess GM, Burnstock G, Claret M, Cocks TM, and Jenkinson DH (1979) Apamin blocks certain neurotransmitter-induced increases in potassium permeability. Nature (Lond) 282: 415-417.[CrossRef][Medline]
Brayden JE (1996) Potassium channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 23: 1069-1076.[Medline]
Cook NS, Weir SW, and Danzeisen MC (1988) Anti-vasoconstrictor effects of the K+ channel opener cromakalim on the rabbit aorta: comparison with the calcium antagonist isradipine. Br J Pharmacol 95: 741-752.[Medline]
Corbin F, Blaise GA, Parent M, Chen H, and Daloze PM (1994) Effect of rapamycin on rat aortic ring vasomotion. J Cardiovasc Pharmacol 24: 813-817.[Medline]
DuBell WH, Lederer LW, and Rogers TB (2000) K(+) currents responsible for repolarization in mouse ventricle and their modulation by FK-506 and rapamycin. Am J Physiol 278: H886-H897.
Fattori R and Piva T (2003) Drug-eluting stents in vascular intervention. Lancet 361: 247-249.[CrossRef][Medline]
Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P, Kaczorowski GJ, and Garcia ML (1990) Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem 265: 11083-11090.
Gardiner SM, March JE, Kemp PA, Fallgren B, and Bennett T (2004) Regional haemodynamic effects of cyclosporine A, tacrolimus and sirolimus in conscious rats. Br J Pharmacol 141: 634-643.[CrossRef][Medline]
Guarda E, Marchant E, Fajuri A, Martinez A, Moran S, Mendez M, Uriarte P, Valenzuela E, and Lazen R (2004) Oral rapamycin to prevent human coronary stent restenosis: a pilot study. Am Heart J 148: e9.[CrossRef][Medline]
Hamilton TC, Weir SW, and Weston AH (1986) Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein. Br J Pharmacol 88: 103-111.[Medline]
Hiatt BL, Carter AJ, and Yeung AC (2001) The drug-eluting stent: is it the Holy Grail? Rev Cardiovasc Med 2: 190-196.[Medline]
Hille B (2001) Ion Channels of Excitable Membranes, Sinauer Assoc., Sunderland, MA.
Hofma SH, van der Giessen WJ, van Dalen BM, Lemos PA, McFadden EP, Sianos G, Ligthart JM, van Essen D, de Feyter PJ, and Serruys PW (2006) Indication of long-term endothelial dysfunction after sirolimus-eluting stent implantation. Eur Heart J 27: 166-170.
Jeanmart H, Malo O, Carrier M, Nickner C, Desjardins N, and Perrault LP (2002) Comparative study of cyclosporine and tacrolimus vs newer immunosuppressants mycophenolate mofetil and rapamycin on coronary endothelial function. J Heart Lung Transplant 21: 990-998.[CrossRef][Medline]
Marks AR (2003) Rapamycin: signaling in vascular smooth muscle. Transplant Proc 35: 231S-233S.[CrossRef][Medline]
Milliard S, Silva A, Blaise G, Chen H, Xu D, Qi S, and Daloze P (1998) Rapamycin's effect on vasomotion in the rat. Transplant Proc 30: 1036-1038.[CrossRef][Medline]
Miura H, Wachtel RE, Loberiza FR Jr, Saito T, Miura M, Nicolosi AC, and Gutterman DD (2003) Diabetes mellitus impairs vasodilation to hypoxia in human coronary arterioles: reduced activity of ATP-sensitive potassium channels. Circ Res 92: 151-158.
Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Hayashi E, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, et al. (2002) A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 346: 1773-1780.
O'Rourke ST (1996a) Effects of potassium channel blockers on resting tone in isolated coronary arteries. J Cardiovasc Pharmacol 27: 636-642.[CrossRef][Medline]
O'Rourke ST (1996b) KATP channel activation mediates nicorandil-induced relaxation of nitrate-tolerant coronary arteries. J Cardiovasc Pharmacol 27: 831-837.[CrossRef][Medline]
O'Rourke ST, Vanhoutte PM, and Miller VM (2005) Biology of blood vessels: vascular pharmacology, in Vascular Medicine: A Companion to Braunwald's Heart Disease (Creager M, Dzau VJ, and Loscalzo J eds) pp 71-98,
Saunders Elsevier, Philadelphia, PA. Rodriguez AE, Rodriguez Alemparte M, Vigo CF, Fernandez Pereira C, Llaurado C, Vetcher D, Pocovi A, and Ambrose J (2005) Role of oral rapamycin to prevent restenosis in patients with de novo lesions undergoing coronary stenting: results of the Argentina single centre study (ORAR trial). Heart 91: 1433-1437.
Schreiber SL (1991) Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science (Wash DC) 251: 283-287.
Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 35: 7S-14S.[CrossRef][Medline]
Sousa JE, Costa MA, Abizaid A, Sousa AG, Feres F, Mattos LA, Centemero M, Maldonado G, Abizaid AS, Pinto I, et al. (2003) Sirolimus-eluting stent for the treatment of in-stent restenosis: a quantitative coronary angiography and three-dimensional intravascular ultrasound study. Circulation 107: 24-27.
Suzuki T, Kopia G, Hayashi S, Bailey LR, Llanos G, Wilensky R, Klugherz BD, Papandreou G, Narayan P, Leon MB, et al. (2001) Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation 104: 1188-1193.
Terashima A, Nakai M, Hashimoto T, Kawamata T, Taniguchi T, Yasuda M, Maeda K, and Tanaka C (1998) Single-channel activity of the Ca2+-dependent K+ channel is modulated by FK506 and rapamycin. Brain Res 786: 255-258.[CrossRef][Medline]
Togni M, Windecker S, Cocchia R, Wenaweser P, Cook S, Billinger M, Meier B, and Hess OM (2005) Sirolimus-eluting stents associated with paradoxic coronary vasoconstriction. J Am Coll Cardiol 46: 231-236.
Wareing M, Bai X, Seghier F, Turner CM, Greenwood SL, Baker PN, Taggart MJ, and Fyfe GK (2006) Expression and function of potassium channels in the human placental vasculature. Am J Physiol 291: R437-R446.
Yu MY, Gao RL, Jiang J, Cheng SJ, Yuan JQ, Wang CN, Zheng JG, Meng L, and Zi ZJ (2004) Pharmacokinetics of rapamycin-eluting stents in miniswine coronary model. Chin Med J (Engl) 117: 1459-1463.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
