Interact CardioVasc Thorac Surg 2008;7:916-918. doi:10.1510/icvts.2008.175281
© 2008 European Association of Cardio-Thoracic Surgery
Brief communication - Cardiac general |
Phenoxybenzamine treatment can lead to loss of endothelial cell viability
Krishnanand R. Paia,
Alan R. Conantb,*,
Paul G. Browningb and
Walid C. Dihmisb
a Department of Cardiothoracic Surgery, Birmingham Heartlands Hospital, Birmingham, UK
b The Cardiothoracic Centre, Liverpool NHS Trust, Thomas Drive, Liverpool, L14 3PE, UK
Received 10 January 2008;
received in revised form 7 May 2008;
accepted 9 May 2008
 This project was funded by the Merseybeat Appeal based at the Cardiothoracic Centre (CTC), Liverpool NHS Trust.
*Corresponding author. Tel.: +44-151-6001345; fax: +44-151-2932283.
E-mail address: conant{at}liv.ac.uk (A.R. Conant).
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Abstract
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Phenoxybenzamine, an irreversible  -adrenoceptor antagonist, is used as a topical treatment against catecholamine-induced contraction in radial artery bypass grafts. Published data suggest that a wide range of phenoxybenzamine doses may be equally effective. This study aimed to investigate whether lower doses of phenoxybenzamine would benefit grafts by better preserving endothelium. To this end human vascular endothelial cells were isolated from sections of radial artery or saphenous vein, and treated with phenoxybenzamine for 30 min. Cells were then washed free of drug and viability assayed using a resazurin-based toxicology assay or returned to culture for assay at 24 h. Phenoxybenzamine treatment showed a dose-dependent effect on cell viability over several clinically employed concentrations. Concentrations above 0.1 mM led to a loss of viability, which became more pronounced with time. The loss of viability was shown to be independent of the carrier used, as results were identical when phenoxybenzamine was dissolved in dimethylsulphoxide, which alone did not affect viability. Changes in pH alone were also not sufficient to affect viability. In conclusion, phenoxybenzamine treatment is likely to cause damage to graft endothelium if employed at concentrations above 0.1 mM (0.03 mg/ml). Phenoxybenzamine may be safely used at lower doses with no potential loss of endothelial cell viability.
Key Words: Coronary artery bypass conduits; Arteries; Endothelium; Vasodilators; Viability
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1. Introduction
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Phenoxybenzamine is an irreversible -adrenoceptor antagonist used to treat radial artery bypass grafts [1]. The recently published results of a prospective, non-randomised, comparative study demonstrated significant benefit to patients receiving radial artery grafts treated with 6 mM phenoxybenzamine, compared with 30 µM verapamil/glyceryl trinitrate [2]. Patients receiving phenoxybenzamine treated grafts had fewer perioperative myocardial events and less evidence of myocardial injury [2]. These data are likely to encourage the use of phenoxybenzamine in the preparation of radial artery bypass grafts.
Since the initial demonstration of the use of 6 mM phenoxybenzamine by Taggart and colleagues [1], later studies by Taggart's group and others have demonstrated that doses as low as 10 µM are equally effective at irreversibly treating grafts against catecholamine-induced vasospasm [3–5]. Although endothelial function was measured in these studies, it was assessed immediately following treatment, thereby potentially missing any slow developing toxicity effects. Our aim was to assess whether phenoxybenzamine does demonstrate any effects on endothelial viability and whether the use of lower doses of phenoxybenzamine would better preserve the viability of vascular endothelial cells.
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2. Material and methods
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2.1. Materials
Phenoxybenzamine solution (50 mg/ml) was obtained from Goldshield Pharmaceuticals Ltd (Croydon, UK) and phenoxybenzamine powder from Calbiochem (Nottingham, UK). All tissue culture reagents were obtained from Invitrogen Ltd (Paisley, UK). All other chemicals and reagents were purchased from Sigma Chemicals (Poole, UK) and VWR International (Leicester, UK). Assays were carried out in Dulbecco's Modified Eagle's tissue culture Medium (DMEM; Invitrogen, UK) or HEPES-buffered-saline (HBS) composed of; NaCl, 145 mM; KCl, 2.5 mM; Na2HPO4, 1 mM; MgSO4, 1 mM; HEPES, 10 mM; D-glucose, 1 mM; CaCl2, 1 mM; pH 7.4 at 37 °C. Results were identical in either buffer. Both DMEM and HBS showed a similar pH dependence to the addition of phenoxybenzamine as heparinized human whole blood up to and including 0.90 mM (0.3 mg/ml). At 2.9 mM (1 mg/ml) the pH dropped to 6.82 in DMEM and 6.60 in HBS compared to a measured pH of 7.02 in blood.
2.2. Endothelial cell isolation and culture
Human vascular endothelial cells were isolated by collagenase digestion from freshly isolated sections of human saphenous vein or radial artery collected excess to coronary artery bypass surgery, with informed patient consent and approval of the Liverpool Research Ethics Committee. Endothelial cells were cultured to confluency in MCDB 131 tissue culture media, supplemented with 20% foetal bovine serum, 2% human AB serum, 15 µg/ml endothelial cell growth supplement, 5 U/ml heparin sulphate, 2 mM L-glutamine and antibiotics, and seeded onto 96 well cell culture microtitre plates. Endothelial identity was confirmed by von-Willebrand factor and CD-31 associated immunofluorescence.
2.3. Measurement of endothelial cell viability
Cells were treated with phenoxybenzamine for 30 min at 37 °C in HBS or DMEM and viability assessed by incubating cells with resazurin (Alamar Blue) for 2 h at 37 °C. Cells were visually inspected following treatment and following resazurin incubation. Treated wells were compared with sham-treated control wells incubated in parallel on the same plate. Non-fluorescent resazurin is converted to fluorescent resorufin by metabolically active cells and has been shown to give comparable results to conventional assays of cell proliferation and cytotoxicity such as thymidine incorporation and tetrazolium reduction but has the advantage that it can be used on non-proliferating or confluent cells [6, 7]. Fluorescence measured at 595 nm using a Wallac Victor Platereader and 550 nm excitation was linearly related to cell number and highly sensitive, being able to measure a minimum number of 200 cells/well.
2.4. Data analysis
Data are presented as mean±standard error of the mean from five independent cell batches unless otherwise stated. Statistical comparisons were undertaken using a one-way ANOVA and a P-value of 0.05 using the program Arcus QuickStat Biomedical (Hearne Scientific Software, Dublin, Ireland).
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3. Results
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Cells were visually inspected immediately following treatment with phenoxybenzamine and at 3 h and at 24 h (Fig. 1). Immediately following treatment very little evidence of cell damage could be observed. At later time points damaged cells, clearly identified by their shrunken and rounded morphology, were visible in high numbers at concentrations >0.1 mM (0.03 mg/ml) (Fig. 1). A more quantitative assessment of cell damage was obtained by measuring cell viability using resazurin. Cells showed a concentration-dependent drop in viability both immediately following treatment and following a recovery period of 24 h (Table 1). However, at 24 h it was apparent that at high concentrations the loss of viability was exacerbated with time.
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Fig. 1. Effect of phenoxybenzamine on radial artery endothelial cells at 3 h and 24 h post-treatment. Confluent cultures of radial artery endothelial cells were treated for 30 min with either buffer alone (a and d), 0.03 mM (b and e) or 0.3 mM (c and f) phenoxybenzamine. Photographs were taken either at 3 h (a–c) or 24 h (d–f) post-treatment under x10 magnification, using a Leica DMIL microscope (Leica Microsystems GmbH, Wetzlar, Germany).
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Phenoxybenzamine is supplied as a 50 mg/ml solution containing ethyl alcohol, hydrochloric acid and propylene glycol. In addition to phenoxybenzamine, each of these constituents could adversely affect endothelial cell viability. Therefore, the toxicity of the clinical formulation was compared against an equivalent laboratory stock solution (50 mg/ml) in dimethylsulphoxide (DMSO). DMSO alone at the highest concentration used did not cause any significant loss of cell viability (99.1±6.2% control). Results obtained using DMSO as solvent were identical to those using the clinical formulation (Table 1), demonstrating that phenoxybenzamine alone was the cause of the measured toxicity. As phenoxybenzamine solutions prepared from both clinical or DMSO stocks are acidic, the effect of this drop in pH on endothelial cell viability was tested using solutions of HBS buffered to give a series of solutions over the pH range 7.4–6.4. No significant loss of cell viability could be measured (n=3).
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4. Discussion
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A functioning endothelium is recognised as a major factor in determining the early, intermediate and late patency of any conduit used for coronary revascularisation [8, 9]. Whereas, an intact endothelium has anti-thrombotic and anti-inflammatory properties, a damaged endothelium is pro-inflammatory, attracting neutrophils, potentially leading to the failure of the new graft [9]. In the radial artery in particular, a damaged endothelium increases the vasoconstrictor response and has been implicated in the development of spasm [8]. Our results show that phenoxybenzamine causes endothelial damage at concentrations above 0.1 mM (0.030 mg/ml). Phenoxybenzamine-induced loss of cell viability developed slowly following drug removal, which could explain why previous studies have not measured significant loss of endothelial function immediately following treatment [1, 3, 4, 10]. The toxicity of phenoxybenzamine was not a result of the carrier solution or acidity but was a direct result of the drug itself. Our conclusions would be that lower concentrations of phenoxybenzamine would better preserve endothelial viability and are likely to benefit the long-term patency of the graft.
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References
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Abstract
1. Introduction
