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(Circulation. 2001;104:2832.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Endothelial Cell Dysfunction in Mice After Transgenic Knockout of Type 2, but Not Type 1, 11ß-Hydroxysteroid Dehydrogenase

Patrick W.F. Hadoke, PhD; Clare Christy, BSc; Yuri V. Kotelevtsev, PhD; Brent C. Williams, PhD; Christopher J. Kenyon, PhD; Jonathan R. Seckl, PhD; John J. Mullins, PhD; Brian R. Walker, MD

From the Endocrinology (P.W.F.H., C.C., B.C.W., C.J.K., J.R.S., B.R.W.), Endothelial Cell Biology (Y.V.K.), and Molecular Physiology (J.J.M.) groups, University of Edinburgh, Edinburgh, UK.

Correspondence to Dr Patrick Hadoke, Endocrinology Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. E-mail phadoke{at}srv0.med.ed.ac.uk

	Abstract

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Background— 11ß-Hydroxysteroid dehydrogenase (11ßHSD) isozymes catalyze the interconversion of active and inactive glucocorticoids, allowing local regulation of corticosteroid receptor activation. Both are present in the vessel wall; here, using mice with selective inactivation of 11ßHSD isozymes, we test the hypothesis that 11ßHSDs influence vascular function.

Methods and Results— Thoracic aortas were obtained from weight-matched male wild-type (MF1x129 cross^+/+), 11ßHSD1^-/-, and 11ßHSD2^-/- mice. mRNA for both isozymes was detected in wild-type aortas by RT-PCR. 11ßHSD activity in aortic homogenates (48.81±4.65% conversion) was reduced in both 11ßHSD1^-/- (6.36±2.47% conversion; P<0.0002) and 11ßHSD2^-/- (24.71±3.69; P=0.002) mice. Functional responses were unaffected in aortic rings isolated from 11ßHSD1^-/- mice. In contrast, aortas from 11ßHSD2^-/- mice demonstrated selectively enhanced constriction to norepinephrine (E_max 4.28±0.56 versus 1.72±0.47 mN/mm; P=0.004) attributable to impaired endothelium-derived nitric oxide activity. Relaxation responses to endothelium-dependent and -independent vasodilators were also impaired. To control for chronic renal mineralocorticoid excess, MF1 mice were treated with fludrocortisone (16 weeks) but did not reproduce the functional changes observed in 11ßHSD2^-/- mice.

Conclusions— Although both 11ßHSD isozymes are present in the vascular wall, reactivation of glucocorticoids by 11ßHSD1 does not influence aortic function. Mice with 11ßHSD2 knockout, however, have endothelial dysfunction causing enhanced norepinephrine-mediated contraction. This appears to be independent of renal sodium retention and may contribute to hypertension in 11ßHSD2 deficiency.

Key Words: hydroxysteroid dehydrogenases • hypertension • aorta • endothelium • vasoconstriction

	Introduction

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The 11ß-hydroxysteroid dehydrogenase (11ßHSD) enzymes catalyze the interconversion of active and inactive glucocorticoids. Two isozymes have been identified: 11ßHSD2^1,2 is a dehydrogenase and converts cortisol (corticosterone in mice) into its inactive metabolite, cortisone (11-dehydrocorticosterone), whereas 11ßHSD1³ is predominantly a reductase in vivo and regenerates cortisol from cortisone (corticosterone from 11-dehydrocorticosterone). 11ßHSD2 is expressed in the distal nephron, colon, and sweat glands, where it protects mineralocorticoid receptors from inappropriate activation by glucocorticoids (reviewed in Reference 4). Deficiency or inhibition of 11ßHSD2 results in activation of renal mineralocorticoid receptors by glucocorticoids, producing the syndrome of "apparent mineralocorticoid excess," with its characteristic sodium retention, hypokalemia, and hypertension. 11ßHSD1 is expressed in many tissues in which mineralocorticoid receptors are not present, including liver and fat; its likely role is to reactivate glucocorticoids and thereby maintain activation of glucocorticoid receptors at sites where they have most influence on metabolism.^4,5

Both 11ßHSD1 and 11ßHSD2 are expressed in the vascular wall, although their exact localization remains unclear. 11ßHSD1 has been demonstrated in rat vascular smooth muscle,⁶ and 11ßHSD2 has been detected in human vascular smooth muscle⁷ and cultured rat endothelial cells.⁸ Glucocorticoid and mineralocorticoid receptors are both expressed in blood vessels, and corticosteroids have diverse actions on vascular function.⁹ The presence of 11ßHSDs suggests that they influence vascular function by regulating local concentrations of active glucocorticoids in the vascular wall. Inhibition of 11ßHSDs potentiates the effects of glucocorticoids in isolated vessels in vitro^10,11 and in dermal and forearm circulation in vivo,^12,13 but it is not clear which 11ßHSD isozyme is responsible for these effects. Isozyme-specific inhibitors of 11ßHSD are not available, and the nonselective inhibitor carbenoxolone damages endothelial cells in vitro.¹⁴

We recently developed genetically manipulated mice deficient in either 11ßHSD1⁵ or 11ßHSD2.¹⁵ We have now used these to test the hypothesis that 11ßHSD isozymes influence vascular function in mice. We aimed to elucidate the respective importance of each isozyme, identify the pathways affected by glucocorticoid metabolism in the vessel wall, and assess the relevance of these changes in vascular function to hypertension.

	Methods

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Mice
11ßHSD-Knockout
Genetic inactivation of 11ßHSD1⁵ and 11ßHSD2¹⁵ isozymes was described previously. We investigated homozygous null (-/-) male mice from both transgenic groups and matched controls (first generation MF1x129 cross). 11ßHSD1^-/- mice (n=10) were age-matched (99±3 versus 100±2 days) and weight-matched (39±2 versus 37±1 g) with controls. 11ßHSD2^-/- mice (n=8) tended to be older (123±11 versus 98±4 days; P=0.07) than weight-matched (37±2 versus 39±2 g) controls.

Chronic Mineralocorticoid Excess
Mineralocorticoid excess was produced in MF1 mice by subcutaneous implantation under halothane anesthesia of a pellet impregnated with fludrocortisone (50 mg as a 1:4 mixture of fludrocortisone acetate and Silastic elastomer (MDX4-4210; Dow Chemical Corp).¹⁶ Six-week-old, male MF1 mice (Bicester, UK) were treated with a single fludrocortisone-impregnated (n=12) or elastomer-only (n=12) pellet for 102±8 days. Three fludrocortisone-treated mice were killed because of wound breakdown, and 1 control mouse was euthanized after injuring a forelimb.

Blood Pressure
Blood pressure in the knockout strains has been reported elsewhere.^5,15 In mice treated with fludrocortisone or vehicle, systolic blood pressure was measured by tail-cuff plethysmography in conscious, restrained, warmed mice¹⁷ from age 13 weeks (30 g body weight).

Tissue Preparation
Mice were killed by cervical dislocation or decapitation, and organs were weighed. The entire thoracic aorta was removed into physiological saline solution (PSS) containing (mmol/L) NaCl 119, KCl 4.7, CaCl₂ 2.5, MgSO₄.7H₂O 1.17, KH₂PO₄ 1.18, NaHCO₃ 25, K₂EDTA 0.026, and D-glucose 5.5). Two rings, 2 mm in length, were taken for immediate functional studies. Sections of tissues from knockout and control mice were frozen in dry ice and stored at -70°C for enzyme activity assays and reverse transcription–polymerase chain reaction (RT-PCR).

11ßHSD Activity
As previously described,^6,18 aortas were homogenized in Krebs-Ringer buffer at pH 7.4, and protein was measured colorimetrically. In vitro, 11ßHSD1 is an NADP(H)-dependent 11ß-oxidoreductase, whereas 11ßHSD2 is exclusively an NAD-dependent 11ß-dehydrogenase. Dehydrogenase activity was determined^6,18 by measurement of the conversion of 100 nmol/L [³H]corticosterone to [³H]11-dehydrocorticosterone in the presence of 200 µmol/L NAD or NADP at 37°C for 240 minutes.

Detection of 11ßHSD mRNA
Total RNA was extracted from aorta, kidney, and liver from MF1 mice with TRIzol (Gibco) and quantified with UV spectroscopy. Total RNA (1 µg) was reverse transcribed with oligo (dT) primers with a commercial kit (Promega). Ten to 100 ng reverse-transcribed RNA was primed with oligonucleotides specific for 11ßHSD 1 (5'-AAAGCTTGTCACWGGGGCCAGCAAA-3' [where W indicates A or T] and 5'-AGGATCCARAGCAAACTTGCTTGC-3') and 11ßHSD 2 (5'-ACCCCTGCTTGGCAGCCTACGGCA-3' and 5'-TCACATTAGTCACTGCAGCTG TCTTGG-3'). RT-PCR was performed as described previously¹⁹ with different annealing conditions for 11ßHSD1 (30 seconds at 56°C) and 11ßHSD2 (1 minute at 62°C). Aliquots of each RT-PCR reaction mixture (40 µL aorta; 5 µL kidney and liver) were electrophoresed on a 2% agarose gel that was stained with ethidium bromide and photographed under ultraviolet light.

Functional Investigations
Two rings from a test (transgenic or fludrocortisone-treated) animal and 2 from a control were analyzed in parallel. Each was suspended on 40-µm wires in a myograph containing PSS at 37°C, perfused with 95% O₂/5% CO₂, for measurement of isometric force. The endothelium was removed from 1 ring from each mouse by rubbing the luminal surface, and the length of each ring was measured.

Normalization was achieved by stepwise incremental stretches of the vessel and application of the LaPlace relation (P=T/r, where P is the transmural pressure, T is the wall tension, and r is the internal radius of the vessel) to determine the internal diameter under an effective intraluminal pressure of 13.3 kPa (l₁₀₀). Arteries were stretched to their optimum resting level (0.9 l₁₀₀). The arteries were contracted twice with NE-K (a mixture of 0.1 µmol/L norepinephrine [NE] in KPSS [PSS containing 125 mmol/L K⁺]), once with NE alone, once with KPSS alone, and finally with NE-K. Cumulative concentration-response curves were obtained for 5-hydroxytryptamine (5-HT; 1 nmol/L to 30 µmol/L), KCl (2.5 to 320 mmol/L), and NE (1 nmol/L to 3 µmol/L). Vasodilator responses were obtained for acetylcholine (ACh; 1 nmol/L to 30 µmol/L) and the nitric oxide (NO) donor 3'-morpholinosydnonimine (SIN-1; 1 nmol/L to 30 µmol/L) in vessels precontracted with 0.3 µmol/L 5-HT. The influence of the endothelium on adrenoceptor-mediated contraction made adrenoceptor agonists unsuitable for precontraction of these vessels. Preliminary investigations indicated that 5-HT was a suitable alternative.

In some vessels, concentration-response curves to NE were repeated after incubation (30 minutes) with the NO synthase (NOS) inhibitor N^G-nitro-L-arginine (10^-4 mol/L).

Drugs
Fludrocortisone acetate was obtained from ICN Biomedicals Inc, SIN-1 from Alexis, [³H]corticosterone from Amersham Life Sciences, high-performance liquid chromatography solvents from Rathburn Chemicals Ltd, and other chemicals from Sigma BDH.

Statistics
Results are mean±SEM for n mice. Enzyme activities were compared by 1-way ANOVA. Contractions are expressed as force per unit length of vessel (mN/mm) and as percentage of the maximum response to KPSS (to control for vessel size). Relaxation is expressed as percentage of precontraction with 5-HT. Sensitivity to agonists is expressed as negative logarithm of the concentration necessary to produce 50% of maximum responses (pD₂ for constrictors; -logIC₅₀ for dilators). Response curves for control and experimental tissues were compared by 2-way ANOVA with Tukey post hoc test. The effects of N^G-nitro-L-arginine were analyzed by Student’s paired t tests.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Detection of mRNA for 11ßHSD Isozymes in Normal Mouse Aorta
RT-PCR amplification products of 450 and 144 bp for 11ßHSD1 and 11ßHSD2, respectively, were detected in aortas (Figure 1a). 11ßHSD2 mRNA was also detected in kidney, but not liver, whereas 11ßHSD1 mRNA was identified in both tissues.

View larger version (31K): [in this window] [in a new window]   Figure 1. 11ßHSD isozymes in mouse aorta. A, 11ßHSD1 and 2 mRNAs in kidney (K), liver (L), and aorta (A) from MF1 mice detected by RT-PCR. B, Enzyme activity. NAD- (open) and NADP- (hatched) dependent 11ßHSD activity as percentage conversion of corticosterone to 11-dehydrocorticosterone in aortic homogenates from wild-type (n=12) and 11ßHSD1-/- (n=10) or 11ßHSD2-/- (n=6) mice. Values are mean±SEM. *P<0.002, **P<0.0005, #P=0.055 vs wild-type by 1-way ANOVA and Tukey post hoc test.

11ßHSD-Knockout
As described previously,¹⁵ 11ßHSD2^-/- mice had enlarged kidneys (1.08±0.05% body weight) compared with controls (0.82±0.05% body weight; P=0.002).

Enzyme Activity
NAD-dependent and NADP-dependent enzyme activities were similar in aortas from wild-type mice (n=12). Enzyme activities were reduced, but not abolished, in 11ßHSD1^-/- mice (Figure 1b, P<0.001; n=10). Activities were also lowered in mice lacking 11ßHSD2 (n=6), but to a much lower degree (Figure 1b), with the reduction in NAD-dependent activity achieving only borderline significance (P=0.055).

Vascular Function in 11ßHSD1^-/- Mice
NE-mediated contraction was similar in intact aortic rings from 11ßHSD1^-/- and wild-type mice (Figure 2a). Both removal of the endothelium (Figure 2a) and inhibition of NOS (not shown) increased the amplitude of this response equally in both groups. NOS inhibition had no effect on denuded arteries (not shown). Contractile responses to 5-HT and KCl (Table 1) and dilator responses to ACh and SIN-1 (Table 2) were also similar in aortas from 11ßHSD1^-/- and wild-type mice.

View larger version (26K): [in this window] [in a new window]   Figure 2. Concentration-response curves to NE in intact (open) and denuded (solid) aortic rings from transgenic (squares) mice and wild-type (circles) controls. Responses were obtained in (a) 11ßHSD1-knockout mice (n=10) and (b) 11ßHSD2-knockout mice (n=8). Each point represents mean±SEM as percentage maximum response to 125 mmol/L potassium (KPSS). *P<0.03 and NS, not significant vs intact arteries from knockout mice by 2-way ANOVA and Tukey post hoc test.

View this table: [in this window] [in a new window]   Table 1. Maximum Contraction (Emax) and Sensitivity (pD2) to NE, 5-HT and KCl in Aortas From 11ßHSD-/- Mice and Controls (+/+)

View this table: [in this window] [in a new window]   Table 2. Maximum Relaxation (Emax) and Sensitivity (-logIC50) to ACh and SIN-1 Obtained in Aortas From 11ßHSD1-/- mice and Matched Controls (+/+)

Vascular Function in 11ßHSD2^-/- Mice
In contrast, in intact aortas from 11ßHSD2^-/- mice, NE-mediated contraction was substantially greater than in wild-type controls and was unaffected by removal of the endothelium (Figure 2b). Thus, responses to NE in intact and denuded aortas from 11ßHSD2^-/- mice were similar to those of denuded vessels from wild-type mice. Aortas from 11ßHSD2^-/- mice also showed enhanced contractile sensitivity to KCl (Table 1), but this was much less substantial than the difference in NE responsiveness and was not endothelium-dependent. Contraction to 5-HT was not different in 11ßHSD2^-/- and wild-type mice (Table 1).

Inhibition of NOS enhanced the response to NE in intact (but not denuded) aortas from wild-type (1.47±0.36 versus 3.54±0.46 mN/mm; P<0.001; n=12) but not 11ßHSD2^-/- (4.25±0.73 versus 4.94±0.73 mN/mm; P=0.29; n=5) mice. Responses to ACh were impaired in aortas from 11ßHSD2^-/- mice (Figure 3a). Responses to SIN-1 were potentiated by removal of the endothelium but were impaired in aortas from 11ßHSD2^-/- mice whether or not the endothelium was present (Figure 3b).

View larger version (22K): [in this window] [in a new window]   Figure 3. Concentration-response curves to (a) ACh and (b) SIN-1 in intact (open) and denuded (solid) aortic rings from 11ßHSD2-/- (squares) and wild-type (circles) mice. Each point represents mean±SEM. *P<0.01 vs knockout mice by 2-way ANOVA and Tukey post hoc test.

Chronic Exogenous Mineralocorticoid Excess
Implantation of fludrocortisone pellets temporarily interrupted weight gain (for 7 days). Fludrocortisone increased heart weight (0.63±0.02% body weight versus 0.50±0.03% body weight; P=0.006) and kidney weight (0.91±0.07% body weight versus 0.77+0.02% body weight; P=0.04) but had no effect on liver, adrenal, and thymus weights. A trend toward higher systolic blood pressure induced by fludrocortisone was not significant (123±7 mm Hg [n=8] versus 116±5 mm Hg [n=9]; P=0.38).

Vascular Function
NE-mediated contraction was not significantly different in aortas from mice receiving fludrocortisone or vehicle (Figure 4), although there was a trend for a small increase in responsiveness to NE with fludrocortisone in intact vessels. Contractile responses to 5-HT and KCl and dilator responses to ACh and SIN-1 were unaffected by fludrocortisone (Table 3).

View larger version (21K): [in this window] [in a new window]   Figure 4. Concentration-response curves to NE in intact (open) and denuded (solid) aortic rings from control (squares) (n=11) and fludrocortisone-treated (circles) (n=9) mice. Each point represents mean±SEM as percentage maximum response to 125 mmol/L potassium (KPSS). NS indicates not significant vs intact arteries from control mice by 2-way ANOVA.

View this table: [in this window] [in a new window]   Table 3. Maximum Response (Emax) and Sensitivity (pD2 or -logIC50) in Aortas From Fludrocortisone-Treated and Control Mice

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This investigation, using unique 11ßHSD^-/- mice, clarifies a number of contentious issues concerning the role of 11ßHSD enzymes in vascular tissue. We conclude that both isozymes are present in the mouse aorta. Furthermore, 11ßHSD2, rather than 11ßHSD1, influences vascular tone in mice. Thus, inhibition of 11ßHSD2 is likely to account for previously observed effects of nonselective 11ßHSD inhibitors on vascular function in vitro^10,11,20 and in vivo.^12,13,21 11ßHSD2 appears to mediate its effect on vascular function directly in the vessel wall, rather than indirectly through its actions in the kidney, because vascular changes in 11ßHSD2 deficiency were not reproduced by chronic renal mineralocorticoid excess induced by fludrocortisone administration. Finally, the altered vascular function associated with 11ßHSD2 deficiency is consistent with impaired inactivation of glucocorticoids in endothelial cells, resulting in inhibition of NO generation and thus enhanced contractility to NE.

Enzyme activities and RT-PCR show both 11ßHSD isozymes in mouse aorta. Their cofactor specificity is not as clear-cut as in rat,¹⁸ and similar activities were observed with NAD and NADP. 11ßHSD1 has been detected in several blood vessels,⁶ but data for 11ßHSD2 are inconsistent.⁷ 11ßHSD1^-/- mice have negligible 11ßHSD activity at sites at which only the type 1 isozyme is expressed (eg, liver) but normal 11ßHSD2 activity (eg, in kidney).⁵ Therefore, the residual activity in aortas from these mice indicates the presence of 11ßHSD2. Moreover, this is not "compensatory" for knockout of 11ßHSD1, because mRNA for both isozymes was detected in wild-type aorta. The residual activity in 11ßHSD1^-/- aortas and the modest effect of 11ßHSD2-knockout on activity are consistent with mouse 11ßHSD2 expression being restricted to endothelial cells,⁸ whereas 11ßHSD1 is expressed in vascular smooth muscle cells.⁶ The small contribution to total activity made by 11ßHSD2 could thus reflect the relative sparsity of endothelial cells. Potent inactivation of glucocorticoids may occur, however, within the endothelium.

Before the cloning of 11ßHSD2^1,2 and recognition of 11ßHSD1 as a predominant reductase in other cells,⁴ the ability of 11ßHSD inhibitors to potentiate the effects of glucocorticoids on vascular function^10–13 was attributed to inhibition of 11ßHSD1 dehydrogenase activity. Recent data also suggest that inhibition of dehydrogenase activity (by either isozyme) is functionally important.^20,21 In vivo dehydrogenase activity of vascular 11ßHSD1 has not been excluded by the present investigation, but the marked contrast between the effects of knockout of the 2 isozymes suggests that 11ßHSD2 provides the major vascular dehydrogenase activity, loss of which results in increased intracellular glucocorticoid levels and impaired action of the endothelium-derived NO system. The role, if any, of 11ßHSD1 in the vessel wall remains obscure.

11ßHSD2 also has important effects elsewhere. Loss of renal 11ßHSD2 results in activation of mineralocorticoid receptors by glucocorticoids.¹⁵ Previous investigations, however, suggest that this is unlikely to account for the changes in vascular function. In hypertensive models, increased contractile responses occur with most agonists and are not endothelium-dependent. Moreover, induction of renal mineralocorticoid excess with deoxycorticosterone acetate and salt in rats resulted in enhanced endothelial NO generation,^22,23 rather than impaired NO activity. Finally, 11ßHSD inhibitors affect vascular function in isolated vessels^10,11 as well as in vivo.^12,13

Possible indirect effects of renal 11ßHSD2 inhibition were addressed by chronic fludrocortisone administration to MF1 mice. This reproduced the cardiac and renal hypertrophy detected in 11ßHSD2^-/- animals, but not the abnormalities of vascular function. Blood pressure in fludrocortisone-treated mice was not as high as in 11ßHSD2^-/- mice; this is not surprising, because induction of mineralocorticoid hypertension in rodents usually requires salt loading and/or partial nephrectomy. It is possible, however, that either the vascular dysfunction is indirectly mediated in 11ßHSD2^-/- mice by more severe hypertension or vascular dysfunction in the 11ßHSD2^-/- mice explains their more severe hypertension. The difference between 11ßHSD2^-/- and fludrocortisone-treated mice also suggests that vascular dysfunction in 11ßHSD2^-/- mice is not mediated by vascular mineralocorticoid receptors and is most likely to be mediated by the glucocorticoid receptor. Whether glucocorticoid receptor expression is altered in the vessel wall in 11ßHSD-knockout animals has not been tested here, but available data suggest very limited dysregulation of receptor expression in other tissues.²⁴

The mechanisms by which glucocorticoids affect vascular tone remain controversial.⁹ Key targets include inhibition of NO synthesis and potentiation of contractile responses to NE.^25,26 In mouse aorta, NE-mediated contraction is characteristically dependent on endothelium-derived NO. The present results are consistent with an enhanced glucocorticoid effect in the endothelium to inhibit NO generation. Responses to 5-HT and KCl (which are not profoundly influenced by the endothelium) were unaffected. The only discrepancy is that responses to SIN-1 were not enhanced in 11ßHSD2 deficiency, as would be expected if basal NO generation were impaired.²⁷ This suggests an additional impairment of soluble guanylate cyclase activity (the target for NO in smooth muscle), which may be a feature of hypertension per se.²⁸ It remains to be seen whether similar impairments are evident in resistance arteries from these animals.

If these findings in mice can be extrapolated to humans, they may have important pathophysiological implications. In conduit vessels, endothelial function may be important in atherogenesis and arterial compliance. There is also evidence that the same mechanisms operate in resistance vessels. A patient with congenital 11ßHSD2 deficiency displayed enhanced vasoconstrictor responses in the forearm and dermal vasculature.¹³ Nonselective 11ßHSD inhibitors also potentiate vasoconstriction in the forearm and dermal resistance vessels of healthy volunteers.^12,13 Most intriguingly, vascular 11ßHSD activity is impaired in several hypertensive animal models,^29,30 and 11ßHSD2 activity is impaired in some patients with essential hypertension.³¹ It remains to be seen whether a defect in vascular inactivation of cortisol contributes to the enhanced vascular responsiveness to glucocorticoids³² and endothelial dysfunction evident in patients with essential hypertension.

	Acknowledgments

 
This research was funded by the British Heart Foundation and Wellcome Trust. Dr Walker is a British Heart Foundation Senior Research Fellow, and Dr Mullins is the recipient of a Wellcome Trust Principal Research Fellowship.

Received July 6, 2001; revision received September 23, 2001; accepted September 26, 2001.

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