Mol. Hum. Reprod. Advance Access originally published online on March 25, 2004
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Molecular Human Reproduction, Vol. 10, No. 5, pp. 331-337, 2004
© European Society of Human Reproduction and Embryology 2004
Aortic adaptation to pregnancy: elevated expression of matrix metalloproteinases-2 and -3 in rat gestation
1Maternal and Fetal Research Unit, Department of Women’s’ Health, Guy’s, King’s and St Thomas’ School of Medicine, 10th Floor St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH and 2Department of Statistical Science, Glaxo SmithKline Pharmaceuticals, Harlow, Essex CM19 5AW, UK
3 To whom correspondence should be sent. e-mail: brenda.a.kelly{at}kcl.ac.uk
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ABSTRACT |
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The maternal aorta undergoes substantial functional and structural adaptation in pregnancy. Both aortic diameter and compliance are increased and studies of animal and human gestation indicate that these changes are initiated in early pregnancy and maintained until delivery. The mechanisms underlying aortic adaptation in normal pregnancy remain largely unknown but matrix metalloproteinase enzymes (MMP) are likely to play a key role. Gene expression of candidate MMP and specific tissue inhibitors of MMP (TIMP) were investigated in non-pregnant, pregnant (days 7, 14, 21) and postpartum (day 7) rat aorta using real-time PCR. Of the gene transcripts studied (MMP-2, -3, -7, -9, -12, -13, MT1MMP, TIMP-1, -2) in rat aorta, only MMP-3 was significantly elevated with a 24-fold increase observed in late gestation compared to virgin control (P = 0.0001). MMP-2 mRNA appeared constitutively expressed and unchanged at time-points studied, but MMP-2 activity as assessed by gelatin zymography suggested further modulation after transcription and/or post-translation in rat aorta with activity increased in early pregnancy (P < 0.01, compared to virgin control). These data suggest that MMP-2 and MMP-3 may contribute to adaptive processes in the maternal rat aorta at different gestations and further support a role for this family of enzymes in physiological vascular remodelling.
Key words: Key words: aorta/MMP/pregnancy/rat/TIMP
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Introduction |
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The maternal cardiovascular system undergoes remarkable adaptive changes in pregnancy to accommodate 40% increases in both blood volume and cardiac output whilst maintaining normal or slightly reduced blood pressure (McLaughlin and Roberts, 1999). Data from both animal and human gestation suggest that reduced vasomotor tone and remodelling of resistance-sized arteries participate in the pregnancy-associated fall in cardiac afterload (McLaughlin and Keve, 1986; McCarthy et al., 1994; Cockell and Poston, 1996, 1997). However, gestational increases in aortic compliance and diameter indicate that large artery structure and function is also altered in pregnancy (Hart et al., 1986; Poppas et al., 1997; Slangen et al., 1997; Edouard et al., 1998) and may also contribute to reduced afterload. Preliminary studies have shown the compliance of maternal aorta to be reduced in pregnancies complicated by hypertension compared to normotensive pregnant women (Hibbard et al., 1998).
Mechanisms underlying adaptation of large arteries in pregnancy have been infrequently addressed. In rat gestation, enhanced endothelium-dependent aortic relaxation is likely to contribute to initial increases in compliance observed in the first trimester (Jain et al., 1998). In addition, histological studies of maternal aorta in animal (Danforth et al., 1964) and human (Manalo-Estrella et al., 1967) gestation clearly demonstrate greatly altered vascular structure with marked disruption of elastic lamellae, reduced proteoglycans and hypertrophy and hyperplasia of vascular smooth muscle. These changes were most pronounced in late gestation. This reorganization of the vascular structure is likely to supplement initial vasoregulatory mechanisms to maintain or augment increases in aortic compliance and diameter (Langille, 1993). Aberrant medial remodelling may contribute to the pathogenesis of aortic dissection, the incidence of which is substantially increased in pregnancy (Anderson et al., 1994; Zeebregts et al., 1997).
The pregnancy-induced turnover of aortic extracellular matrix (ECM) in normal gestation strongly suggests a role for matrix metalloproteinases (MMP) in maternal aortic adaptation. This family of enzymes is central to numerous physiological remodelling processes including embryogenesis and angiogenesis (Woessner, 1994) and we have recently demonstrated gestational regulation of MMPs and tissue inhibitors of MMP (TIMPs) in rat uterine artery (Kelly et al., 2003). Studies of the expression and role of MMP in large arteries are, however, limited to pathological vascular remodelling with enhanced MMP-2 activity, with or without alteration in MMP-3, -9 and -12 expression, implicated in medial reorganisation contributing to aortic aneurysm degeneration (Newman et al., 1994; Curci et al., 1998; Crowther et al., 2000; Pyo et al., 2000) and to aortic dissection (Ishii et al., 2000).
The aim of this investigation was to elucidate the expression of MMPs and TIMPs contributing to physiological remodelling of aorta that occurs during normal pregnancy. In this study, the gestational profile of the expression of these enzymes was investigated in rat aorta before, during and after pregnancy using real-time PCR and substrate zymography. It was hypothesized that pregnancy would be associated with enhanced gene expression in MMPs and TIMPs previously implicated in aortic remodelling and that expression would be greatest in late gestation consistent with a role in structural adaptation maintaining increases in compliance and diameter.
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Materials and methods |
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Animals and tissue collection
All animal procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. Female Sprague–Dawley (SD) rats (225–250 g; B and K Universal Ltd, UK) were housed under controlled conditions of temperature and humidity with a 12 h light/dark cycle and free access to tap water and rat chow. For mating, an adult male and a virgin female rat were housed in a single cage. Successful mating was confirmed by the presence of a vaginal plug and designated day 0. Delivery usually occurred on day 22. Aortas were harvested from virgin control (estrus day of estrus cycle), day 7, 14 or 21 pregnant and day 7 postpartum SD rats culled by cervical dislocation, then snap-frozen in liquid nitrogen and stored at –80°C until use.
RNA extraction and reverse transcription
All reagents for RNA extraction, DNase treatment and reverse transcription (RT) were purchased from Invitrogen (Paisley, UK). Total RNA was extracted from each aorta (n = 8 per group) using Trizol according to manufacturers’ instructions. The resultant pellet was resuspended in molecular biology grade water and RNA concentration determined in triplicate by A260 measurement. Following DNase treatment, first strand cDNA was synthesized from triplicate RNA (1 µg) samples using SuperscriptTM II reverse transcriptase kit as described previously (Kelly et al., 2003). cDNA triplicates synthesized were then diluted (10 ng starting RNA/µl), aliquoted and stored at –20°C.
Relative quantification of MMP and TIMP gene expression using real time PCR
Real-time PCR assays for each gene target (MMPs-2, -3, -7, -9, -12, MT1MMP TIMPs-1 and -2) GAPH were performed on cDNA triplicates from each animal (n = 8 animals per group) in 96-well optical plates using an ABI Prism 7700 Sequence Detection system (‘TaqMan’; Applied Biosystems). For each 25 µl TaqMan reaction, 5 µl of cDNA was mixed with 12.5 µl of 2xTaqMan Universal Master Mix, 0.75 µl of each forward and reverse primer (10 µmol/l; final concentration 300 nmol/l), 1 µl TaqMan probe (5 mmol/l; final concentration 250 nmol/l) and 5 µl of molecular biology grade water. Forty PCR cycles were then performed under standard thermal cycle conditions. Details of primer and probe oligonucleotide sequences used have been published previously (Kelly et al., 2003). Additional reactions were performed on each 96-well plate using known dilutions of highly sheared rat genomic DNA to allow construction of a standard curve (see below; Harrison et al., 2000; Macdonald et al., 2001; Kelly et al., 2003). Wells were also included on each plate where no template was added and parallel assays were performed to assess for genomic DNA contamination in each sample where reverse transcriptase enzyme had previously been omitted. Test gene values were then interpolated from the standard curve and expressed as arbitrary units of gene expression.
Standard curve generation
Primers and probes were not purposefully designed to target intron/exon boundaries, as the genomic structure of many of the MMP investigated was unknown at the time of commencing this study. In preliminary PCR assays, a single product of appropriate size was detected for each gene of interest by gel electrophoresis, indicating that primers were designed within exons (data not shown). The advantage of within-exon primer and probe design was that serial dilutions of highly sheared genomic DNA of known concentration could be used to generate standard curves on each plate for quantification purposes (Harrison et al., 2000; Macdonald et al., 2001; Kelly et al., 2003). Any stock RNA or DNA containing the appropriate amplicon sequence can be used to prepare standards (ABI User Bulletin 2, Applied Biosystems). For the purposes of this study, preliminary experiments were undertaken to ensure equivalent amplification efficiency between genomic DNA standards and tissue cDNA. Briefly, real-time PCR assays were performed for a number of MMP/TIMP genes using serial dilutions of pooled rat spleen/placenta cDNA (positive control) and highly sheared genomic DNA. Comparison of slopes of standard curves generated showed similar amplification efficiency between cDNA and genomic DNA (within 0.03–0.08; Table I and Figure 1) and validated subsequent use of genomic DNA standard curves in relative quantification of target cDNA.
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Protein extraction
All reagents were supplied by Sigma (UK). Aortas (n = 4 or 5 per group) were pulverized under liquid nitrogen. Protein was extracted in a two-step sequential process first using a salt/detergent extraction buffer [phosphate-buffered saline (pH 7.4), 0.1% sodium dodecyl sulphate (SDS), 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.02% sodium azide and EDTA-free Protease Inhibitor Cocktail V (Calbiochem, UK), 5 µl/mg tissue; ‘detergent fraction’], then by a second urea-based buffer consisting of TNC buffer [50 mmol/l Tris–HCl (pH 7.5), 150 mmol/l NaCl, 10 mmol/l CaCl2, 0.05% (w/v) Brij35, 0.02% sodium azide] containing 8 mol/l urea and EDTA-free Protease Inhibitor Cocktail V (5 µl/mg starting tissue; ‘urea fraction’), as described previously (Kelly et al., 2003). This was to ensure removal of both soluble and matrix-bound MMP (Woessner, 1995). For each extraction step, aortic protein was extracted in parallel from each of the five groups under identical experimental conditions. Total protein concentration for each fraction was determined in triplicate using Bio-Rad DC protein assay.
Gelatin zymography
Equal amounts of protein (15 µg) from virgin control, day 7, 14 and 21 pregnancy and day 7 postpartum aortas from each of the two extractions were mixed with sample loading buffer, then resolved by electrophoresis in 10% SDS–polyacrylamide electrophoretic gels containing 1 mg/ml gelatin. Samples were run alongside purified recombinant human proMMP-2 (200 pg, loaded to standardize signal between gels; Calbiochem, UK) and proMMP-2/9 (positive control, kindly donated by Dylan Edwards, University of East Anglia, Norwich, UK). After electrophoresis, proteins in the gel were renatured by incubation in 2.5% Triton X-100, washed in TNC buffer (15 min) then incubated at 37°C in TNC buffer overnight. Bands of lytic activity were visualized as zones of clearing in Coomassie Blue-stained gels after a single stain/destain step (Leber et al., 1997). Gel images were captured by FluorS MultiImager and the mean optical density product of lytic bands in each sample calculated using MultiAnalyst image analysis software and standardized to that obtained for the MMP-2 standard on that zymogram.
To verify MMP activity, control substrate gels loaded with vessel extract were incubated under the same conditions in TNC buffer containing 25 mmol/l EDTA.
Data analyses
In the study of MMP and TIMP gene expression, mean values were obtained for each set of cDNA triplicates. Data were log-transformed (base 10) to meet the requirements of normality and homogeneity of variation and analyses of variance (ANOVA) and covariance (ANCOVA) were performed (Bond et al., 2002) using Genstat software V5.4.1 (Lawes Agricultural Trust, Harpenden, UK). Post hoc pairwise comparisons between virgin control and remaining groups were performed using Dunnett t-test with a value of P < 0.05 considered significant.
Densitometric data from gelatin zymograms were generated through analysis of four or five individual aortas per time-point with each assay carried out in duplicate. Mean data were log-transformed (base 10) to meet the requirements of normality and homogeneity of variation. ANOVA was performed with post hoc pairwise comparisons between virgin control and remaining groups using Dunnett’s t-test with a value of P < 0.05 considered significant.
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Results |
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Gestational MMP and TIMP gene expression in rat aorta
No amplification was observed in wells containing sample RNA that had not been reverse-transcribed or in wells where no template had been added.
Preliminary univariate analysis without adjustment to a reference gene indicated no significant gestational alteration in GAPDH expression compared to virgin controls (P = 0.2). GAPDH was then adopted as a steady-state control (internal reference gene) to control for sample-to-sample variation in quality and quantity of mRNA. After adjustment to GAPDH, only MMP-3 expression was significantly elevated (P = 0.0001) with a 24.6-fold increase (95% CI: 4.4–139) in late gestation compared to virgin controls (Figure 2). The trend towards enhanced TIMP-1 gene expression in late gestation did not reach significance (1.7-fold increase 95% CI: 0.9–3.2, P = 0.08). The expression of remaining MMP and TIMP genes was not significantly altered in pregnancy (Figure 3).
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MMP-2 activity in rat aorta
Gelatin zymography of aortic extracts revealed two well-separated gelatinolytic bands at 72 and 65 kDa across gestation and in virgin and postpartum aorta. These bands were observed in both salt/detergent and urea fractions (Figure 4a, b). No significant gelatinolytic activity was isolated by additional extraction of the pellet (Figure 4c). Based on co-migration with recombinant MMP-2 standards and on inhibition with EDTA (Figure 4d), these bands corresponded to proMMP-2 and active MMP-2 respectively. A third gelatinase (78 kDa), present in each reproductive stage, was observed only in the detergent fraction, and is likely to represent a glycosylated form of MMP-2 (Fernandez-Patron et al., 1999a).
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The majority of gelatinolytic activity was isolated in the first round extraction with total MMP-2 (78, 72 and 65 kDa) activity greatest at day 7 pregnancy (P < 0.01, compared to virgin controls, Figure 5a). Total MMP-2 activity was not significantly altered in day 14 or 21 pregnant or at day 7 postpartum aortas compared to virgin aorta. No significant differences in total gelatinolytic activity were observed between reproductive stages after urea extraction (Figure 5b). However, in both detergent and urea fractions, a greater proportion of total MMP-2 activity was isolated as active 65 kDa species at day 7 gestation (Figure 5c). Gelatinolytic bands consistent with rat aorta gelatinase MMP-9 were not detected.
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P < 0.01) and in urea fraction at day 7 (**P < 0.01) and at day 14 (*P < 0.05) compared to virgin controls. |
Discussion |
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In order to investigate potential mechanisms underlying pregnancy-related remodelling of rat aorta, the expression profiles of seven MMPs and two TIMPs were studied before, during and after pregnancy using real-time PCR. mRNA expression of all but one of these enzymes was unaffected by pregnancy. The striking up-regulation of MMP-3 gene expression in late pregnancy occurred without significant alteration in the profile of its endogenous inhibitor, TIMP-1, and in the absence of appreciable change in expression of other MMP and TIMP-2 genes. These data contrast with those obtained for MMP and TIMP gene expression in rat uterine artery in late pregnancy, where all MMP and TIMP transcripts were significantly up-regulated (Kelly et al., 2003).
MMP-3 and vascular remodelling
MMP-3 is one of the most versatile members of the metalloproteinase family. Possessing broad substrate specificity, this enzyme can digest collagens III, IV, IX and X, proteoglycans, elastin, fibronectin, laminin, aggrecan, versican and perlecan and several other ECM proteins that play an important role in maintaining structural integrity of the vascular wall (Woessner and Nagase, 2000a). MMP-3 also makes a major contribution to the pericellular MMP activation cascade by cleaving other MMP proenzymes (Woessner and Nagase, 2000b). Additionally, MMP-3 has effects unrelated to matrix protein degradation (McCawley et al., 2001) as it is able to degrade proteins such as insulin-like growth factor binding protein-1 (IGFBP-1; Westwood et al., 2003) and IGFBP-3 (Fowlkes et al., 1994) and in doing so increased growth factor bioavailability.
The gestational increase in MMP-3 expression may indicate a role in remodelling events which could increase aortic diameter. This is supported by studies of abdominal aortic aneurysm (AAA) progression in man in which the lumen of the aorta is grossly distended, a process in which MMP-3 up-regulation has been implicated. Carrell et al. (2002) recently examined differences in profile of MMP gene expression between patients with aortic aneurysm and patients with aortic atherosclerosis but without aneurysm. Of 14 MMP tested, only MMP-3 was increased in aortic aneurysm samples, showing a 40-fold increase in expression. Additional studies specifically suggest a link with the MMP-3 genotype and increased risk of AAA. A two-allele polymorphism involving either a run of five or six adenosines (5A/6A) has been identified in the MMP-3 gene promoter (Ye et al., 1996) and homozygosity for the 5A allele correlates with genetic predisposition for aortic (Yoon et al., 1999) and coronary artery (Lamblin et al., 2002) aneurysmal formation. In vitro studies of promoter strength show the 5A allele to express higher transcriptional activity than the 6A allele, resulting in 2-fold higher protein expression in both cultured fibroblasts and VSMC (Ye et al., 1996). Further evidence implicating MMP-3 in the aberrant expansive remodelling that occurs in AAA is the reduced incidence of aneurysm formation in ApoE-null mice with MMP-3 gene inactivation and increased frequency of aneurysms in MMP-3 +/+ mouse aortas (Silence et al., 2001).
MMP-3 could also participate in maintaining increases in aortic compliance, but the influence of MMP-3 expression on elastic properties has not yet been directly assessed. However, recent preliminary data specifically linking the 5A/5A MMP-3 genotype with age-associated aortic stiffening (Kingwell et al., 2001) suggest that factors other than MMP-3 may contribute to altered compliance in late gestation.
Regulation of MMP-3 gene expression
Expression of MMP-3 is primarily regulated at the level of transcription. With growing evidence of an intravascular inflammatory response occurring in association with maternal vascular adaptation in pregnancy (Redman et al., 1999), it may be relevant that MMP-3 transcription is enhanced by a number of proinflammatory cytokines including IL-1 (Quinones et al., 1994) and growth factors such as EGF (McDonnell et al., 1990) and PDGF (Diaz-Meco et al., 1991). Although estradiol, which rises in late gestation in the rat, is reported to reduce MMP-3 gene expression in vitro (Schatz et al., 1994; Kapila and Xie, 1998), the addition of relaxin to estrogen-primed cells markedly enhanced MMP-3 mRNA (Kapila and Xie, 1998). Since maternal plasma concentrations of both relaxin and estradiol peak just before delivery in the rat (Gibori and Sridaran, 1981; Honda et al., 1998), synergy between these hormones could contribute to MMP-3 mRNA enhancement in late pregnant rat aorta.
Altered MMP-2 activity in rat aorta
Elevated plasma MMP-2 levels have recently been reported in pre-eclamptic women in late pregnancy (Narumiya et al., 2001), and it has been postulated that this metalloprotease could contribute to vascular dysfunction in this syndrome. Prior to the present study, the potential role of MMP-2 in aortic adaptation during normal pregnancy had not been addressed. In contrast to our recent findings in rat uterine artery (Kelly et al., 2003), MMP-2 mRNA in rat aorta was not gestationally regulated. However, although most MMP are regulated primarily at the level of transcription, MMP-2 is often constitutively expressed by vascular smooth muscle and endothelial cells and may be modulated post-transcription, through inducible effects on mRNA stability (Overall et al., 1991) and/or post-translation via a unique mechanism of enzyme activation (Strongin et al., 1995). For this reason, the gestational profile of MMP-2 activity was also assessed. A two-step sequential protein extraction was employed in order to recover both soluble and membrane-bound enzyme as well as enzyme that may have been bound to matrix. Most gelatinolytic activity was recovered in the first, salt/detergent, extraction, in which total MMP-2 enzyme (pro and active forms) was significantly elevated at day 7 gestation. By further extracting the remaining pellet with a second, more chaotropic, urea-based buffer and by showing no pregnancy-related change in total MMP-2 activity, we could conclude that this increase was not due to gestation-induced alteration in extractability of enzyme.
Disparity between total MMP-2 protein/activity and message levels has been described in cultured aortic VSMC (Palumbo et al., 2000) and intact arteries (Karwowski et al., 1999) in response to altered flow in addition to VSMC subjected to mechanical stretch (Grote et al., 2003). Whilst these studies have not commented on this disparate pattern, one possible interpretation of our data is that MMP-2 in pregnant rat aorta is partly modulated at the level of translation. Indeed MMP-9, the closest homologue of MMP-2, can be regulated through altered translational efficiency as shown in a prostatic carcinoma cell line (Jiang and Muschel, 2002). Our results also suggest that MMP-2 in early pregnant rat aorta is subject to further regulation post-translation as a greater fraction of total MMP-2 was recovered as active 65 kDa enzyme at day 7 pregnancy in both extractions, consistent with increased proMMP-2 activation.
Possible role of MMP-2 in gestational regulation of vascular reactivity
The biological significance of enhanced MMP-2 activity at day 7 gestation must be speculative. An increase in aortic MMP-2 has been implicated in matrix degradation contributing to aberrant aortic remodelling in AAA progression (Knox et al., 1997; Crowther et al., 2000) and to medial changes observed in ageing aorta (Wang and Lakatta, 2002). It is also possible that MMP-2 could contribute to the previously reported reduction in vasomotor tone (Jain et al., 1998) and increase in aortic compliance (Slangen et al., 1997) in early pregnant rat aorta as MMP-2 has been shown to induce rapid vasodilation of preconstricted rat mesenteric arteries at picomolar concentrations (Fernandez-Patron et al., 2000). MMP-2 may also mediate vasodilatory effects of other vasoactive agents, since MMP-2 inhibition abrogates both relaxin- and thrombin-induced vasorelaxation in rat resistance arteries (Fernandez-Patron et al., 2000; Jeyabalan et al., 2003). A mechanism for MMP-2-induced vasodilatation has recently been elucidated in an elegant series of experiments. Vascular MMP-2 (Fernandez-Patron et al., 1999b) but not thrombin (Fernandez-Patron et al., 2000) is able to cleave big endothelin-1 (ET-1) to ET-11–32, which is a potent activator of the endothelial ‘vasodilatory’ ETB receptor, suggesting that MMP-2-mediated vasorelaxation may occur through activation of ETB receptors. A similar mechanism may underlie relaxin-induced MMP-2-mediated vasodilatation in renal arteries, a pathway also dependent on ETB receptor activation (Jeyabalan et al., 2003).
In summary, this study has examined for first time regulation of MMPs and TIMPs in maternal aorta during pregnancy. The data presented strongly support a role for MMP-3 in aortic adaptation in late gestation. Although MMP-2 mRNA appeared to be constitutively expressed in rat aorta, enhanced levels of MMP-2 activity were observed in early gestation. This protease may contribute to altered lumen diameter and compliance through recently reported effects on vascular tone as well as more established routes of matrix degradation. Aberrant regulation of these enzymes may contribute to the pathogenesis of vascular disorders in pregnancy including pre-eclampsia and aortic dissection.
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Acknowledgements |
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We gratefully acknowledge the advice given by Susan Pickering (King’s College London) and Paul Murdock (Glaxo SmithKline Pharmaceuticals, Harlow, UK) on real-time PCR and Mike Mitchell (Haemophilia Unit, St Thomas’ Hospital, London, UK) for use of the TaqMan. We thank Yoshifumo Itoh and Hideaki Nagase for advice on protein extraction. This research was funded by the British Heart Foundation (B.A.K., Clinical PhD Studentship, FS99044) and by Tommy’s the Baby Charity (registered charity no. 106058).
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Submitted on December 29, 2003; accepted on January 26, 2004.
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