Victoria L Bills
email: victoria.bills@bristol.ac.uk

Julia Varet

Ann Millar

Steven J Harper

Peter W Soothill

Dr David O Bates
email: dave.bates@bris.ac.uk

Failure to upregulate VEGF 165 b in maternal plasma is a first trimester predictive marker for pre-eclampsia

Keywords: vascular endothelial growth factor 165 b, pre-eclampsia, splice variant, plasma marker, vascular permeability, angiogenesis

Pre-eclampsia is a pregnancy related condition characterised by hypertension, proteinuria and endothelial dysfunction. VEGF 165 b, formed by alternative splicing of vascular endothelial growth factor (VEGF) pre-mRNA inhibits VEGF 165 mediated vasodilatation and angiogenesis, but has not been quantified in pregnancy. Enzyme-linked immunoassays (ELISA) were used to measure mean±SEM plasma VEGF 165 b, soluble endoglin (sEng) and soluble Flt1 (sFlt-1). At 12 weeks gestation the plasma VEGF 165 b concentration was significantly upregulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group 4.90 ±1.6 ng/ml, p<0.01 Mann-Whitney U test) compared with non-pregnant women (0.40±0.22ng/ml). In contrast, in patients who later developed pre-eclampsia VEGF 165 b levels were lower than in the normotensive group (0.467 ±0.21 ng/ml) but no greater than non-pregnant women.

At term plasma VEGF 165 b concentrations was greater than normal in both pre-eclamptic (3.75 ±2.24 ng/ml) and normotensive pregnancies (10.6ng/ml±3.84 ng/ml p>0.1 compared with pre-eclampsia). Patients with a lower than median plasma VEGF 165 b at 12 weeks, had elevated soluble fms-like tyrosine kinase receptor 1 (sFlt-1) and soluble endoglin (sEng) pre-delivery. Concentrations of sFlt-1 (1.20±0.07 ng/ml and 1.27±0.18ng/ml) and sEng (4.4±0.18 vs 4.1±0.5) were similar at 12 weeks gestation in the normotensive and pre-eclamptic groups, respectively. Plasma VEGF 165 b levels were elevated in pregnancy, but this elevation is delayed in women that subsequently develop pre-eclampsia. Low VEGF 165 b may therefore be a clinically useful first trimester plasma marker for increased pre-eclampsia risk.

Introduction

Pre-eclampsia, the pregnancy related disease of hypertension, proteinuria and oedema is responsible for around 12% of the world's annual 514,000 maternal deaths [START_REF] Khan | WHO analysis of causes of maternal death: a systematic review[END_REF]. Aside from maternal and fetal death, the condition may also result in intra-uterine growth restriction, seizures (eclampsia), renal or liver failure, and placental abruption. Despite much investigation, the pathological processes underlying this disease are still largely undiscovered. Recent investigation had focussed on defective placental implantation as an important aetiological factor, with the resulting release of placentally derived circulating factors, which cause endothelial dysfunction [START_REF] Zhou | Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome?[END_REF][START_REF] Maynard | Soluble Fms-like tyrosine kinase 1 and endothelial dysfunction in the pathogenesis of preeclampsia[END_REF][START_REF] Levine | Circulating angiogenic factors and the risk of preeclampsia[END_REF]. At the microvascular level, there is a state of vasoconstriction from smooth muscle contraction, increased vascular permeability and anti-angiogenesis [START_REF] Baumwell | Pre-eclampsia: clinical manifestations and molecular mechanisms[END_REF], which correspond to the clinical findings of high blood pressure, oedema and a characteristically small placenta at delivery of the baby.

The vascular endothelial growth factor (VEGF) family is thought to be one of the important molecular systems involved in the pathogenesis of pre-eclampsia.

Conventional VEGF; also known as VEGF-A, is made up of 6 different isoforms formed from alternative exon splicing resulting in proteins of varying amino acid length, termed VEGF xxx . VEGF 165 is the most common isoform of VEGF xxx , and consists of 165 amino acids. VEGF 165 acts via its receptor VEGFR-2 to increase vascular permeability, vasodilatation and angiogenesis [START_REF] Ferrara | Vascular endothelial growth factor: basic science and clinical progress[END_REF]. Endogenous alternative splicing of the VEGF receptor results in soluble VEGFR-1 (also known as soluble fms-like tyrosine kinase 1 or A c c e p t e d M a n u s c r i p t sFlt-1), which binds to VEGF and inhibits its function [START_REF] Ferrara | Vascular endothelial growth factor: basic science and clinical progress[END_REF]. High levels of sFlt-1 have been documented in pre-eclampsia [START_REF] Maynard | Excess placental soluble fmslike tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia[END_REF].

VEGF levels in pre-eclampsia have been measured by a number of techniques, with conflicting results according to the technique used. When measured by commercial sandwich Enzyme Linked ImmunoSorbent Assays (ELISAs) which has been proposed to measure only the free, unbound forms of VEGF, levels appear to be reduced in preeclampsia [START_REF] Livingston | Reductions of vascular endothelial growth factor and placental growth Clinical Science Immediate Publication[END_REF]9]. When measured by radioimmunoassay (RIA) or competitive enzyme immuno assay (cEIA), VEGF levels are shown to substantially increase. This discrepancy has been proposed to be due to these latter 2 methods not being affected by circulating binding proteins [10,11].

In 2002, an alternative family of VEGF-A isoforms were identified, termed VEGF xxx b.

These are the same size as conventional VEGF-A but are alternatively spliced in exon 8

Endoglin and sFlt-1 ELISAs

ELISAs for soluble endoglin and sFlt-1 were carried out on maternal plasma samples using commercial ELISA kits from R&D Systems (DNDG00 and DVR100B respectively) according to the manufacturer's instructions.

Results

During the study period, 100 patients were recruited. 25 patients had pre-eclampsia, and 45 remained normotensive. Of the 30 recruits who were excluded from the study, 5

developed pregnancy induced hypertension, 1 developed idiopathic fetal growth restriction, 9 patients chose not to attend follow up appointments due to social reasons, 2 patients experienced intrauterine deaths at 21 and 28 weeks gestation, 3 patients experienced preterm labour in the absence of pre-eclampsia, and in 10 patients with preeclampsia no first trimester blood sample was available.

Discussion

There have been a number of studies investigating the VEGF family of proteins in preeclampsia [START_REF] Levine | Circulating angiogenic factors and the risk of preeclampsia[END_REF]14,20], which have suggested that they may play a role in its pathophysiology [21,22]. The total VEGF levels measured by EIA are consistent with those previously measured using this assay methodology [11], and by those using an independent method, the radioimmunoassay [10]. In contrast the ELISA results from the same samples gave much lower readings, consistent with previous ELISA reports of plasma VEGF [START_REF] Nadar | Plasma markers of angiogenesis in pregnancy induced hypertension[END_REF]. These experiments therefore highlight the previously reported discrepancy between measurements of total circulating VEGF in plasma by commercial ELISAs compared with cEIA, or RIA [START_REF] Anthony | Variation in detection of VEGF in maternal serum by immunoassay and the possible influence of binding proteins[END_REF]. The antibodies used in the ELISA are two monoclonals raised against the VEGF peptide sequence and thus may be raised against a similar or identical epitope.

The ELISA appears to yield artificially low results, presumably as VEGF is bound by agents in plasma which prevent its detection by both antibodies simultaneously. sFlt-1 does not affect this ELISA when given as recombinant protein [START_REF] Varey | VEGF(165)b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro-and antiangiogenic VEGF-A isoforms has implications for therapy[END_REF], but the effect of endoglin or other plasma constituents have not been tested.

The discrepancy was particularly striking after measurement of VEGF 165 b levels, using an ELISA that detects plasma VEGF 165 b using two antibodies that have epitopes on Of the VEGF family, VEGF 165 the most widely studied form [START_REF] Ferrara | Vascular endothelial growth factor: basic science and clinical progress[END_REF] is known to increase vascular leakage, induce vasodilatation and promote angiogenesis. Although this isoform is up-regulated in pre-eclampsia, its metabolic activities may be blocked by other proteins, which bind to VEGF and inhibit its function. sFlt-1 and sEng both bind to VEGF and prevent it from exerting its physiological effects [15] . sFlt-1 is an antiangiogenic molecule that is able to induce a pre-eclamptic-like syndrome of hypertension and proteinuria when administered to pregnant rats [START_REF] Maynard | Excess placental soluble fmslike tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia[END_REF]. sEng is an anti-angiogenic protein that inhibits transforming growth factor β

Clinical

  Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270

[

  12], This alternative splice site selection results in an alternate 6 amino acid C terminus, which affects the property of the isoforms. VEGF 165 b is the most widely studied of these isoforms. VEGF 165 b has been shown to inhibit the effects of VEGF 165 by binding to its principal receptor VEGFR-2 and preventing it from exerting its physiological effects such as endothelial cell proliferation and migration. VEGF 165 b also binds to and activates Flt-1 (VEGFR-1) resulting in a transient increase in capillary hydraulic conductivity, but no sustained increase in permeability, in contrast with VEGF 165 [13]. A previous study of VEGF 165 b in term placenta detected a decrease in VEGF xxx b expression in pre-eclamptic placenta compared with control placenta, and an uncoupling of the splicing link between VEGF 165 b and VEGF 165 . To determine whether VEGF 165 b may play a role in the Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t pathogenesis of pre-eclampsia, we have investigated the expression of VEGF 165 b in maternal plasma from normotensive and pre-eclamptic pregnancies. Methods Pregnant subjects were recruited from St Michael's Maternity Hospital, Bristol between June 2006 and December 2007. 18 non-pregnant females aged between 20 and 39 years were recruited from the University of Bristol. The protocol for this study was granted ethical approval by Central and South Bristol Research Ethics Committee. 50 subjects were recruited from routine antenatal clinics in the first trimester of pregnancy. Subjects were aged between 17 and 42 years. Blood was taken from subjects for VEGF 165 b quantification at recruitment, and at a further 3 times at 28, 34 and 37 weeks gestation.Pre-eclampsia was defined as BP ≥140/90mmHg on ≥ 2 occasions measured 6 hours apart and ≥300mg of proteinuria/24 hours, in the absence of a urinary tract infection, occurring after 20 weeks gestation. 5 patients who later developed pre-eclampsia had further blood taken at disease diagnosis.Following venepuncture, blood was immediately centrifuged at 4000rpm for 10 minutes, the supernatant removed and stored at -80 °C until protein quantification. Subjects also received fetal growth ultrasound scans at 28, 34 and 37 weeks gestation to screen for intra-uterine growth restriction secondary to pre-eclampsia. During the study period, a further 20 patients who developed pre-eclampsia in the third trimester were recruited into the study at disease diagnosis, and received fetal growth scans at the point of recruitment into the study. In these cases plasma from their first trimesters was obtained from aliquots of frozen plasma stored under the same standard blood storage conditions by the hospital's virology department. Sample size was calculated to see an 80% change in mean VEGF 165 b levels

Total

  VEGF ELISA and EIA Total circulating VEGF was quantified by commercial ELISA (Alpco Diagnostics, 45-VEGFH-0111) and by competitive enzyme immunoassay (Calbiochem, QIA69). The EIA measures both bound and free forms of VEGF. Competitive EIAs for total VEGF quantification have not been commercially available since 2006 and we had access to only a single 96 well EIA. For this reason total VEGF quantification was possible in only Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t 10 patients. For each plasma sample, VEGF concentration was determined both by ELISA and EIA.

  Patients with a lower than median plasma VEGF 165 b at 12 weeks, had elevated sFlt-1 and sEng just before delivery. Concentrations of sFlt-1 (1.20±0.07 ng/ml and 1.27±0.18ng/ml) and sEng (4.4±0.18 vs. 4.1±0.5ng/ml) were similar at 12 weeks gestation in the normotensive and pre-eclamptic groups, respectively [Figure2a]. Therefore, at 12 weeks gestation, neither sFlt-1 nor sEng were able to predict the onset of pre-eclampsia later in the pregnancy [Figure5b and 5c]. At disease diagnosis however, both sEng and sFlt-1 were significantly up-regulated in comparison with healthy patients [p<0.001, Mann Whitney U test, Figures2b and 2c]. VEGF 165 b predicts sFLT-1 and sEndoglin The reduced first trimester levels of VEGF 165 b are able to predict the elevated sFLT-1, which occurs with the onset of pre-eclampsia[p=0.028, Mann-Whitney U Test]. Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t However, VEGF 165 b concentration in the first trimester does not correlate with the elevated sEndoglin of pre-eclampsia. Commercial total VEGF ELISAs underestimate total VEGF levels. Total circulating VEGF was quantified in the same plasma samples both by commercial ELISA and EIA. When quantified by ELISA, VEGF concentrations were on average 2500 fold lower than when quantified by EIA [p<0.0001, Figure 3a and 3b].VEGF 165 b accounts for the majority of total circulating VEGF in the third trimester in pre-eclamptic pregnancy.In 5 patients from each group we were able to quantitate VEGF 165 b and total VEGF in the same samples. VEGF 165 b expression increased in both pre-eclampsia and normotensive pregnancy with increasing gestational age. At 12 weeks gestation, VEGF 165 b accounts for 10.5±20% of total plasma VEGF in patients that went on to develop pre-eclampsia, compared with 18.1±10% in control patients (figure5). With the onset of pre-eclampsia, VEGF 165 b accounted for the majority of total circulating VEGF, comprising 69.3±21% of total plasma VEGF in the patient group and 49±12% in the control group.VEGF 165 b levels at 12 weeks predict pre-eclampsia.To determine which of VEGF 165 b, sFlt1 and endoglin are more accurate prognostic factors, receiver operating characeteristic (ROC curves) were generated by calculating sensitivity (proportion of times that the test predicts pre-eclampsia) and specificity (proportion of times that the test excluded pre-eclampsia). Thus a high sensitivity value Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t would include all patients, but if not discriminatory, would provide a low specificity value (would include false positives). Thus non discriminatory test would give a straight line with a slope of 1 and area under the curve of 0.5 (dotted line). A perfect discriminatory test would have an area under the curve of 1.0. It can be seen from figure 5A that VEGF 165 b levels have an area under the curve significantly greater than 0.5, in contrast to sflt and sEng.

Clinical

  Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t completely separate parts of the antigen (VEGF) molecule. It is therefore rather disturbing that the cEIA is no longer commercially available and was withdrawn from sale by all known suppliers between 2006 and 2007.

  1 and β 3 signalling and increases the severity of pre-eclampsia occurring in pregnant rats treated with sFlt-1 [16]. However, neither molecule can be used clinically as a first trimester marker of pre-eclampsia as sFlt-1 levels are seen to rise only 5 weeks before the onset of the clinical disease [17], and sEng concentrations become elevated at 17 weeks gestation [15]. In 2002 VEGF 165 b was identified in normal renal cortex, and subsequently shown to be present in many different tissues, and forms the majority of VEGF in tissues such as human colon [25], and vitreous [26]. VEGF 165 b is relatively downregulated in many conditions, including prostate, renal, bowel, and skin cancers [12,18,25,27], diabetic retinopathy [26], Denys Drash Syndrome [28] and in the placenta of patients with preeclampsia [29]. The mechanisms underlying these changes in expression are still under investigation, but the reduction is associated with excess angiogenesis. VEGF 165 b has been shown to be anti-angiogenic in animal models of VEGF 165 induced blood vessel Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t growth in the cornea [30], mouse subcutaneous tissue [31] and rat mesentery [18] and inhibits physiological [32], and pathological [25, 30,32] angiogenesis. Studies have also shown that VEGF 165 b transiently, but not chronically increases hydraulic conductivity [18]. The results shown here indicate that VEGF 165 b fails to be upregulated in the first trimester in those pregnancies that will later be complicated by pre-eclampsia. It can be concluded that VEGF 165 b may be a clinically useful first trimester marker for increased pre-eclampsia risk, providing for instance a guide to commencement of first trimester oral aspirin therapy, as this decreases the incidence of pre-eclampsia by 15% [19]. It is not clear what mediates the upregulation of VEGF 165 b in early pregnancy, nor what prevents it in women who will develop pre-eclampsia, and further work must be done to investigate this finding. The failure of upregulation may be reflective of the aetiology, or could be contributory to the subsequent pre-eclampsia. For instance, in the first trimester the reduced anti-angiogenic VEGF 165 b compared with normal pregnancy may reflect a maternal vasculature response to try and correct the defective implantation processes underlying the disease, or the failure to upregulate VEGF 165 b may contribute to defective implantation. Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t factor concentrations in severe preeclampsia. Am J Obstet Gynecol. Dec;183(6):1554-7. 9 Lyall F, Young A, Boswell F, Kingdom JC, Greer IA. (1997) Placental expression of vascular endothelial growth factor in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth restriction does not support placental hypoxia at delivery. Placenta. May;18(4):269-76. 10 McKeeman GC, Ardill JE, Caldwell CM, Hunter AJ, McClure N. (2004) Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is increased throughout gestation in patients who have preeclampsia develop. Am J Obstet Gynecol. Oct;191(4):1240-6. 11 Lee ES, Oh MJ, Jung JW, Lim JE, Seol HJ, Lee KJ, Kim HJ. (2007) The levels of circulating vascular endothelial growth factor and soluble Flt-1 in pregnancies complicated by preeclampsia. J Korean Med Sci. Feb;22(1):94-8. 12 Bates DO, Cui TG, Doughty JM et al. (2002) VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma. Cancer Res. Jul 15;62(14):4123-31. 13 Glass CA, Harper SJ, Bates DO. (2006) The anti-angiogenic VEGF isoform VEGF 165 b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo. J Physiol. Apr 1;572(Pt 1):243-57. 14 Brockelsby J, Hayman R, Ahmed A, Warren A, Johnson I, Baker P. (1999) VEGF via VEGF receptor-1 (Flt-1) mimics preeclamptic plasma in inhibiting uterine blood vessel relaxation in pregnancy: implications in the pathogenesis of preeclampsia. Lab Invest. Sep;79(9):1101-11. Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 A c c e p t e d M a n u s c r i p t Levine RJ, Lam C, Qian C et al. (2006) Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med. Sep 7;355(10):992-1005. 16 Venkatesha S, Toporsian M, Lam C et al. (2006) Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat Med. Jun;12(6):642-9. 17 Levine RJ, Maynard SE, Qian C et al. (2004) Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med. Feb 12;350(7):672-83. 18 Woolard J, Wang WY, Bevan HS et al. (2004) VEGF 165 b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res. Nov 1;64(21):7822-35. 19 Duley L, Henderson-Smart D, Knight M, King J. (2001) Antiplatelet drugs for prevention of pre-eclampsia and its consequences: systematic review. BMJ Feb 10;322(7282):329-33. 20 Sgambati E, Marini M, Zappoli Thyrion GD et al. (2004) VEGF expression in the placenta from pregnancies complicated by hypertensive disorders. BJOG. 2004 Jun;111(6):564-70. 21 Zhihe Li, Ying Zhang, Jing Ying Ma et al. (2007) Recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia. Hypertension. Oct;50(4):686-92.

Figure 4 .

 4 Figure 4. Total VEGF was quantified both by EIA (shown in grey) and ELISA (shown in black) in maternal plasma from normotensive and pre-eclamptic pregnancies (n=10). Detectable levels are 2500 fold lower when measured by ELISA compared to EIA, p<0.0001.

Figure 5 .

 5 Figure 5. The increase in VEGF levels seen during pregnancy are primarily due to increased VEGF 165 b. (A). At 12 weeks, only a small proportion of total VEGF (10-18%) was VEGF 165 b(n=10). (B). In contrast at term, approximately half the VEGF was VEGF 165 b in normals, and In pre-eclampsia, 70% of total VEGF was VEGF 165 b.

Figure 6 .

 6 Figure 6. Receiver-operating characteristic (ROC) curves for first trimester VEGF 165 b, sFlt-1 and sEng in the prediction of pre-eclampsia. Area under the curve (AUC) was highest for VEGF 165 b (0.705, p=0.0047 compared with random (0.5)). AUC for sEng and sFlt-1 were 0.59 (p=0.34) and 0.56 (p=0.43) respectively, not different from random (0.5).

  Figure 3

  Figure 4

  Figure 5

  Figure 6

  concentrations to ensure values were in the range of the ELISA, in triplicate. This sandwich ELISA measures total circulating VEGF 165 b. It has been shown not to detect VEGF 165 and sFlt-1 is known not to interfere due to the use of antibodies against the

	Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270
	at p<0.05 with a power of >90% given a standard deviation equivalent to the mean
	(calculated using G Power).
	VEGF 165 b ELISA
	A c c e p t e d M a n u s c r i p t Anti-VEGF xxx b antibody (MAB3045, clone 56/1, R&D systems) was coated onto the surface of a sterile Immulon-2HB 96 well plate at a concentration of 200µg/ml overnight. This antibody recognises an epitope within a 9 amino acid sequence at the C terminus of human VEGF 165 b. The plate was washed 3 times with PBS-Tween 0.05%, 100µl/well. The plate was blocked for 12 hours with Superblock (250µl/well, Pierce 37515). Serial dilutions of recombinant VEGF 165 b standards (R&D systems) diluted in PBS/BSA up to a concentration of 16ng/ml were then added to the wells in triplicate (200µl/well). Plasma samples were also added in triplicate (200µl/well). Plates were then incubated at room temperature with shaking for 2 hours and then washed as above. 50ng/ml of anti-human VEGF biotinylated affinity purified polyclonal antibody (BAF293, R&D systems) was added (200µl/well) and incubated at room temperature with shaking in foil for 2 hours as a detection reagent. Following a further wash, 100µl HRP-streptavidin diluted 1 in 200 in PBS was added for 20 minutes protected from light, and then substrates A and B (100µl/well) were added following washing. After 25 minutes the colour change was stopped on addition of 1M H 2 SO 4 (50µl/well) and plates were read immediately at a wavelength of 450nm using a plate photospectrometer (Dynex Technologies). Revelation Quicklink 4.25 software was used to construct a standard curve from mean absorbance values of VEGF 165 b standards, which enabled estimation of VEGF 165 b concentration in A c c e p t e d M VEGF 165 b molecule with epitopes at different parts of the molecule 25 . The coefficients of variation (CV) of this assay in quantifying VEGF xxx b was 17% for within subject variation (samples taken at least a week apart), and 7% for within sample variation, a n u s c r i p t whereas between sample CV was >200%, indicating consistency of assay, and significant	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270
	plasma samples. VEGF 165 b sample concentrations were quantified at multiple different

variation amongst the population. VEGF 165 b concentration in maternal plasma was quantified at 8-12 weeks, 28, 34 and 37 weeks gestation in 45 normotensive subjects and 4 subjects recruited in the first trimester who later developed pre-eclampsia in the third trimester. VEGF 165 b concentration was also quantified in 21 pre-eclamptic patients at 12 weeks gestation, and again in the third trimester at disease diagnosis. This ELISA is now available as a DuoSet Kit from R&D Systems.

Table 1

 1

	Characteristic Maternal age (yrs)	Normotensive (n=45) 30±0.8 M a n u s c r i p t Pre-eclampsia (n=25) 30±1.3	1042/CS20080270
	Gestational age at diagnosis (wks) Gestational age at birth (wks) Systolic BP (mmHg) Diastolic BP (mmHg) Proteinuria (g/24 hours) Primiparous (%) Birth weight (g) Platelet count (10 9 /l) Creatinine (mmol/l) A c c e p t e d NA 39+3 ±0.17 <140 <90 <0.3 58 3495±481 259±10 60±1.3	34+5±0.6 36+3 ±0.47 151±3.1 98±1.7 1.3±0.17 52 2513±166 206±17 79±2.5

shows the clinical characteristics of the study participants. Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.

Table 1

 1 Clinical characteristics of the study participants. Values shown are mean±SEM.

	were 30±0.8 and 30±1.3 years respectively. There were no differences in smoking status
	or ethnicity between the groups. Within the pre-eclampsia group, the mean gestational
	age at diagnosis was 34+5±0.6 weeks, the mean proteinuria was 1.3 ± 0.17g/24 hours,
	and the mean blood pressure was 151/98 ±3.1/1.7mmHg. Mean birth weight within the

NA, not applicable.

The mean maternal age within the normotensive (n=45) and pre-eclamptic (n=25) groups pre-eclamptic and normotensive groups was 2513±166g and 3495±481g respectively. Of the 25 pre-eclamptic patients, 6 developed early-onset pre-eclampsia (<34 weeks gestation) and 12 developed pre-eclampsia between 34 and 37 weeks gestation. The remaining 7 patients developed pre-eclampsia at full term. 5 of the 25 pre-eclamptic patients developed severe pre-eclampsia (according to the Royal College of Obstetricians and Gynaecologists criteria: systolic BP >169mmHg or diastolic BP >109mmHg with > 1 gram proteinuria in 24 hours; or the occurrence of HELLP syndrome). 5 of the 25 fetuses born to pre-eclamptic mothers had growth restriction (ultrasonically defined as estimated fetal weight < 10 th percentile for gestational age with further evidence of placental insufficiency such as oligohydramnios or abnormal umbilical artery Dopplers 33 ). Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.

Reduced first trimester VEGF 165 b in patients who later develop pre-eclampsia

At 12 weeks gestation the plasma VEGF 165 b concentration was significantly lower in patients who later developed pre-eclampsia (0.467±0.209 ng/ml) compared with plasma from normotensive pregnancies [p<0.0047, as determined by Mann-Whitney U test, 165 b concentrations between pre-eclamptic (3.75±2.24 ng/ml) and normal pregnancies (10.58 ±3.74 ng/ml). Thus, pre-eclampsia was associated with an 8±1.8 fold increase in plasma VEGF 165 b from first trimester to pre-delivery, compared with a 2±0.3 fold increase in normotensive plasma [p<0.0012, Mann-Whitney U test, Figure

1B

].

  Schumacher VA, Jeruschke S, Eitner F et al. (2007) Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome. J Am Soc Figure 1. Measurement of VEGF 165 b levels in human plasma. A. At 12 weeks gestation VEGF 165 b was increased in plasma from pregnant women who went on to have normotensive pregnancies (n=45). This was not the case in patients who subsequently developed severe, early onset and non severe pre-eclampsia (n=25),. One way anova, Kruskal Wallis test p=0.0003. Subgroup analysis of severe/early onset pre-eclampsia (n=9) vs normotensive patients also showed that VEGF 165 b is significantly lowered, Mann-Whitney U test, p=0.008. B. VEGF 165 b levels in both pre-eclamptic and normal patients was increased in the third trimester (n=25). Mann-Whitney U Test, p=0.0012.Figure2. First trimester sFlt-1 and sEng do not predict increased risk of preeclampsia. At 12 weeks gestation, healthy subjects and subjects who later developed pre-eclampsia had similar levels of both sFlt-1 and sEng. Neither plasma marker was able to predict pre-eclampsia at 12 weeks gestation. Preeclampsia is associated with up-regulation of maternal plasma levels of B. sFlt-1 and C, sEng relative to first trimester levels. In normotensive pregnancy plasma levels of both molecules increase with advancing gestational age by 2.8 fold (sEng) and 5.3 fold (sFlt-1). Mann-Whitney U Test, p<0.001. Figure 3. Lack of upregulation of VEGF 165 b in the first trimester is able to predict the elevated sFlt-1 concentration occurring with the onset of pre-eclampsia. Mann-Whitney U test, 0.028. However, first trimester VEGF 165 b does not correlate with sEndoglin concentration at pre-eclampsia diagnosis.

	Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270 Clinical Science Immediate Publication. Published on 01 Oct 2008 as manuscript CS20080270
	Nephrol. Mar;18(3):719-29.
	A c c e p t e d M a n u s c r i p t 29 Bates DO, MacMillan PP, Manjaly JG et al. (2006) The endogenous anti-angiogenic family of splice variants of VEGF, VEGFxxxb, are down-regulated in pre-eclamptic placentae at term. Clin Sci (Lond). May;110(5):575-85. 30 Konopatskaya O, Churchill AJ, Harper SJ, Bates DO, Gardiner TA. (2006) VEGF165b, an endogenous C-terminal splice variant of VEGF, inhibits retinal neovascularization in mice. Mol Vis. May 26;12:626-32. 31 Rennel ES, Waine E, Guan H et al. (2008) The endogenous anti-angiogenic VEGF isoform, VEGF(165)b inhibits human tumour growth in mice. Br J Cancer. Apr 8;98(7):1250-7. 32 Qiu Y, Bevan H, Weeraperuma S et al. (2008) Mammary alveolar development during lactation is inhibited by the endogenous antiangiogenic growth factor isoform, VEGF165b. FASEB J. Apr;22(4):1104-12. 33 Alberry M, Soothill PW. (2007) Management of fetal growth restriction. Arch Dis Child Fetal Neonatal Ed. Jan;92(1):F62-7. A c c e p t e d M a n u s c r i p t	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080270

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society


MD Gjin Ndrepepa
email: ndrepepa@dhm.mhn.de

Siegmund Braun

Raisuke Iijima

Dritan Keta

Robert A Byrne

Stefanie Schulz

Julinda Mehilli

Albert Schömig

Adnan Kastrati

percutaneous coronary intervention

Keywords: Acute coronary syndrome, mortality, myocardial infarction, statins, unstable angina, white blood cells Clinical Science Immediate

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Considerable evidence exists that elevated white blood cell (WBC) count -a nonspecific marker of inflammation -is a risk factor for future cardiovascular events in individuals apparently free of cardiovascular disease [START_REF] Friedman | The leukocyte count as a predictor of myocardial infarction[END_REF][START_REF] Mänttäri | Leukocytes as a coronary risk factor in a dyslipidemic male population[END_REF] and a prognostic marker for patients with already cardiovascular disease. [START_REF] Schlant | The natural history of coronary heart disease: prognostic factors after recovery from myocardial infarction in 2789 men. The 5-year findings of the coronary drug project[END_REF][START_REF] Lowe | White blood cell count and haematocrit as predictors of coronary recurrence after myocardial infarction[END_REF] In patients with acute coronary syndromes (ACS), an elevated baseline WBC count predicts mortality in those with ST-segment elevation acute myocardial infarction [START_REF] Hajj-Ali | Relation of the leukocyte count to recurrent cardiac events in stable patients after acute myocardial infarction[END_REF][START_REF] Furman | Effect of elevated leukocyte count on in-hospital mortality following acute myocardial infarction[END_REF][START_REF] Barron | The association between white blood cell count and acute myocardial infarction mortality in patients ≥65 years of age: findings from the cooperative cardiovascular project[END_REF], non-ST-segment elevation acute myocardial infarction [START_REF] Sabatine | Relationship between baseline white blood cell count and degree of coronary artery disease and mortality in patients with acute coronary syndromes: a TACTICS-TIMI 18 (Treat Angina with Aggrastat and determine Cost of Therapy with an Invasive or Conservative Strategy-Thrombolysis in Myocardial Infarction 18 trial) substudy[END_REF][START_REF] Mueller | White blood cell count and long term mortality after non-ST elevation acute coronary syndrome treated with very early revascularisation[END_REF], and unstable angina. [START_REF] Sabatine | Relationship between baseline white blood cell count and degree of coronary artery disease and mortality in patients with acute coronary syndromes: a TACTICS-TIMI 18 (Treat Angina with Aggrastat and determine Cost of Therapy with an Invasive or Conservative Strategy-Thrombolysis in Myocardial Infarction 18 trial) substudy[END_REF][START_REF] Cannon | Association of white blood cell count with increased mortality in acute myocardial infarction and unstable angina pectoris. OPUS-TIMI 16 Investigators[END_REF] However, several aspects of the association between elevated WBC and cardiovascular disease require further investigation. First, although an association between elevated WBC count and mortality was almost uniformly confirmed by various studies, the independence of association, i.e., whether elevated WBC count retains its prognostic power after full adjustment for other cardiovascular risk factors has been inadequately studied [START_REF] Madjid | Leukocyte count and coronary heart disease: implications for risk assessment[END_REF] or is controversial. [START_REF] Camargo | Effect of leukocytosis at initial examination on prognosis in patients with primary unstable angina[END_REF][START_REF] Haim | Predictive value of elevated white blood cell count in patients with pre-existing coronary heart disease: the Bezafibrate Infarction Prevention Study[END_REF] Second, a comparative assessment of prognostic value of elevated WBC and C-reactive protein has not been performed. Third, it is not known whether WBC retains its prognostic potential (capacity) in patients under treatment with statin therapy.

The aim of the present study was three-fold: first, to test the association between WBC count and one-year mortality across the entire spectrum of patients with ACS after adjustment for traditional cardiovascular risk factors; to investigate the relationship between WBC count and C-reactive protein and compare their prognostic discriminatory power in patients with ACS; and third to assess the influence of statin therapy on the relationship between elevated WBC and one-year mortality in patients with ACS. Methods Patients This prospective registry included 4329 patients with ACS who underwent diagnostic coronary angiography and percutaneous coronary intervention (PCI) in the Deutsches Herzzentrum München between September 1997 and December 2006: 1059 patients with STsegment elevation acute myocardial infarction, 1753 patients with non-ST-segment elevation acute myocardial infarction and 1517 patients with unstable angina. Patients with acute inflammatory states, known malignancies and advanced kidney disease were not included in the present study. The diagnosis of ST-segment elevation acute myocardial infarction was based on the presence of chest pain lasting ≥20 min associated with typical changes on surface ECG (ST-segment elevation of ≥0.1 mV in ≥2 limb leads or ≥0.2 mV in ≥2 contiguous precordial leads, pathological Q waves, or complete left bundle branch block of new onset). Unstable angina was diagnosed when characteristic chest pain (with an accelerating pattern or prolonged [>20 minutes] or recurrent episodes at rest or with minimal effort) was associated with either ST segment depression of ≥0.1 mV and/or T-wave inversion in two continuous leads in the electrocardiogram and with documentation of significant coronary artery disease in the coronary angiography. Diagnosis of non-ST-segment elevation acute myocardial infarction required clinical and electrocardiographic criteria similar to those of unstable angina plus elevated troponin T (troponin T level >0.03 µg/L) or creatine kinase or creatine kinase-myocardial band (CK-MB). Arterial hypertension was defined in the presence of active treatment with antihypertensive medications or documentation of a systolic blood pressure of ≥140 mmHg and/or diastolic blood pressure of ≥90 mmHg in at least two separate occasions. Hypercholesterolemia was defined as a documented total cholesterol concentration of ≥240 mg/dl. Current smokers were defined as those currently smoking tobacco. Diabetes mellitus was defined if patients were under active treatment with insulin or oral hypoglycemic agents. For those on diet alone, documentation of an abnormal fasting blood glucose or glucose tolerance test was required. The study has been carried out in accordance with the Declaration of Helsinki and has been approved by the institutional ethics committee. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

WBC Count AND C-Reactive Protein Measurement

Blood samples were obtained at the time of admission (before angiographic examination) for WBC count, high-sensitivity C-reactive protein, and other biochemical parameters. For patients with ST-segment elevation and non-ST-segment elevation acute myocardial infarction, time interval (median [25th-75th percentiles]) from the pain onset to admission was 7.8 [3.0-19.0] hours. Total WBC count was counted in EDTA-anticoagulated whole blood specimens using the Sysmex XE 2100 automated haematology analyser. Measurement of WBC count was performed by flow cytometry using a semiconductor laser to detect forward-and side-scattered light information after red cell lysis. The plasma concentrations of high-sensitivity C-reactive protein were measured with a fully automated latex-enhanced immunoturbidometric assay on a Cobas Integra (Roche Diagnostics, Mannheim, Germany). The C-reactive protein assay has an analytical sensitivity of 0.085 mg/L and a measuring range up to 160 mg/L. The upper limit of the reference range of CRP in healthy adults is 5 mg/L. Other biochemical parameters were measured using routine tests. Laboratory personnel were unaware of the results of clinical diagnosis or other study details.

Angiographic Evaluation and Stent Implantation

Coronary angiography was performed according to standard criteria. Angiographic data were analyzed using the same Quantitative Angiographic Core Laboratory. Coronary artery disease was confirmed by the presence of coronary stenoses ≥50% lumen obstruction in at least one of the three main coronary arteries. A culprit lesion was described in the presence of an acute occlusion, intraluminal filling defects (or thrombus), ulcerated plaques with contrast-filled pocket protruding into plaque with or without delayed contrast wash-out, extraluminal contrast, dissection or intraluminal flaps. [START_REF] Kerensky | Revisiting the culprit lesion in non-Q-wave myocardial infarction. Results from the VANQWISH trial angiographic core laboratory[END_REF] Offline analysis of digital angiograms was performed in the core laboratory using an automated edge detection system (CMS; Medis Medical Imaging Systems, Neuen, the Netherlands). The initial and post-procedural blood flow in the artery owning the culprit lesion was quantified using the Thrombolysis in Myocardial Infarction (TIMI) grading system. [START_REF]Definitions used in TIMI trials[END_REF] Global left ventricular ejection fraction was determined in left ventricular angiograms by using the area-length method.

Stent implantation and periprocedural care were performed according to standard criteria. Bare metal stents were mostly used. Postinterventional antiplatelet therapy consisted of ticlopidine (500 mg/day) or clopidogrel (300 mg or 600 mg as a loading dose followed by 75 mg/day for at least 4 weeks to 6 months) and aspirin (200 mg/day administered orally and continued indefinitely).

Endpoint and Follow-up

The primary outcome of this analysis was 1-year mortality. Occurrence of myocardial infarction, major bleeding and stroke was also assessed. The follow-up protocol after discharge consisted of a phone interview at one month after the procedure, a visit at six months and a phone interview at 12 months. Information about death was obtained from hospital records, death certificates, or phone contact with relatives of the patient or attending physician. The diagnosis of myocardial infarction, major bleeding and stroke was made according to the TIMI criteria. [START_REF]Definitions used in TIMI trials[END_REF] Patients were advised to present to the outpatient clinic or their referring physicians if they developed chest pain or other cardiac symptoms. In case of symptoms, at least one clinical, laboratory and electrocardiographic check-up was performed.

Statistical Analysis

Data are presented as median (with 25 th and 75 th percentiles) or counts and proportions (percentages). The distribution of the data was analyzed with one-sample Kolmogorov-Smirnov test. Categorical data were compared with chi-square test. Continuous data were compared with Kruskal-Wallis rank sum test. One-year mortality was estimated by applying the Kaplan-Meier method and log rank test which allowed the calculation of odds ratios (95% confidence intervals and respective P values) associated with being in various tertiles of the WBC count. Heterogeneity in risk prediction by WBC count in various subgroups was assessed by using the Breslow-Day test. Receiver operating characteristic (ROC) curves were constructed to assess the accuracy of WBC count and C-reactive protein to predict one-year mortality. Differences between the areas under curve were assessed for statistical significance according to the method by DeLong et al. [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF] The Cox proportional hazards model was used to assess the association WBC count and one-year mortality while adjusting for potential confounders: age, sex, diabetes, arterial hypertension smoking, hypercholesterolemia, prior myocardial infarction, prior coronary artery bypass surgery, ACS type, cardiogenic shock, serum creatinine, C-reactive protein, left ventricular ejection fraction, multivessel disease, baseline TIMI flow grade, post-PCI TIMI flow grade and WBC count. Analyses were performed using S-plus statistical package (S-PLUS, Insightful Corp, Seattle, Washington). A P value < 0.05 was considered to indicate statistical significance.

Results Baseline Characteristics

Patients were divided into 3 groups according to tertlies of WBC count: first tertile (WBC count <6800 cells/mm 3 , n=1392 patients), second tertile (WBC count 6800-9400 cells/mm 3 , n=1466 patients) and third tertile (WBC count >940 cells/mm 3 , n=1471 patients). Baseline demographic and clinical characteristics are shown in Table 1. Patients in the 3 rd tertile of WBC count were younger, had a higher proportion of women and of current smokers and had less often arterial hypertension, hypercholesterolemia, prior myocardial infarction and prior coronary artery bypass surgery than patients in lower tertiles. They presented more often with acute myocardial infarction and cardiogenic shock and had higher levels of troponin and creatine kinase. Angiographic characteristics are shown in Table 2. Patients in the 3 rd tertile of WBC count had lower left ventricular ejection fraction and a lower proportion of patients with multivessel disease that patients in other 2 tertiles (see atable 1 and table 2 for the remaining characteristics).

WBC Count and Clinical Outcome

Thirty-day clinical outcome is shown in Table 3. With the increase in the tertiles of WBC count from 1 to 3 there was a significant increase in the death rate, major bleeding and stroke.

At one year, 345 patients (8%) died: 45 patients in the 1 st tertile, 93 patients in the 2 nd tertile and 207 patients in the 3 rd tertile of WBC count (Kaplan-Meier estimates of mortality, 3.2%, 6.4% and 14.1%, respectively; odds ratio [OR] =2.42, 95% confidence interval [CI] 1.78-3.12, P<0.001 for tertile 3 vs tertile 2 and OR=1.99, 95% CI 1.77-2.25, P<0.001 for tertile 2 vs tertile 1; Figure 1). WBC count was 10800 [7900-14900] cells/mm 3 in patients who died versus 7800 [6200-10100] cells/mm 3 in those who survived one-year follow-up (P<0.001). There were no significant differences in the one-year occurrence of myocardial infarction according to WBC count tertiles: Kaplan-Meier estimates of 2.5% in the 1 st tertile (n=34), 3.9% in the 2 nd tertile (n=57) and 3.3% in the 3 rd tertile of WBC count (n=48).

Subgroup analyses of relationship between WBC count and one-year mortality are shown in Figure 2. As seen, WBC count predicted mortality across all the analyzed subgroups with the exception of patients with cardiogenic shock. Of note, WBC count predicted oneyear mortality in patients with ST-segment elevation acute myocardial infarction OR=4.22, 95% CI 1.52-11.73; P<0.001), non-ST-segment elevation acute myocardial infarction (OR=3.65, 95% CI 2.31-5.79); P<0.001) and unstable angina (OR=2.71 [1.21-6.08]; P=0.01).There was no interaction between WBC count and any analyzed subgroups regarding one-year mortality.

ROC curves were constructed to assess the accuracy of WBC and C-reactive protein regarding one-year mortality. Area under ROC curve [95% confidence interval] was 0.69 [95% CI 0.65-0.72], P<0.001 for WBC count and 0.65 [95% CI 0.62-0.69], P<0.001 for Creactive protein (P=0.09; Figure 3).

Results of Multivariable Analysis

The Cox proportional hazards model was applied to the independence of association between WBC count and one-year mortality while adjusting for potential confounders (see methods for variables entered into the model). The model showed that WBC count was an independent correlate of one-year mortality with an increase of 5% of the adjusted risk for every 1000 cell/mm 3 increase in WBC count. C-reactive protein was not independently associated with one-year mortality (hazard ratio [HR=1.09, 95% confidence interval [CI] 0.92-1.36; P=0.20). Significant correlates of one-year mortality are shown in Table 4. We repeated the Cox proportional hazards model with WBC count and C-reactive protein entered as tertiles. Again WBC count was independently associated with one-year mortality with a HR of 1.33, 95% CI 1.09-1.63; P=0.006 for 1 tertile increase in the WBC count; conversely, C-reactive protein was not independently associated with the one-year mortality (HR=1.13, 95% CI 0.95-1.34; P=0.15 for 1 tertile increase in the CRP level).

WBC Count and One-Year Mortality in Patients With and Without Statins

Statin therapy was prescribed in 3898 patients (90%): 1301 patients in the 1 st tertile, 1338 in the 2 nd tertile and 1259 patients in the 3 rd tertile of WBC count. At one year there were 29 deaths among patients of the 1 st tertile (2.2%), 56 deaths among patients of 2 nd tertile (4.2%) and 71 deaths (5.6%) among patients of the 3 rd tertile of WBC count (OR=1.91, 95% CI 1.21-3.02; P=0.004 for tertile 2 vs tertile 1 and OR=2.62, 95% CI 1.69-4.07; P<0.001 for tertile 3 vs tertile 1). In 431 patients statins were not prescribed: 91 patients in the first WBC count tertile, 128 patients in the second WBC count tertile and 212 patients in the third WBCcount tertile. Since in patients dying early after acute event (mostly due to cardiogenic shock), statins were not prescribed we analyzed association between WBC count and one-year mortality in patients who survived the first 30 day after acute event. In patients who survived the first 30 days after acute event one year mortality was 9.6% (8 of 83 patients) among patients in the first WBC count tertile, 9.9% (10 of 101 patients) among patients in the second WBC count tertile and 20.8% (20 of 96 patients) among patients in the third WBC tertile (OR=2.46, 95% CI 1.02-5.94; P=0.04 for third vs. first WBC count tertile).

Discussion

In this study, we investigated the association between WBC count and one year-mortality using data of a large prospective registry of patients with ACS treated with PCI. The main findings of this study are as follows: 1) In patients with ACS treated with PCI (mostly coronary stenting) elevated WBC is associated with increased risk of one-year mortality. This association is independent of cardiovascular risk factors including other markers of inflammatory status (C-reactive protein). We found that for every 1000 cells/mm 3 increase in the WBC count adjusted one-year mortality was increased by 5%. Moreover, elevated WBC count was associated with higher rates of 30-day mortality, major bleeding and stroke. 2) Comparative analysis of WBC and C-reactive protein in the multivariable model (the Cox proportional hazards model) showed that WBC count but not C-reactive protein was an independent correlate of one-year mortality. 3) WBC count was an independent predictor of one-year mortality in patients with ACS receiving statin therapy.

Multiple studies have demonstrated an association between elevated WBC count and extent of coronary artery disease [START_REF] Sabatine | Relationship between baseline white blood cell count and degree of coronary artery disease and mortality in patients with acute coronary syndromes: a TACTICS-TIMI 18 (Treat Angina with Aggrastat and determine Cost of Therapy with an Invasive or Conservative Strategy-Thrombolysis in Myocardial Infarction 18 trial) substudy[END_REF][START_REF] Amaro | Leukocyte count as a predictor of the severity ischaemic heart disease as evaluated by coronary angiography[END_REF] or increased short-term or long-term risk of death in patients with various presentations of coronary artery disease. [START_REF] Hajj-Ali | Relation of the leukocyte count to recurrent cardiac events in stable patients after acute myocardial infarction[END_REF][START_REF] Furman | Effect of elevated leukocyte count on in-hospital mortality following acute myocardial infarction[END_REF][START_REF] Barron | The association between white blood cell count and acute myocardial infarction mortality in patients ≥65 years of age: findings from the cooperative cardiovascular project[END_REF][START_REF] Sabatine | Relationship between baseline white blood cell count and degree of coronary artery disease and mortality in patients with acute coronary syndromes: a TACTICS-TIMI 18 (Treat Angina with Aggrastat and determine Cost of Therapy with an Invasive or Conservative Strategy-Thrombolysis in Myocardial Infarction 18 trial) substudy[END_REF][START_REF] Mueller | White blood cell count and long term mortality after non-ST elevation acute coronary syndrome treated with very early revascularisation[END_REF][START_REF] Cannon | Association of white blood cell count with increased mortality in acute myocardial infarction and unstable angina pectoris. OPUS-TIMI 16 Investigators[END_REF] A host of mechanisms through which elevated WBC count may impact coronary artery disease progression and mortality has been proposed and recently reviewed by Madjid et al. [START_REF] Madjid | Leukocyte count and coronary heart disease: implications for risk assessment[END_REF] In the Global Registry of Acute Coronary Events (GRACE), association between elevated WBC and hospital death or development of congestive heart failure was consistent across the entire spectrum of ACS. [START_REF] Furman | Elevated leukocyte count and adverse hospital events in patients with acute coronary syndromes: findings from the Global Registry of Acute Coronary Events (GRACE)[END_REF] Although an association between elevated WBC and mortality is undisputed, the independence of association from other potential confounders remains A c c e p t e d M a n u s c r i p t controversial. [START_REF] Camargo | Effect of leukocytosis at initial examination on prognosis in patients with primary unstable angina[END_REF][START_REF] Haim | Predictive value of elevated white blood cell count in patients with pre-existing coronary heart disease: the Bezafibrate Infarction Prevention Study[END_REF] In a study by Lloyd-Jones et al. [START_REF] Camargo | Effect of leukocytosis at initial examination on prognosis in patients with primary unstable angina[END_REF] association between elevated WBC and one-year mortality in patients with unstable angina was markedly diminished after adjustment for other covariates. In the same line, a recent study of patients with existing coronary artery disease showed that adjustment for coronary risk factors totally eliminated association between WBC count and cardiac mortality. [START_REF] Haim | Predictive value of elevated white blood cell count in patients with pre-existing coronary heart disease: the Bezafibrate Infarction Prevention Study[END_REF] Other studies assumed that the impact of WBC on mortality may be mediated by smoking [START_REF] Kostis | Association between leukocyte count and the presence and extent of coronary atherosclerosis as determined by coronary arteriography[END_REF] or diabetes. [START_REF] Rasouli | Total and differential leukocytes counts, but not hsCRP, ESR, and five fractioned serum proteins have significant potency to predict stable coronary artery disease[END_REF] Of note, in the majority of reported studies no adjustment for C-reactive protein has been performed. Thus as recently emphasized by Madjid et al. [START_REF] Madjid | Leukocyte count and coronary heart disease: implications for risk assessment[END_REF] there is a need to determine the degree to which WBC count is independent to other risk factors.

In the present study of patients with ACS, we performed an adjustment for traditional coronary risk factors, clinical presentation of ACS, extent of coronary artery disease, baseline and post-procedural TIMI flow grade and C-reactive protein. We found that in patients with ACS treated with PCI elevated WBC count predicted one-year mortality independent of all above-mentioned coronary risk factors and clinical covariates. Furthermore, the association between WBC count and one-year mortality was consistent in patients with unstable angina, non-ST-segment elevation or ST-segment elevation acute myocardial infarction. In this aspect our data dovetail with those of GRACE study [START_REF] Furman | Elevated leukocyte count and adverse hospital events in patients with acute coronary syndromes: findings from the Global Registry of Acute Coronary Events (GRACE)[END_REF] in reporting a consistent ability of WBC count to predict mortality across the entire spectrum of ACS. Of interest is the finding that elevated WBC was associated with a higher incidence of major bleeding and stroke within the first 30 days following PCI which may imply that elevated WBC count may be involved in the genesis of these serious adverse events in patients with ACS undergoing coronary intervention. In the present study, an association between elevated WBC count and occurrence of myocardial infarction could not be demonstrated. With regard to the lack of association between WBC count and future occurrence of myocardial infarction, our study is in agreement with a recent study Mueller et al. [START_REF] Mueller | White blood cell count and long term mortality after non-ST elevation acute coronary syndrome treated with very early revascularisation[END_REF] in patients with non-ST-segment elevation ACS. In fact, inability of WBC to predict cardiac mortality has been reported [START_REF] Haim | Predictive value of elevated white blood cell count in patients with pre-existing coronary heart disease: the Bezafibrate Infarction Prevention Study[END_REF] and the current finding may be, at least, one of the putative explanations. Of particular interest was the finding that after adjustment in the multivariable model, WBC count but not C-reactive protein was an independent correlate of one-year mortality. Recent studies have also shown that WBC count outperformed the C-reactive protein in predicting severity of coronary artery disease [START_REF] Rasouli | Total and differential leukocytes counts, but not hsCRP, ESR, and five fractioned serum proteins have significant potency to predict stable coronary artery disease[END_REF] and a list of other inflammatory markers such as serum amyloid A, fibrinogen and interleukin-6 in predicting 5-year mortality in patients with ACS. [START_REF] Zairis | The impact of hs C-reactive protein and other inflammatory biomarkers on long-term cardiovascular mortality in patients with acute coronary syndromes[END_REF] These data clearly demonstrate that WBC count, a universally available test, is one of the best markers to assess the inflammatory status and its impact on mortality across the entire spectrum of patients with coronary artery disease.

The impact of statin therapy on the association between WBC count and mortality is largely unknown. In the Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) study, WBC count predicted cardiac mortality in patients randomized to placebo, but not in those who received pravastatin. [START_REF] Stewart | White blood cell count predicts reduction in coronary heart disease mortality with pravastatin[END_REF] In the present study 90% of patients with ACS received statins within the first year following PCI. A separate analysis in these patients demonstrated that WBC predicted one-year mortality in patients receiving statins with odds ratios quite similar to those in the whole group of patients. Reasons for these discrepancies regarding impact of statin therapy on the power of WBC count to predict mortality remain unknown.

The present study does not provide information on WBC count differential. It has recently been reported that neutrophil-to-lymphocyte ratio may be superior to total WBC count in predicting subsequent mortality. [START_REF] Horne | Which white blood cell subtypes predict increased cardiovascular risk?[END_REF][START_REF] Núñez | Usefulness of the neutrophil to lymphocyte ratio in predicting long-term mortality in ST segment elevation myocardial infarction[END_REF] However, other studies have shown that total WBC was a better correlate of long-term outcome (mortality) than individual subtypes of WBC count. [START_REF] Gurm | Impact of preprocedural white blood cell count on long term mortality after percutaneous coronary intervention: insights from the EPIC, EPILOG, and EPISTENT trials[END_REF] In conclusion, the present study demonstrated that elevated WBC count but not Creactive protein predicts one-year mortality independent of risk factors for coronary artery Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298

Clinical

  Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  disease across the entire spectrum of ACS. Statin therapy did not abolish the association between WBC count and one-year mortality in these patients. Clinical Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Clinical

  Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ClinicalFigure 1 .

 1 Figure 1. Kaplan-Meier Curves of One-year Mortality According to Tertiles of WBC Count

FalseFigure 3 .

 3 Figure 3. Receiver Operating Characteristic (ROC) Curves Showing Discriminatory Power of White Blood Cells (WBC) and C-reactive Protein (CRP) Regarding One-Year Mortality

  Clinical Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298

Table 1 .

 1 Baseline Characteristics According to Tertiles of White Blood Cell Count Clinical Science Immediate Publication. Published on 14 Oct 2008 as manuscript CS20080298 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080298 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

	Characteristic

Table 2 .

 2 Angiographic Characteristics According to Tertiles of White Blood Cell Count

	Characteristic

Table 3 .

 3 Thirty-day Outcome According to Tertiles of White Blood Cell Count

	Outcome

TableTable 4 .

 4 Correlates of One-year Mortality and Hazard Ratios Calculated by Cox Proportional Regression Analysis Left ventricular ejection fraction (for 10 % decrease) 1.37 [1.24 -1.52] <0.001 Post-intervention TIMI flow grade (for 1 grade increase) 0.61 [0.50 -0.74] <0.001 Serum creatinine (for 1 mg increase in concentration) 1.45 [1.19 -1.78] <0.001 Acute coronary syndrome presentation STEMI vs Unstable angina NSTEMI vs Unstable angina NSTEMI Non-ST-segment elevation myocardial infarction; STEMI = STsegment elevation myocardial infarction; TIMI = Thrombolysis in Myocardial Infarction; WBC = White blood cell.

	Variable

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Odds ratio

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Konstantinos Kantartzis

Fausto Machicao

Jürgen Machann

Fritz Schick

Andreas Fritsche

Hans-Ulrich Häring

MD Norbert Stefan
email: norbert.stefan@med.uni-tuebingen.de

Hans- Ulrich Häring

Insulin Dgat2

Fatty Resistance

Liver

The DGAT2 gene is a

Keywords: Diabetes, fatty liver, DGAT, insulin resistance, lifestyle intervention, obesity

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Nonalcoholic fatty liver disease represents a strong predictor of insulin resistance, the metabolic syndrome, cardiovascular disease and type 2 diabetes [START_REF] Loria | Is liver fat detrimental to vessels?: intersections in the pathogenesis of NAFLD and atherosclerosis[END_REF][START_REF] Roden | Mechanisms of Disease: hepatic steatosis in type 2 diabetes--pathogenesis and clinical relevance[END_REF][START_REF] Targher | Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients[END_REF][START_REF] Targher | Non-alcoholic fatty liver disease and increased risk of cardiovascular disease[END_REF][START_REF] Shibata | Nonalcoholic fatty liver disease is a risk factor for type 2 diabetes in middle-aged Japanese men[END_REF]. However, despite the strong association of fatty liver with insulin resistance [START_REF] Kelley | Fatty liver in type 2 diabetes mellitus: relation to regional adiposity, fatty acids, and insulin resistance[END_REF][START_REF] Utzschneider | Review: The role of insulin resistance in nonalcoholic fatty liver disease[END_REF][START_REF] Kotronen | Fatty liver: a novel component of the metabolic syndrome[END_REF], subjects can be identified who, for the same amount of liver fat, have very high and low insulin resistance. When accumulation of liver fat is not accompanied by respective increase in insulin resistance, the term "dissociation" is used [9;10] Recent animal data suggest that a possible explanation for this dissociation between fat accumulation in the liver and insulin resistance may be variation in the expression and/or activity of the enzyme acyl-CoA: diacylglycerol acyltransferase (DGAT). This enzyme catalyzes the final step of triglyceride synthesis [11;12]. There are two isoforms of the enzyme, DGAT1 and DGAT2. DGAT2 is specific for triglyceride synthesis and is predominantly expressed in the liver [START_REF] Stone | Lipopenia and skin barrier abnormalities in DGAT2-deficient mice[END_REF]. Mice overexpressing DGAT2 develop fatty liver but, unexpectedly, not insulin resistance under a high-fat diet [START_REF] Monetti | Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver[END_REF]. In agreement, treatment with antisense oligonucleotide causing a reduction in the expression of DGAT2 gene (DGAT2) resulted in an improvement of hepatic steatosis without change in insulin sensitivity [START_REF] Yu | Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice[END_REF]. In another study with similar design in rats insulin sensitivity improved as well, but this may have been due to the observed reduction in body weight [START_REF] Choi | Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance[END_REF].

With the present study we set out to delineate whether DGAT2 plays a role in the dissociation of fatty liver and insulin resistance in humans. We studied the relationships of common single nucleotide polymorphisms (SNPs) in DGAT2 with liver fat and insulin sensitivity during a lifestyle intervention.

Precise phenotyping methods as magnetic resonance tomography (MRT) to measure total body fat and fat content in the visceral as well as in the subcutaneous depots and 1 HMR spectroscopy to determine fat deposition in ectopic tissues as liver and skeletal muscle were used. [15;16]. Individuals were included in the study when they fulfilled at least one of the following criteria: a family history of type 2 diabetes, a BMI > 27 kg/m 2 , a previous diagnosis of impaired glucose tolerance and/or of gestational diabetes. They were considered healthy according to a physical examination and routine laboratory tests. Subjects had no history of liver disease and did not consume more than 2 alcoholic drinks per day. They had measurements at baseline and after a mean of 9 months of lifestyle intervention. After the baseline measurements, individuals underwent dietary counseling and had up to ten sessions with a dietician. During each visit participants presented a 3-day food diary and discussed the results with the dieticians. Diet composition was determined with a validated computer program using 2 representative days of a 3-day diary (DGE-PC 3•0, Deutsche Gesellschaft für Ernährung, Bonn, Germany). Counseling was aimed to reduce body weight by ≥5%, to reduce the intake of calories from fat to < 30% and particularly the intake of saturated fat to ≤ 10% of energy consumed and to increase the intake of fibers to at least 15 g/1000 kcal. Individuals were asked to perform at least 3 hours of moderate sports per week. Aerobic endurance exercise (e.g. walking, swimming) with an only moderate increase in the heart rate was encouraged. Participants were seen by the staff on a regular basis to ensure that these recommendations were accomplished. The research has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Informed written consent was obtained from all participants and the local medical ethics committee approved the protocols.

Body fat distribution, liver fat and intramyocellular fat

Waist circumference was measured at the midpoint between the lateral iliac crest and lowest rib. Total body-and visceral fat were measured by MR tomography, and liver fat and intramyocellular fat of the tibialis anterior muscle by 1 H-MR spectroscopy as previously described [START_REF] Stefan | Polymorphisms in the gene encoding adiponectin receptor 1 are associated with insulin resistance and high liver fat[END_REF].

Oral glucose tolerance test

All individuals underwent a 75 g oral glucose tolerance test (OGTT). We obtained venous plasma samples at 0, 30, 60, 90 and 120 minutes for determination of plasma glucose and insulin. Glucose tolerance was determined according to the 1997 World Health Organization diagnostic criteria [START_REF]Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus[END_REF].

Insulin sensitivity from the OGTT was calculated as proposed by Matsuda and DeFronzo (10 000 / √(mean insulin x mean glucose) x (fasting insulin x fasting glucose) [START_REF] Matsuda | Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp[END_REF].

Euglycemic, hyperinsulinemic clamp

A subgroup (n=142 at baseline and n=46 at follow-up) additionally underwent a euglycemic, hyperinsulinemic clamp. Insulin sensitivity was determined with a primed insulin infusion at a rate of 40 mU•m -2 •min -1 for 2 hours as previously described [START_REF] Stefan | Polymorphisms in the gene encoding adiponectin receptor 1 are associated with insulin resistance and high liver fat[END_REF]. In these subgroups of individuals, insulin sensitivity calculated from the OGTT was strongly correlated with insulin sensitivity measured during the clamp (r=0.72, p<0.0001 at baseline and r=0.78, p<0.0001 at follow-up).

Genotyping

SNPs that are representative in a region of the genome with high linkage disequilibrium (the nonrandom association of alleles at two or more loci -tagging SNPs) allow identifying genetic variation without genotyping every SNP in a chromosomal region. Tagging SNPs for DGAT2 were selected from SNPs (rs10899116 and rs1944438) had a minor allele frequency (MAF) ≥0.05 and covered 100% of the alleles with a D' of 1 (Haploview Program). Thus, these two SNPs are representative for all SNPs in DGAT2 which have a relevant frequency in the general population. These SNPs were genotyped in our population by direct sequencing and using the TaqMan assay (Applied Biosystems, Foster City,CA, USA).

Statistical analyses

For statistical analyses, non-normally distributed parameters were logarithmically transformed.

Relationships between parameters at baseline and at follow-up were tested using the paired t-test. To test the effect of genotype on the metabolically relevant parameters, multivariate linear regression models were used with the parameter to be considered set as the dependent variable. In cross-sectional analyses differences in parameters at baseline between genotypes were tested. Body weight, waist circumference and total body fat were adjusted for age and gender. The other parameters were additionally adjusted for total body fat. In longitudinal analyses, changes in parameters during the intervention between genotypes were tested. Change in body weight, waist circumference and total body fat were adjusted for the respective baseline values, gender and age at follow-up. Changes in the other parameters were additionally adjusted for body fat at baseline and follow-up. The genotype was included in both analyses as an independent nominal variable. For each dependent variable two models were applied. In the additive model the effects of all possible genotypes on the dependent variable were compared; in the dominant model homozygotes for the major allele were compared to heterozygotes and homozygotes of the minor allele. A p-value ≤ 0.05 was considered statistically significant. The statistical software package JMP 4.0 (SAS Institute Inc, Cary, NC, USA) was used.

Results

Cross-sectional analyses

The anthropometrics and metabolic characteristics of the subjects at baseline are shown in table 1. All parameters at baseline covered a wide range. Seventy-one subjects (38%) were obese (BMI > 30 kg/m 2 ) and 49 (26.2%) had impaired glucose tolerance (IGT) at baseline. According to the cut-off of liver fat 5.56% [START_REF] Szczepaniak | Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population[END_REF], 54 individuals were found to have fatty liver at baseline.

Liver fat was higher in males than females (median [interquartile range]; 4.28 [2.48-11.19] vs. 2.26 [1,17-4.46] %, p<0.0001) and correlated significantly with age (r=0.15, p=0.04), total body fat (r=0.28, p=0.0001), visceral fat (r=0.62, p<0.0001), intramyocellular fat (r=0.18, p=0.02), and inversely with insulin sensitivity (OGTT, r=-0.54, p<0.0001, clamp, r=-0.58, p<0.0001).

Insulin sensitivity was higher in females (OGTT, 12.97 [8.47-18.04] vs. 9.37 [6.81-14.12] arb. units, p=0.02; clamp, 0.066 [0.047-0.090] vs. 0.054 [0.037-0.071] µmol•kg -1 •min -1 •pM -1 , p=0.009) and correlated with total body fat (OGTT, r=-0.39, p<0.0001; clamp, r=-0.39, p<0.0001), visceral fat (OGTT, r=-0.47, p<0.0001; clamp, r=-0.51, p<0.0001) and intramyocellular fat (OGTT, r=-0.28, p=0.0004; clamp, r=-0.27, p=0.002).

Both SNPs, rs10899116 and rs1944438, were in Hardy-Weinberg equilibrium (χ 2 -test; p≥0.75). The minor allele frequency (MAF) of the SNPs rs10899116 C>T and rs1944438 C>T were 0.09 and 0.39, respectively. The SNP rs10899116 was not associated with anthropometrics or metabolic characteristics. In a dominant model the SNP rs1944438 was not associated with age or gender. No relationship of this SNP with insulin sensitivity, visceral-, liver-or intramyocellular fat, all adjusted for age, gender and total body fat, were observed (all p≥0.13, table 2). Carriers of the minor T allele of SNP rs1944438 had higher total body fat, adjusted for age and gender, compared to homozygous carriers of the C allele (p=0.04).

Longitudinal analyses

During the 9 months of intervention there were significant mean decreases in body weight (-3.1%), total body-(-12.1 %), visceral-(-18.7%) and liver fat (-29.1%). Over baseline, significantly fewer subjects were still obese (59 subjects, 31.6%), had IGT (40 subjects, 21.4%) and fatty liver (39 subjects, 20.9%) at follow-up (all p<0.0001). Insulin sensitivity estimated from the OGTT increased by 15.5%, and insulin sensitivity measured by the clamp increased by 11% (table 1). Change in insulin sensitivity correlated inversely with changes in total body fat, visceral fat and intramyocellular fat (OGTT: all p≤0.046; clamp: all ≤0.02). Furthermore, changes in insulin sensitivity correlated inversely with change in liver fat (OGTT: r=-0.32, p<0.0001; clamp:r=-0.32, p=0.03).

The SNP rs10899116 was not associated with any changes in the parameter tested (all p≥0.38). The SNP rs1944438 was also not associated with changes in total body-(figure, panel A), visceral-, or intramyocellular fat (all p≥0.39, table 2). However, subjects carrying the minor T allele had less decrease in liver fat, independently of age, gender, liver fat at baseline and total body fat at baseline and at follow-up compared to homozygous carriers of the C allele (table 3, figure, panel B). In contrast, change in adjusted insulin sensitivity calculated from the OGTT was not associated with the SNP (table 3, figure, panel C). Even in the small subgroup of individuals with measurements of insulin sensitivity by the clamp (n=46), similar results were found for the relationships of the SNP rs1944438 with changes in liver fat (p=0.0021) and insulin sensitivity (p=0.53).

In the group of subjects who had fatty liver at baseline the results were analogous to the larger group. In the smallest group of subjects in our analyses who underwent the clamp (n=46) we had a power of 81% in the additive model to detect an effect of the DGAT genotype on change in liver fat. The least significant number of subjects to detect the effect at the α-level of 0.05 was 30. In the dominant model the power was 89% and the least significant number of subjects to detect the effect at the α-level of 0.05 was 20. In this group we had a power of 98% to detect an effect size of 20% in change of insulin sensitivity at the α-level of 0.05 in the dominant model and of 97% in the additive model, respectively.

Discussion

In agreement with animal data [9;13] we found that dissociation between fat accumulation in the liver and insulin resistance mediated by DGAT2 may also exist in humans. During the lifestyle intervention subjects carrying the minor T allele of SNP rs1944438 had less decrease in liver fat compared to homozygous carriers of the C allele. Importantly, changes in total body-, visceral-and intramyocellular fat did not differ between the genotypes, which is in agreement with a predominant effect of DGAT2 on hepatic steatosis [START_REF] Monetti | Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver[END_REF]. A recent study, which applied mutation screen in DGAT2, also failed to show an important role of common genetic variation in the gene for the development of obesity [START_REF] Friedel | Mutation screen and association studies in the diacylglycerol O-acyltransferase homolog 2 gene (DGAT2), a positional candidate gene for early onset obesity on chromosome 11q13[END_REF].

Furthermore, the SNP rs1944438 of DGAT2 was not associated with change in insulin sensitivity, a finding that is in line with the data in mice [9;13]. Interestingly, the relationships of the SNP with change in liver fat and insulin sensitivity were also observed in the much smaller group of participants who underwent the clamp, supporting that a type 1 error most probably can be excluded.

The dissociation between change in liver fat and insulin sensitivity, which was attributable to the SNP, was apparent in subjects with fatty liver who are at very high risk for metabolic diseases [2;8]. This finding is important particularly since in the treatment of fatty liver moderate lifestyle intervention, as it was used in the present study, is considered the first line therapy [22;23]. It, therefore, underscores the relevance for considering DGAT2 activity during a lifestyle intervention to reduce hepatic steatosis and increase insulin sensitivity. In addition, it remains to be established whether DGAT2 is also important in determining the metabolic effect of pharmacological intervention, e.g. with thiazolidinediones [START_REF] Belfort | A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis[END_REF], in the treatment of fatty liver.

Mechanisms involved in this dissociation of fatty liver and insulin resistance are not fully understood.

As suggested by the animal studies [9;13;14] altered amounts of fatty acid metabolites regulating insulin sensitivity, such as diacylglycerol [25;26], long-chain fatty acyl-CoAs and ceramides [START_REF] Kim | Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance[END_REF] A c c e p t e d M a n u s c r i p t be important in this aspect. Furthermore, unsaturated long-chain fatty acyl-CoA were found to have a protective function against saturated acyl-CoAs-induced lipotoxicity and cellular apoptosis by promoting triglyceride accumulation [14;27]. DGAT has the same effect, catalyzing triglyceride synthesis, but whether the SNP rs1944438 of DGAT2 is also associated with such a protective effect in liver in humans, needs to be determined.

In conclusion, our findings support that dissociation between fatty liver and insulin resistance exists in humans. Since during a lifestyle intervention in subjects with fatty liver, the reduction in liver fat does not always result in a beneficial effect on insulin sensitivity, this finding may be of clinical relevance in the prevention and treatment of type 2 diabetes. Genetic screening for this polymorphism prior to the intervention may help to early apply alternative strategies to improve insulin sensitivity. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

3

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t Methods Subjects Data of 187 Caucasians, 72 men and 115 women, without type 2 diabetes from the southern part of Germany were analyzed. These individuals participated in an ongoing study to reduce adiposity and prevent type 2 diabetes

4

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t

6

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t

7

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t

  Subjects with fatty liver carrying the minor T allele of SNP rs1944438 had less decrease in adjusted liver fat compared to homozygous carriers of the C allele (p=0.02). No relationship was found between the SNP and change in insulin sensitivity calculated from the OGTT (p=0.52) in these individuals. 8 Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

9

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t

may 10 Clinical

 10 Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306

11 Clinical

 11 Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Figure legend

 legend Figure legend

  Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t

5

  Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 HapMap (HapMap Public Release no. 22). Applying a pairwise tagging SNP r 2 threshold of 0.8, 2

	A c c e p t e d M a n u s c r i p t	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306

Table 1

 1 Subject characteristics at baseline and after 9 months of follow-up

	Baseline	Follow-up	p value

LDL-cholesterol (mg/dl) 119 (101-139) 116 (95-137) 0.001

	Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306
	Fasting glucose (mM) 2 h glucose (mM) NGT / IGT Fasting insulin (pM) 2 h insulin (pM) Insulin sensitivity OGTT (arb. units) ISI Clamp (µmol•kg -1 •min -1 •pM -1 ) Values represent median (interquartile range). For statistical analyses, non-normally distributed parameters were 5.17 (4.85-5.56) 5.11 (4.83-5.44) 0.008 6.61 (5.72-8.11) 6.33 (5.61-7.56) 0.014 138 / 49 147 /40 <0.0001 # 50 (33-76) 42 (30-61) <0.0001 382 (225-594) 303 (195-495) <0.0001 11.8 (7.6-17.3) 13.5 (9.1-19.3) <0.0001 0.058 (0.042-0.082) § 0.065 (0.047-0.085) § § 0.037 A c c e p t e d M a n u s c r i p t	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306

log transformed. The paired t-test was then applied. # χ 2 -test IMCL, intramyocellular lipids, *available in 175 subjects; NGT, normal glucose tolerance; IGT, impaired glucose tolerance; ISI, insulin sensitivity index, § available in 142 subjects at baseline and § § 46 at follow-up.

Table 2

 2 Associations of the rs1944438 C/T SNP in DGAT2 with subjects' demographic and metabolic characteristics at baseline and follow-up.Values represent median (interquartile range). For statistical analyses, non-normally distributed parameters were log transformed. + p<0.05, ‡ p<0.01 and † p<0.001 for paired differences between baseline and follow-up. # χ 2 -test. The genotype effect at baseline and during the intervention was tested using an additive and a dominant model. Body weight, waist circumference and total body fat at baseline were adjusted for age and gender. The other parameters were additionally adjusted for total body fat. Change in body weight, waist circumference and total body fat were adjusted for the respective baseline values, gender and age at follow-up. Changes in the other parameters were additionally adjusted for body fat at baseline and follow-up. IMCL, intramyocellular lipids, *available in 149 subjects; ISI, insulin sensitivity index, § available in 142 subjects (C/C n=55; C/T

		C/C		C/T		T/T		p baseline	p for change between genotypes
		Baseline	Follow-up	Baseline	Follow-up	Baseline	Follow-up	additive dominant additive dominant
	Demographics
	Gender (males / females)	24 / 43	40 / 54	8 / 18		0.47 #	0.57 #
	Age (years)	49 (41-55)	50 (42-56) †	46 (36-54)	47 (37-55) †	43 (39-53)	44 (40-53) †	0.26	0.17
	Body composition
	Body weight (kg)	83.4 (72.5-90.7) 81.2 (67.0-88.8) † 88.3 (75.0-99.7) 85.5 (71.0-97.2) † 89.8 (77.3-97.1) 84.6 (74.1-97.8) ‡ 0.14	0.09	0.85	0.97
	Waist circumference (cm)	94.0 (85.5-102) 89.0 (82.5-97.0) † 98.0 (88.0-106.0) 96.0 (85.0-104) † 97.5 (91.5-105) 94.5 (83.5-102) † 0.09	0.052	0.051	0.03
	Total body fat (kg)	22.6 (17.2-26.6) 20.3 (14.3-25.6) † 24.9 (17.6-31.8) 21.8 (16.1-29.7) ‡ 27.4 (20.9-36.3) 25.5 (19.0-32.3) + 0.08	0.04	0.78	0.93
	Visceral fat (kg)	2.46 (1.40-3.79) 1.80 (1.22-3.23) † 3.01 (1.48-4.30) 2.24 (1.10-3.82) † 2.91 (1.51-4.52) 2.41 (1.13-3.64) † 0.94	0.91	0.41	0.63
	IMCL tibialis anterior (arb.units) *	3.64 (2.54-4.37) 3.64 (2.54-4.37) 3.90 (2.89-4.89) 3.51 (2.49-4.92) 4.28 (2.70-5.84) 3.51 (2.69-4.45) + 0.37	0.16	0.39	0.39
	Liver fat (%)	2.49 (1.41-3.82) 1.62 (0.95-3.03) † 3.96 (1.63-9.32) 2.68 (1.14-6.01) † 3.12 (1.13-5.69) 2.09 (0.91-6.43) 0.07	0.18	0.023	0.008
	Metabolic characteristics
	Fasting glucose (mM)	5.22 (4.83-5.53) 5.17 (4.83-5.53) 5.17 (4.88-5.68) 5.11 (4.83-5.44) ‡ 5.10 (4.82-5.42) 5.09 (4.78-5.40) 0.22	0.77	0.33	0.14
	2 h glucose (mM)	6.61 (5.94-7.72) 6.44 (5.61-7.67) 6.67 (5.82-8.17) 6.17 (5.49-7.43) ‡ 6.31 (5.24-7.56) 6.45 (5.57-7.76) 0.12	0.77	0.65	0.65
	Fasting insulin (pM)	39 (31-73)	40 (29-69) +	58 (36-79)	43 (32-60) †	39 (32-80)	42 (30-64)	0.19	0.59	0.73	0.45
	2 h insulin (pM)	310 (205-519) 281 (165-433) ‡	456 (260-683) 312 (197-502) † 267 (158-564) 401 (194-536) 0.006	0.35	0.16	0.38
	Insulin sensitivity OGTT (AU)	14.2 (8.4-19.2) 14.3 (9.2-21.4) +	9.6 (6.9-13.6) 13.1 (8.9-18.0) † 15.3 (8.1-20.9) 14.2 (9.2-21.0) 0.003	0.13	0.93	0.76
	ISI Clamp (µmol•kg -1 •min -1 •pM -1 ) § .071 (.051-.097) .073 (.057-.115) .053 (.039-.068) .050 (.038-.076) .059 (.044-.088) .082 (.068-.110) 0.051	0.17	0.26	0.53

n=67, T/T n=20) at baseline and 46 (C/C n=16; C/T n=24, T/T n=6) at follow-up. Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306 A c c e p t e d M a n u s c r i p t

Table 3

 3 Determinants of change in liver fat and insulin sensitivity calculated from the (OGTT) in Clinical Science Immediate Publication. Published on 03 Nov 2008 as manuscript CS20080306 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080306

	multivariate regression models
	Covariates	F Ratio	p
	Change in liver fat (additive model) Liver fat at baseline Female sex Age at follow-up Total body fat at baseline Total body fat at follow-up DGAT2 rs1944438 Change in liver fat (dominant model) Liver fat at baseline Female sex Age at follow-up Total body fat at baseline Total body fat at follow-up DGAT2 rs1944438 Change in insulin sensitivity (additive model) M a n u s c r i p t 31.66 < 0.0001 0.009 0.93 0.015 0.90 0.086 0.77 5.170 0.024 3.863 0.023 33.46 < 0.0001 0.009 0.92 0.030 0.86 0.067 0.80 5.326 0.022 7.210 0.008
	Insulin sensitivity at baseline Female sex Age at follow-up Total body fat at baseline Total body fat at follow-up DGAT2 rs1944438 Change in insulin sensitivity (dominant model) Insulin sensitivity at baseline Female sex Age at follow-up Total body fat at baseline Total body fat at follow-up DGAT2 rs1944438 A c c e p t e d	42.86 0.770 0.677 0.802 16.62 0.068 44.61 0.774 0.702 0.845 16.67 0.090	< 0.0001 0.38 0.41 0.37 < 0.0001 0.93 < 0.0001 0.38 0.40 0.36 < 0.0001 0.76
			17

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

The study was supported by a grant from the Deutsche Forschungsgemeinschaft (KFO 114) and the European Community's FP6 EUGENE6 (LSHM-CT-2004-512013). Dr. Stefan is currently supported by a Heisenberg-Grant of the Deutsche Forschungsgemeinschaft (STE-1096/1-1).