Britt-Marie Iresjö

Ulla Körner

Anders Hyltander

David Ljungman

Professor Kent Lundholm
email: kent.lundholm@surgery.gu.se

INITIATION FACTORS FOR TRANSLATION OF PROTEINS IN THE RECTUS ABDOMINIS MUSCLE FROM PATIENTS ON OVERNIGHT STANDARD PARENTERAL NUTRITION BEFORE OPERATION Short title: TPN and initiation factors

Keywords: Protein synthesis, initiation, translation, TPN

EDTA-Ethylenediamintetraacetic acid EGTA-Ethylene-bis[oxyethylenenitrilo]tetraacetic acid DTT-Dithiothreitol AEBSF-4-(2-aminoethyl)benzenesulfonyl fluoride SDS-sodium dodecyl sulfat KCl-potassium chloride NaF-sodiumfluoride FFA-free fatty acids AA-amino acids BCAA-branched chain amino acids IGF-1 insulin-like growth factor 1 eIF4E-eukaryotic initiation factor 4E eIF4G-eukaryotic initiation factor

INTRODUCTION

Mechanisms regulating protein balance in skeletal muscles have been extensively evaluated in human and animal models [START_REF] Waterlow | Protein turnover in mammalian tissues and in the whole body[END_REF]. Our previous investigations have indicated rapid activation of protein synthesis in skeletal muscles during oral refeeding of murines, a phenomenon that is independent of muscle innervation and circulating insulin [START_REF] Svanberg | The role of diet components, gastrointestinal factors, and muscle innervation on activation of protein synthesis in skeletal muscles following oral refeeding[END_REF]. Intravenous infusion by stepwise increased loads of amino acids to unselected hospitalized patients and healthy volunteers have indicated that amino acids are involved in the process of initiation of muscle protein translation [START_REF] Lundholm | Transport kinetics of amino acids across the resting human leg[END_REF] as also shown in numerous studies by others, while carbohydrates and fat lacked such effects [START_REF] Svanberg | The role of glucose, long-chain triglycerides and amino acids for promotion of amino acid balance across peripheral tissues in man[END_REF]. Also, there was no indication that glutamine had stimulatory effects in vivo in human muscles [START_REF] Svanberg | The effect of glutamine on protein balance and amino acid flux across arm and leg tissues in healthy volunteers[END_REF], while branched chain amino acids (BCAA) stimulate incorporation of labeled amino acids into muscle proteins [START_REF] Lundholm | Protein synthesis in human skeletal muscle tissue: influence of insulin and amino acids[END_REF] and phosphorylate proteins involved in translation of mRNAs [START_REF] Tang | Amino acid-induced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation[END_REF][START_REF] Gebauer | Molecular mechanisms of translational control[END_REF] Accordingly, direct and indirect evidence have been reported in numerous publications on amino acid stimulation of muscle protein synthesis in a variety of clinical conditions [START_REF] Flaim | The role of amino acids in the regulation of protein synthesis in perfused rat liver. II. Effects of amino acid deficiency on peptide chain initiation, polysomal aggregation, and distribution of albumin mRNA[END_REF][START_REF] Garlick | Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Effect of branched-chain amino acids[END_REF][11][START_REF] Watt | Stimulation of protein synthesis in pig skeletal muscle by infusion of amino acids during constant insulin availability[END_REF]. However, we and others have also reported that classical experiments with labeling of proteins in assessment of protein synthesis at steady-state conditions may sometimes give erroneous results even in the presence of flooding tracee conditions [START_REF] Watt | Stimulation of protein synthesis in pig skeletal muscle by infusion of amino acids during constant insulin availability[END_REF][START_REF] Iresjo | Reevaluation of amino acid stimulation of protein synthesis in murine-and human-derived skeletal muscle cells assessed by independent techniques[END_REF] Therefore, there is a need of additional methods to conduct analysis of activation of protein synthesis in vivo [START_REF] Greiwe | Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle[END_REF][START_REF] Liu | Amino acids stimulate translation initiation and protein synthesis through an Akt-independent pathway in human skeletal muscle[END_REF][START_REF] Gingras | Hierarchical phosphorylation of the translation inhibitor 4E-BP1[END_REF]. The aim of the present study was therefore to apply recent methods for evaluation of changes of initiation of protein translation of skeletal muscle proteins following an overnight constant infusion of standard total parenteral nutrition (TPN).

MATERIAL AND METHODS

Patients and study design

12 patients were included in the study (Table 1). All patients underwent surgery of the upper gastrointestinal tract. Nine patients were operated for cure due to local malignancy and three patients had benign disease. On the day before operation they were randomized to receive overnight infusions of either saline or total parenteral nutrition supplied as all in one bag prior to surgery (30 kcal/kg/day, 0.16 gN/kg/day, Kabiven Perifer, Fresenius Kabi AB, Uppsala, Sweden). Measurements for nutritional status and baseline blood samples were taken before start of infusions indicating manifest or incipient malnutrition. Infusions started in the afternoon the day before operation between 4 and 5 p.m. and continued with constant rate until muscle biopsies were taken from the rectus abdominis muscles directly after anesthesia induction (15-16 hour later) [START_REF] Sandstrom | The effect on energy and nitrogen metabolism by continuous, bolus, or sequential infusion of a defined total parenteral nutrition formulation in patients after major surgical procedures[END_REF]. This procedure was chosen for practical reasons and due to the fact that artificial nutrition should provide protein anabolism within 12 hours if being clinically efficacious. Blood samples were drawn at the same time for analysis of amino acids and substrate levels in the peripheral circulation. Inclusion criterion was patients who may be candidates for intravenous nutritional support post surgery without having diabetes or steroid medications.

Analytical methods

Blood Samples

Arterial blood samples were collected and immediately centrifuged at + 4°C. Serum and plasma was stored at -80°C until analysis. For amino acid analysis 0.5 ml plasma was deproteinized with 125 μl sulfosalisylic acid (10 %). Para-fluoro-phenylalanine (20 umol/ml plasma) was added for internal standard use. After 30 minutes, precipitates were centrifuged and supernatants were used for amino acid analysis by a reversed phase HPLC method [START_REF] Georgi | High-performance liquid chromatographic determination of amino acids in protein hydrolysates and in plasma using automated pre-column derivatization with o-phthaldialdehyde/2-mercaptoethanol[END_REF].

All other plasma and serum analyses were standard analyses at the Clinical Chemistry Laboratory, Sahlgrenska University Hospital.

Muscle Biopsies

Muscle biopsies from the rectus abdominis muscle were cut out with scissors with a minimum of handling and immediately put in RNAlater solution [START_REF] Lundholm | Protein synthesis in human skeletal muscle tissue: influence of insulin and amino acids[END_REF][START_REF] Lundholm | Protein synthesis in isolated human skeletal muscle tissue: evaluation of an experimental model[END_REF][START_REF] Lundholm | Protein degradation in human skeletal muscle tissue: the effect of insulin, leucine, amino acids and ions[END_REF] (Ambion, Cambridgeshire, UK). The samples were kept at +4°C for 24 hours in RNAlater and then stored at -20°C until analysis; a procedure, which did not interfere with the phosphorylation state of protein confirmed in pre-study experiments. Biopsies were homogenized in seven volumes of ice- Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. cold buffer A (20 mM Hepes, pH 7.4, 100 mM KCL, 0,2 mM EDTA, 2mM EGTA, 1 mM DTT, 50 mM NaF, 50 mM β-glycerofosfat, 0,1 mM AEBSF, 1 mM bensamidine, 0,5 mM sodiumvanadate,). The homogenate was centrifuged at 10 000 x g for 10 min at 4ºC and aliquots of the supernatant were used as follows:

Analysis of eIF4G⋅ eIF4E complex and 4E-BP1⋅ eIF4E complex Aliquots of supernatant were mixed with an agarose conjugate of a mouse monoclonal anti-eIF4E antibody (sc-9976 Santa Cruz Biotechnology, Santa Cruz, USA) and samples were rotated over night at +4°C. Next day samples were centrifuged at 1000 x g and supernatant was removed. The pellets were washed with 1 ml ice-cold buffer A. Washing procedure was repeated five times with a centrifugation step between each wash. After final wash, pellets were resuspended in 2x SDS electrophoresis sample buffer and boiled for 3 minutes. Samples were centrifuged at 1000 x g to pellet the agarose beads and equal volumes of supernatant were thereafter used for immunoblotting. Samples were electrophoresed in either a 15% polyacrylamid gel (4E-BP1) or a 7.5% polyacrylamid gel (eIF4G). Proteins were transferred to PVDF membranes. Membranes were incubated over night at +4°C with a rabbit anti rat 4E-BP1 antibody, or 90 min at + 20 °C with a rabbit anti human eIF4G antibody (sc-6936 and sc-11373, Santa Cruz biotechnology, Santa Cruz, USA) after blocking in 5% nonfat dry milk in Tris-buffered Saline-Tween 20. The blots were then washed, incubated with secondary antibodies and developed using an ECL Western Blotting Kit according to the manufacturers´ description (Amersham Biosciences, UK). For detection, membranes were exposed against Hyperfilm ECL (Amersham Biosciences, UK). After detection of signals, antibodies were removed by 30 minutes incubation at +50°C in stripping buffer (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfat ( SDS), 62.5 mM Tris-HCl pH 7.6).and membranes were thereafter reprobed for measurement of eIF4E by incubating membranes overnight at +4°C with a rabbit anti human eIF4E antibody (sc-13963, Santa Cruz biotechnology, Santa Cruz, USA) Quantification of signals was carried out with GS-710 imaging densitometer and Quantity One software (Bio-Rad Laboratories AB, Sundbyberg, Sweden). Measured optical density was expressed as arbitrary units in all analyses. All samples were analyzed on two gels. Two lanes were loaded with MagicMark XP Western Protein Standards (Invitrogen) on each gel.

The average optical density for the standard bands, with similar molecular size as measured proteins, was used to normalize signal intensity between blots. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Analysis of 4E-BP1 phosphorylation state.

An aliquot of the homogenate supernatant was boiled for 5 minutes, cooled on ice and thereafter centrifuged at 10 000 x g for 30 minutes at +4°C. Four volumes of supernatant were mixed with 1 volume of 5x SDS electrophoresis sample buffer. Samples were boiled for 3 minutes and equal volumes of sample were electrophoresed in a 15% polyacrylamid gel.

Western blots were performed as described above. Membranes were incubated over night at +4°C with rabbit anti rat 4E-BP1 antibody (Sc-6936 Santa Cruz Biotechnologies).

Analysis of p70S6kinase phosphorylation state.

Aliquots of the homogenate supernatant were mixed with equal volumes of 2x SDS electrophoresis sample buffer. Samples were electrophoresed in 7.5% polyacrylamide gel.

Western Blots were performed as described above except that membranes were incubated with rabbit anti rat p70 S6 kinase antibody for 60 minutes at room temperature (sc-230, Santa Cruz Biotechnologies).

This study was approved by the Ethics Committee at the Medical Faculty of Göteborg University (no. S328-3). All patients were provided written informed consent.

Statistics

Results are presented as mean ±SEM. Comparisons between groups were performed by twotailed t-test. P ≤0.05 was considered statistically significant. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

Amino acids and substrate levels in peripheral blood

Total plasma amino acids increased significantly during TPN compared to saline infusions (15-16 hours). Methionine, phenylalanine and threonine increased significantly in the group of essential amino acids, while tryptophan and lysine did not change. Only isoleucine increased among the branch chain amino acids (Table 2). Of non-essential amino acids alanine, arginine, aspartic acid, glycine and histidine increased significantly.

Plasma glucose and serum insulin, glycerol, triglycerides and S-FFA did not change statistically significantly by TPN compared to saline infusions (Table 3).

Effects of total parenteral nutrition on 4E-BP1⋅ eIF4E complex and eIF4G⋅ eIF4E complex formation

Provision of overnight TPN increased the formation of the active eIF4G•eIF4E complex (p<0.05). The optical density of the complex increased from 1.16±0.26 in saline infused patients to 2.45±0.45 in TPN treated patients (Fig. 1A, Fig. 2). The inhibitory 4E-BP1•eIF4E complex decreased from 4.06±0.9 in the saline group to 1.94±0.41 in the TPN group (p<0.06) (Fig. 1B).

Effects of total parenteral nutrition on 4E-BP1 phosphorylation

Phosphorylation of total 4E-BP1 protein was analyzed as a complement to the analysis of 4E-BP1 ⋅ eIF4E complex, by determination of different isoforms following electrophoretic separation. The least phosphorylated forms migrate faster and more extensively phosphorylated forms migrate at slower rate through the gel. The least phosphorylated forms are attached to eIF4E. Increased phosphorylation makes the protein disassociate from eIF4E so eIF4G binding can occur. Quantification of free and bound forms of the protein was performed by comparing the amount protein present in the most phosphorylated form to the total amount of protein. Provision of overnight nutrition increased the amount of 4E-BP1 in the most phosphorylated form to 72% compared to 60% in patients with only saline infusions (p<0.05)( Fig. 1C). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Effects of total parenteral nutrition on p70S6 kinase phosphorylation

DISCUSSION

It is well established that malnutrition hits both muscle mass and function with deteriorations that may explain reduced survival and increased morbidity in both patients and undernourished individuals [21,[START_REF] Lundholm | Palliative nutritional intervention in addition to cyclooxygenase and erythropoietin treatment for patients with malignant disease: Effects on survival, metabolism and function. A randomized prospetive study[END_REF]. Numerous, experimental and clinical studies have been provided to illustrate effects of exercise, hormones and nutrition on protein synthesis and breakdown of skeletal muscles [START_REF] Waterlow | Protein turnover in mammalian tissues and in the whole body[END_REF]. Unanimous findings are that reduced food intake will cause protein imbalance explained by decreased synthesis of mixed muscle protein and sometimes by elevated breakdown to support vital organs [START_REF] Lundholm | Efflux of 3-methylhistidine from the leg in cancer patients who experience weight loss[END_REF][START_REF] Rennie | Depressed protein synthesis is the dominant characteristic of muscle wasting and cachexia[END_REF]. Thus, refeeding initiates protein translation to support re-synthesis of both globular and contractile proteins [START_REF] Svanberg | The role of diet components, gastrointestinal factors, and muscle innervation on activation of protein synthesis in skeletal muscles following oral refeeding[END_REF].

However, protein synthesis is not an unanimous activity but a highly differentiated process with selective translation of specific proteins [START_REF] Lundholm | Protein synthesis in liver tissue under the influence of a methylcholanthreneinduced sarcoma in mice[END_REF]. Thus, the choice of methods for measurements of protein synthesis should be a matter of the question under test. In clinical studies it has usually been questioned whether overall mixed protein synthesis is low, unchanged or increased [START_REF] Rennie | Depressed protein synthesis is the dominant characteristic of muscle wasting and cachexia[END_REF]. Accordingly, most studies have relied on measurements of net balance of amino acids across vascular beds or on labeling of mixed muscle proteins following bolus or primed constant infusions of labeled amino acids [START_REF] Bennegard | Flux of amino acids across the leg in weight-losing cancer patients[END_REF][START_REF] Emery | Protein synthesis in muscle measured in vivo in cachectic patients with cancer[END_REF], techniques that suffer from both advantages and limitations. Results from dynamic and static evaluations of protein synthesis should therefore be interpreted with caution, since required prerequisites are not always fulfilled [START_REF] Iresjo | Reevaluation of amino acid stimulation of protein synthesis in murine-and human-derived skeletal muscle cells assessed by independent techniques[END_REF]. Thus, information may not always indicate what we conclude emphasized by discrepancies between incorporation rates of amino acids and phosphorylation/dephosphorylation of regulatory proteins [START_REF] Carroll | Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion[END_REF]. Both under-and overestimation of protein synthesis rates may occur when labeling techniques are used [START_REF] Iresjo | Reevaluation of amino acid stimulation of protein synthesis in murine-and human-derived skeletal muscle cells assessed by independent techniques[END_REF], and measurements of net balance of amino acids across resting or exercising muscles may not fully account for exchanges among various tissue components in organ compartments [START_REF] Lundholm | Transport kinetics of amino acids across the resting human leg[END_REF][START_REF] Lundgren | Leg exchange of amino acids during exercise in patients with arterial insufficiency[END_REF]. This situation may appear particularly uncertain if extracellular amino acids are controlling initiation of translation without prior equilibration with intracellular amino acids [START_REF] Bohe | Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study[END_REF][START_REF] Lundholm | Incorporation of leucine into human skeletal muscle proteins[END_REF]. The present investigation took advantage of a technique measuring alterations in phosphorylation of proteins reflecting induction and inhibition of initiation of protein translation independent of muscle steady state conditions and tracer equilibration [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF].

Alterations in protein synthesis is reflected by changes in eIF4G⋅eIF4e complex and the binding of 4E-binding protein 1(4E-BP1) to eIF4E. A limiting step for initiation of translation in protein syntheses involves the assembly of eIF4F. This complex is composed of eIF4E-4G and -4A. During initiation of protein translation the mRNA 5´ cap binds to eIF4E with subsequent binding of eIF4G and -4A to form complex that allows translation to proceed.

The binding of eIF4G to eIF4E are controlled in part by the 4E-BP1. This binding protein can prevent the assembly of the eIF4F complex by binding to eIF4E at the site of eIF4G binding.

The 4E-BP1 protein is controlled by phosphorylation at multiple sites. When 4E-BP1 becomes multiply phosphorylated it can not bind to eIF4E. Free eIF4E for binding to eIF4G becomes therefore a limitation in formation of eIF4F-complex [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF][START_REF] Gingras | Hierarchical phosphorylation of the translation inhibitor 4E-BP1[END_REF]. Translation can also be regulated by activation of p70s6k by phosphorylation, a kinase that is associated with increased translation of mRNAs containing a 5´ oligopyrimidine tract encoding components of the translational machinery [32]. This concept has recently been challenged but activation of p70s6k was necessary for muscle fibers to achieve normal size during development observed in cells null for the gene [START_REF] Tang | Amino acid-induced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation[END_REF][START_REF] Ohanna | Atrophy of S6K1(-/-) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control[END_REF].

Studies by isotopic infusions in our laboratory indicated that conventional TPN decreased net whole body protein degradation while stimulation of whole body synthesis demanded considerably high substrate infusion rates [START_REF] Hyltander | Effect on whole-body protein synthesis after institution of intravenous nutrition in cancer and non-cancer patients who lose weight[END_REF]. Amino acid balance across resting forearm and legs indicated also that high arterial concentrations of amino acids were required to switch net efflux of amino acids to influx [START_REF] Lundholm | Transport kinetics of amino acids across the resting human leg[END_REF], which should be seen in the light of observations that incorporation of labeled amino acids into mixed muscle proteins increased by 30% [START_REF] Bohe | Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study[END_REF], even when extracellular amino acids were only slightly increased comparable to levels observed in the present study (Table 2). Therefore, different techniques may imply various results. Analyses of controlling factors as insulin, IGF-1 and exercise have implied that both individual and groups of amino acids stimulate mixed human muscle protein synthesis both in vitro and in vivo [START_REF] Lundholm | Protein synthesis in human skeletal muscle tissue: influence of insulin and amino acids[END_REF][START_REF] Iresjo | Reevaluation of amino acid stimulation of protein synthesis in murine-and human-derived skeletal muscle cells assessed by independent techniques[END_REF][START_REF] Lundholm | Protein synthesis in isolated human skeletal muscle tissue: evaluation of an experimental model[END_REF][START_REF] Lundholm | Protein degradation in human skeletal muscle tissue: the effect of insulin, leucine, amino acids and ions[END_REF][START_REF] Sandstrom | The effect of recombinant human IGF-I on protein metabolism in post-operative patients without nutrition compared to effects in experimental animals[END_REF][START_REF] Moller-Loswick | Insulin selectively attenuates breakdown of nonmyofibrillar proteins in peripheral tissues of normal men[END_REF]. A recent investigation on ICU patients on nutritional support indicated that conventional i.v. nutrition did not increase individual amino acids in arterial blood to the extent observed in orally fed healthy volunteers [START_REF] Iresjo | Appearance of individual amino acid concentrations in arterial blood during steadystate infusions of different amino acid formulations to ICU patients in support of whole-body protein metabolism[END_REF], although severely injured ICU patients were kept alive for long time despite a lack of clear cut indices of improved muscle protein balance [START_REF] Rennie | Urinary excretion and efflux from the leg of 3-methylhistidine before and after major surgical operation[END_REF]. Such discrepancies and uncertainties suggest that available clinical programs for nutrition are still suboptimal or that applied methods for measurements of protein synthesis are insensitive or even inaccurate at some conditions [START_REF] Bohe | Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study[END_REF]. By contrast, the present study implies that overall initiation of translation of muscle proteins in the rectus abdominis muscles in patients who received overnight standard TPN was activated and remained so as long as the infusion continued. For practical reasons previous studies have examined different muscles in humans as vastus lateralis and soleus muscles, since needle biopsies are usually applied. Such studies have however not provided unanimous conclusions, since different muscles fiber composition may be related to various phosphoprotein status and mechanisms by which such proteins control protein dynamics. In the present study we used the rectus abdominis muscle based on its significance in abdominal surgical procedures. This muscle is also sensitive to nutritional alterations due to its type I fiber composition [START_REF] Lundholm | Metabolic studies in human skeletal muscle tissue. Evaluation of an in vitro preparation intended for clinical metabolic studies[END_REF]40]. A previous study examining the response to 3 hours infusion of amino acids in both soleus and vastus lateralis muscle from young healthy humans observed no effect on phosphorylation of either 4E-BP1 at Thr 37/46 or p70s6 kinase at Thr 789 compared to basal values, but observed both higher total amount and more phosphorylated 4E-BP1 in soleus muscle fibers [START_REF] Carroll | Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion[END_REF]. Our patients displayed variations in the total amount of 4E-BP1 while other measured proteins as p70s6k or eIF4E did not show similar variations. These observations imply the importance to measure initiation factor complex as eIF4G⋅eIF4E, since it provides more direct information of the active complex formation. Present investigation is the first study in humans reporting eIF4G⋅eIF4E complex levels in skeletal muscles.

It may be questioned to what extent phosphoprotein status reflects protein synthesis activity.

Data from rats, mouse and pigs are available, which show that changes of initiation factors with increasing eIF4G⋅eIF4E and decreasing 4E-BP1⋅eIF4E complexes are related to elevated protein synthesis. Exceptions to these reports occur where increased and decreased protein syntheses were not reflected by changes of initiation factors [START_REF] Vary | Insulin fails to stimulate muscle protein synthesis in sepsis despite unimpaired signaling to 4E-BP1 and S6K1[END_REF][START_REF] Anthony | Orally administered leucine enhances protein synthesis in skeletal muscle of diabetic rats in the absence of increases in 4E-BP1 or S6K1 phosphorylation[END_REF][START_REF] Anthony | Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine[END_REF]. Similar studies on human skeletal muscles are limited, but infusion of BCAA increased the amount of 4E-BP1 in the gamma form and increased phosphorylation of p70s6k without increased protein synthesis [START_REF] Liu | Branched chain amino acids activate messenger ribonucleic acid translation regulatory proteins in human skeletal muscle, and glucocorticoids blunt this action[END_REF]. However, infusion of mixed amino acids caused similar changes in 4E-BP1 phosphorylation simultaneously with increased protein synthesis [START_REF] Liu | Amino acids stimulate translation initiation and protein synthesis through an Akt-independent pathway in human skeletal muscle[END_REF]. Therefore, it remains uncertain to what extent alterations in phosphoporotein status reflect dynamics in protein synthesis. The present study used a constant low dose amino acid infusion (~ 43 mg/kg/hr), which compares well with a low dose used by Bohé et al (43.5 mg/kg/hr) inducing a 30% increased mixed muscle protein synthesis [START_REF] Bohe | Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study[END_REF]. However, another study by Bohé et al indicated that protein synthesis may became refractory despite continuous availability of amino acids [START_REF] Bohe | Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids[END_REF]. By contrast, our present results showed sustained activation of proteins for initiation of translation during 16 hours infusion of a low dose of amino acids. This difference may be explained by amino acid overloading with high dose infusion indicated by appearing Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

ureagenesis [START_REF] Bohe | Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids[END_REF]. Also, it is likely that activated phosphoproteins reflect continuously changing recruitment of various mRNAs for translation reflected by sustained formation of initiation complexes. Therefore, selection of investigative methods should always be done with the specific aim in mind, where quantification of initiation complexes seems ideal for determination of directional changes of protein synthesis in non-steady state conditions.

In conclusion, the present study demonstrates that overnight preoperative constant infusion of standard TPN alters phosphorylation status of proteins indicating activation of initiation of protein translation in the rectus abdominis muscle. 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. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  Provision of TPN over night increased the total amount of p70S6 kinase and the phosphorylation of the protein. The least phosphorylated forms migrate faster while more S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  extensively phosphorylated forms migrated at slower rate through the electrophoretic gel.Blots were quantified by measuring total optical density of all bands (α+β+γ), and by determination of the ratio between the least phosphorylated form (α) compared to the total (α+β+γ) amount. In the group receiving TPN total p70S6k increased 30% compared to saline treated patients (p<0.05). The relative amount of the protein present in the least phosphorylated α band fell from 63 to 49 % which indicates activation of the kinase by phosphorylation (p<0.01, Fig.1D, Fig.2). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

  FIGURE LEGENDSFig 1A-D Complexes of eIF4G eIF4E and 4E-BP1 eIF4E as well as phosphorylation state of 4E-BP1 and p70s6k proteins in skeletal muscles from patients who received overnight constant TPN infusion ( ) compared to patients who received saline infusion ( ) only. The amount of eIF4G was corrected for eIF4E recovered from immunprecipitation. No difference in eIF4E content was found between the groups. Antibodies recognizing multiple phosphorylation forms of 4E-BP1 and p70s6k were used as described in Material and Methods (Mean ± SEM. n=6 patients in each group).

Fig. 2

 2 Fig. 2 Western blot analysis of initiation factors in rectus abdominis muscle biopsies from patients who received overnight infusion of saline (S) or TPN (T). Proteins were immunoprecipitated (IP) with anti-eIF4E and then immunoblotted (IB) with anti-eIF4G, -4E-BP1 or -eIF4E for measurement of eIF4G•eIF4E-and 4E-BP1•eIF4E complexes. Results are optical density of eIF4G or 4E-BP1/optical density of eIF4E. Increased phosphorylation of p70s6k and total 4E-BP1 was measured as relative change in mobility. S t a g e 2 ( a ) P O S T -P R I N T

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 15 Nov 2007 as manuscript CS20070359 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359

Table 1 .

 1 Clinical characteristics of patients randomized to receive preoperative infusions with either total parenteral nutrition (TPN) or saline as described in Methods.

	Saline	TPN

Table 2 .

 2 Amino acid concentrations in peripheral blood from patients treated with either total parenteral nutrition (TPN) or physiological saline at constant rate as described in Methods.

		Saline	TPN	p<
			μmol/l
	Essential aa
	Isoleucine	64 ± 10	95 ± 8	0.05
	Leucine	117 ± 14	129 ± 9	NS
	Valine	234 ± 32	316 ± 28	NS
	BCAA	416 ± 54	541 ± 43	NS
	Lysine	143 ± 12	163 ± 13	NS
	Methionine	28 ± 3	72 ± 5	0.001
	Phenylalanine	46 ± 4	86 ± 5	0.001
	Threonine	116 ± 12	195 ± 17	0.01
	Tryptophan	9 ± 1	12 ± 1	NS
	Essential AA	760 ± 72	1070 ± 67	0.05
	Non essential aa
	Alanine	318 ± 47	481 ± 48	0.05
	Arginine	77 ± 15	135 ± 14	0.05
	Aspartic acid	4 ± 1	9 ± 1	0.001
	Asparagine	30 ± 4	18 ± 2	NS
	Citrulline	13 ± 3	16 ± 2	NS
	Glutamic acid	103 ± 12	132 ± 23	NS
	Glutamine	432 ± 38	466 ± 26	NS
	Glycine	195 ± 21	326 ± 24	0.01
	Histidine	70 ± 7	120 ± 6	0.01
	Serine	121 ± 15	145 ± 16	NS
	Taurine	40 ± 6	46 ± 7	NS
	Tyrosine	51 ± 6	43 ± 3	NS
	Ornitine	59 ± 7	78 ± 14	NS
	α-Aba	9 ± 1	15 ± 2	0.05
	Total AA	2284 ± 160	3105 ± 173	0.01
	Mean ±SEM
	BCAA = Branched chain amino acid

Table 3 .

 3 Substrate levels in peripheral blood when muscle biopsies were taken during constant infusion of total parenteral nutrition (TPN) or saline as described in Methods.

	Saline	TPN	p<

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070359 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Supported in parts by grants from the Swedish Cancer Society (2014), the Swedish Research Council (08712), Tore Nilson Foundation, Assar Gabrielsson Foundation (AB Volvo), Jubileumskliniken foundation, IngaBritt & Arne Lundberg Research Foundation, Swedish and Göteborg Medical Societies and the Medical Faculty, Göteborg University, VGR 19/00, 1019/00, Swedish Nutrition Foundation


MD Terho Lehtimäki
email: terho.lehtimaki@uta.fi

Nina Hutri-Kähönen

Mika Kähönen

Jukka Hemminki

Vera Mikkilä

Marika Laaksonen

Leena Räsänen

Nina Mononen

Nina Hutri-Kähönen §

Vera Mikkilä #

Marika Laaksonen #

Leena Räsänen #

Markus Juonala

Jukka Marniemi ¶

Jorma Viikari

Olli Raitakari

the cardiovascular risk in young Finns study

Keywords: adult-type hypolactasia, carotid artery, intima-media thickness, brachial artery, flowmediated vasodilatation, LCT

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.

INTRODUCTION

Adult-type hypolactasia (ATH), also known as lactase deficiency or lactase persistence/nonpersistence (please see OMIM, http://www.ncbi.nlm.nih.gov/sites/entrez), is a genetically defined condition affecting approximately one half of the world' s population [START_REF] Sahi | Recessive inheritance of adult-type lactose malabsorption[END_REF][START_REF] Jussila | Prevalence of lactose malabsorption in a Finnish rural population[END_REF][START_REF] Sahi | Lactose malabsorption in Finnish children of school age[END_REF][START_REF] Rasinperö | A genetic test which can be used to diagnose adult-type hypolactasia in children[END_REF]. Today, ATH can be diagnosed by using a simple genetic test (SNP rs4988235) [START_REF] Rasinperö | A genetic test which can be used to diagnose adult-type hypolactasia in children[END_REF][START_REF] Enattah | Identification of a variant associated with adult-type hypolactasia[END_REF][START_REF] Enattah | Genetic variant of lactase-persistent C/T-13910 is associated with bone fractures in very old age[END_REF][START_REF] Lehtimäki | The effects of adult-type hypolactasia on body height growth and dietary calcium intake from childhood into young adulthood: a 21-year follow-up study--the Cardiovascular Risk in Young Finns Study[END_REF].

The single nucleotide C/T-13910 polymorphism (SNP rs4988235) in the LCT gene in chromosome 2 defines the ATH status, leading to several times higher expression of lactasephlorizin hydrolase in T/T homozygotes than is found in C/C genotype subjects [START_REF] Kuokkanen | Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia[END_REF][START_REF] Olds | Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element[END_REF]. LCT C/T-13910 heterozygotes have intermediate levels of lactase, as measured by enzyme activity [START_REF] Enattah | Correlation of intestinal disaccharidase activities with the C/T-13910 variant and age[END_REF] and mRNA levels, but they still produce enough lactase to be asymptomatic when given a 50-gram dose of lactose in connection with a standard physiological test for lactose tolerance. Enattah et al. [START_REF] Enattah | Identification of a variant associated with adult-type hypolactasia[END_REF] reported that C/T-13910 polymorphism completely associates with biochemically verified lactase non-persistence in Finnish families and a sample set of 236 individuals from four different populations. After the original discovery of the ATH-causing LCT C/T-13910 genotype, molecular epidemiological studies in more than 40 countries have confirmed that the prevalence of the C/C-13910 genotype is consistent with previously published epidemiological data on ATH [START_REF] Rasinperö | A genetic test which can be used to diagnose adult-type hypolactasia in children[END_REF][START_REF] Lehtimäki | The effects of adult-type hypolactasia on body height growth and dietary calcium intake from childhood into young adulthood: a 21-year follow-up study--the Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Lember | Lactase non-persistence and milk consumption in Estonia[END_REF][START_REF] Anthoni | Molecularly defined adult-type hypolactasia among working age people with reference to milk consumption and gastrointestinal symptoms[END_REF]. Therefore, ATH persistence (genotypes TT and CT) and non-persistence (CC genotype) status can be reliably defined by this genetic test.

During the last decades, the consumption of milk has decreased while that of the other milk products, such as cheese and yoghurt, has increased in the general Finnish population [START_REF] Männistö | The National FINDIET 2002 study[END_REF]14].

However, ATH defined by C/C -13910 genotype limits the use of dairy products, in addition to regulating the intake of dietary fat and calcium in Finnish subjects and in subjects from several other populations [START_REF] Enattah | Genetic variant of lactase-persistent C/T-13910 is associated with bone fractures in very old age[END_REF][START_REF] Lehtimäki | The effects of adult-type hypolactasia on body height growth and dietary calcium intake from childhood into young adulthood: a 21-year follow-up study--the Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Lember | Lactase non-persistence and milk consumption in Estonia[END_REF][START_REF] Anthoni | Molecularly defined adult-type hypolactasia among working age people with reference to milk consumption and gastrointestinal symptoms[END_REF]15]. Moreover, some previous studies have shown that in subjects with ATH, serum levels of insulin, glucose and triglycerides are lower and high-density lipoprotein (HDL) cholesterol levels higher than in subjects who do not have ATH [16,[START_REF] Russo | Hypolactasia and metabolic changes in post-menopausal women[END_REF]. It is not known whether these biochemical differences in subjects with and without ATH [16,[START_REF] Russo | Hypolactasia and metabolic changes in post-menopausal women[END_REF] might eventually lead to differences in the early markers of atherosclerosis.

Carotid intima-media thickness (CIMT), carotid artery compliance (CAC) and brachial artery flow-mediated dilatation (FMD) are all accurately quantifiable and reproducible markers of early atherosclerosis [START_REF] Blacher | Carotid arterial stiffness as a predictor of cardiovascular and all-cause mortality in end-stage renal disease[END_REF][START_REF] Burke | Arterial wall thickness is associated with prevalent cardiovascular disease in middle-aged adults. The Atherosclerosis Risk in Communities (ARIC) Study[END_REF][START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF][START_REF] Chan | The prognostic importance of endothelial dysfunction and carotid atheroma burden in patients with coronary artery disease[END_REF][START_REF] Gokce | Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function: a prospective study[END_REF][START_REF] Juonala | Geographic origin as a determinant of carotid artery intima-media thickness and brachial artery flow-mediated dilation: the Cardiovascular Risk in Young Finns study[END_REF][START_REF] O'leary | Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group[END_REF][START_REF] Oliver | Noninvasive assessment of arterial stiffness and risk of atherosclerotic events[END_REF][START_REF] Raitakari | Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Tounian | Presence of increased stiffness of the common carotid artery and endothelial dysfunction in severely obese children: a prospective study[END_REF][START_REF] Yufu | Brachial arterial stiffness predicts coronary atherosclerosis in patients at risk for cardiovascular diseases[END_REF]. Of these markers, increased CIMT correlates with cardiovascular risk factors [START_REF] Raitakari | Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study[END_REF] as well as the severity of coronary atherosclerosis [START_REF] Burke | Arterial wall thickness is associated with prevalent cardiovascular disease in middle-aged adults. The Atherosclerosis Risk in Communities (ARIC) Study[END_REF] and predicts cardiovascular events in population groups [START_REF] O'leary | Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group[END_REF]. FMD as well as CAC offer a new means to study early functional changes caused by atherosclerosis [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF][START_REF] Oliver | Noninvasive assessment of arterial stiffness and risk of atherosclerotic events[END_REF][START_REF] Corretti | Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force[END_REF]. Impaired FMD is a marker of endothelial dysfunction, which also relates to the prevalence and extent of coronary atherosclerosis and predicts cardiovascular events in patient groups [START_REF] Chan | The prognostic importance of endothelial dysfunction and carotid atheroma burden in patients with coronary artery disease[END_REF][START_REF] Gokce | Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function: a prospective study[END_REF].

Given the established relationship between ATH and dietary intake of milk and dairy products and thus dietary fat and calcium, it may be hypothesized that variation at the ATH gene locus (rs4988235) directly, or indirectly by modifying biochemical risk factors, translates into differences in the early markers of atherosclerosis. Therefore, in the present study, we investigated the association of ATH status and early markers of atherosclerosis in a young population-based cohort of otherwise healthy individuals participating in the Cardiovascular Risk in Young Finns Study [START_REF] Raitakari | Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

SUBJECTS AND METHODS

Subjects

The Cardiovascular Risk in Young Finns Study (http://vanha.med.utu.fi/cardio/youngfinnsstudy) is an on-going follow-up study of the precursors of atherosclerosis in Finnish children and adolescents. The first cross-sectional survey was conducted in 1980 when 3,596 participants aged 3, 6, 9, 12, 15 and 18 years were randomly chosen from the national population register to represent each (geographical) area in Finland [START_REF] Akerblom | Atherosclerosis precursors in Finnish children and adolescents. I. General description of the cross-sectional study of 1980, and an account of the children's and families' state of health[END_REF]. The sample was representative of the entire Finnish population. In 2001, we re-examined 2,283 of these individuals, now aged 24 to 39 years. Complete data on the LCT gene C/T-13910 polymorphism as well as carotid and brachial artery ultrasound examinations were available for 2,109 subjects [START_REF] Juonala | Geographic origin as a determinant of carotid artery intima-media thickness and brachial artery flow-mediated dilation: the Cardiovascular Risk in Young Finns study[END_REF][START_REF] Raitakari | Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study[END_REF]. The study was carried out in accordance with the Declaration of Helsinki 2000 of the World Medical Association and approved by local Ethics committees, and all subjects gave their written informed consent.

Clinical characteristics and risk factors

Height and weight were measured, and body mass index (BMI) was calculated [START_REF] Juonala | The 21-year follow-up of the Cardiovascular Risk in Young Finns Study: risk factor levels, secular trends and east-west difference[END_REF]. In 2001, a random zero sphygmomanometer was used for blood pressure measurements. The average of three measurements was used in the analysis. In adults, smoking habits and history of diabetes were assessed with a questionnaire. The physical activity index was calculated as previously described [START_REF] Telama | Physical activity from childhood to adulthood: a 21-year tracking study[END_REF].

For the determination of serum lipoprotein levels, venous blood samples were drawn after an overnight fast. Lipid concentrations were measured using standard methods [START_REF] Juonala | The 21-year follow-up of the Cardiovascular Risk in Young Finns Study: risk factor levels, secular trends and east-west difference[END_REF][START_REF] Lehtimäki | Apolipoprotein E phenotypes in Finnish youths: a cross-sectional and 6-year follow-up study[END_REF]. Low density lipoprotein (LDL) cholesterol concentration was calculated with the Friedewald formula.

The fasting plasma high-sensitive C-reactive protein concentrations (CRP) were analyzed by the latex turbidometric immunoassay (Wako Chemicals GmbH, Neuss, Germany). The lower detection limit reported for the assay was 0.06 mg/l, and the coefficient of variation in repeated measurements was 3.3%. Plasma glucose concentrations were analyzed enzymatically (Olympus Diagnostica GmbH, Hamburg, Germany). Serum insulin concentrations were measured with microparticle enzyme immunoassay kits (Abbott Laboratories, Diagnostic Division, Dainabot, Japan).

Assessment of dietary variables

Food consumption was assessed with dietary questionnaires on habitual eating behaviour and food choices, including questions on the habitual frequency of the consumption of selected foods and food groups. On the basis of these data, we also constructed an index variable representing the Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

subjects' usual daily consumption of milk and other dairy products, such as cheese, sour milk, yoghurt and ice cream. Similarly, we calculated the daily consumption of fresh vegetables and the weekly consumption of fish. The consumption of alcohol is presented as a sum of weekly portions of beer, wine and hard liquor (grams/week). The details of the dietary study included in the project have been described previously [START_REF] Mikkila | Longitudinal changes in diet from childhood into adulthood with respect to risk of cardiovascular diseases: The Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Mikkila | Consistent dietary patterns identified from childhood to adulthood: the cardiovascular risk in Young Finns Study[END_REF].

Ultrasound measurements

Ultrasound examinations were performed using Sequoia 512 ultrasound mainframes (Acuson, Mountain View, CA, USA) with a 13.0 MHz linear array transducer, as previously described [START_REF] Raitakari | Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study[END_REF].

In brief, the image in the CIMT measurements was focused on the posterior (far) wall of the left carotid artery. A magnified image was recorded from the angle showing the greatest distance between the lumen-intima interface and the media-adventitia interface. A minimum of four measurements of the common carotid far wall were taken 10 mm proximally to the bifurcation in order to derive mean CIMT. The between-visit (2 visits 3 months apart) coefficient of variation (CV) for CIMT measurements was 6.4% [START_REF] Raitakari | Cardiovascular risk factors in childhood and carotid artery intima-media thickness in adulthood: the Cardiovascular Risk in Young Finns Study[END_REF].

For CAC measurements, the left carotid artery was scanned by physicians and ultrasound technicians following standard protocol [START_REF] Juonala | Geographic origin as a determinant of carotid artery intima-media thickness and brachial artery flow-mediated dilation: the Cardiovascular Risk in Young Finns study[END_REF]. In brief, several five-second moving image clips of the beginning of the carotid bifurcation and the common carotid artery were acquired and stored in digital format on optical discs for subsequent off-line analysis. All digitally stored ultrasound scans were manually analyzed by a single reader blinded to the subjects' details (M.J.). The analyses were performed using ultrasonic calipers. From the five-second clips, the best-quality cardiac cycle was selected. The carotid diameter was measured at least twice in end-diastole and end-systole, respectively. The mean of the measurements was used as the end-diastolic or end-systolic diameter.

Blood pressure was measured during the ultrasound study with an automated sphygmomanometer (Omron M4, Omron Matsusaka Co., Ltd., Japan) from the non-dominant brachial artery. The mean of two measurements was used in the analysis. CAC [%/10 mmHg] was calculated with the equation ([D s -D d ]/D d )/(P s -P d ), with D d representing the diastolic diameter, D s the systolic diameter, P s systolic blood pressure and P d diastolic blood pressure. To assess the reproducibility of the CAC measurements, we re-examined 60 subjects 3 months after the initial visit (2.5% random sample).

The between-visit coefficient of variation (CV) for the end-diastolic carotid diameter was 2.7% and for carotid diameter change 13.1% [START_REF] Juonala | Geographic origin as a determinant of carotid artery intima-media thickness and brachial artery flow-mediated dilation: the Cardiovascular Risk in Young Finns study[END_REF].

To assess brachial artery FMD, the left brachial artery diameter was measured both at rest and during reactive hyperemia, as described previously [START_REF] Juonala | Geographic origin as a determinant of carotid artery intima-media thickness and brachial artery flow-mediated dilation: the Cardiovascular Risk in Young Finns study[END_REF]. In brief, increased flow was induced by inflation of a pneumatic tourniquet placed around the forearm to a pressure of 250 mmHg for 4.5 minutes, followed by a release. Three measurements of arterial diameter were performed at enddiastole at a fixed distance from an anatomic marker at rest as well as 40, 60 and 80 seconds after cuff release. The vessel diameter in scans after reactive hyperemia was expressed as a percentage relative to the resting scan (100 percent). The average of three measurements at each point in time was used to derive maximum FMD (the greatest value between 40 to 80 seconds). We have previously reported a short-term (2-hour) between-study coefficient of variation of 9% for FMD measurements [START_REF] Järvisalo | Enhanced endothelium-dependent vasodilation in subjects with Proline7 substitution in the signal peptide of neuropeptide Y[END_REF]. The long-term between-visit variation CVs of brachial measurements, achieved through re-examining 57 subjects 3 months after the initial visit (2.5% random sample), were 3.2% for brachial artery diameter measurements and 26.0% for FMD measurements.

Lactase-phlorizin hydrolase genotyping

The LCT C/T -13910 single nucleotide polymorphism (SNP rs4988235) was measured for 2,281 participants in 2001. Genomic DNA was extracted from peripheral blood leukocytes using a commercially available kit (Qiagen Inc., Hilden, Germany). LCT C/T-13910 genotyping was performed by employing the 5'-nuclease assay with fluorogenic allele-specific TaqMan probes and primers [START_REF] Lehtimäki | The effects of adult-type hypolactasia on body height growth and dietary calcium intake from childhood into young adulthood: a 21-year follow-up study--the Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Livak | Allelic discrimination using fluorogenic probes and the 5' nuclease assay[END_REF], using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). As quality control we used known control samples which were run in parallel with unknown DNA samples and random blind duplicates.

Statistical analysis

Statistical analyses were carried out using SPSS version 14.0 for Windows software (SPSS Inc., Chicago, Illinois, USA).

The levels of continuous (normally distributed) variables were compared between the ATH status groups (no/yes) using the t-test. These analyses were performed separately for males and females. The compliance of genotype data with the Hardy-Weinberg equilibrium was assessed with the χ 2 -test.

In multivariate linear regression analysis, we used the vascular end points, i.e., CIMT, CAC or FMD, as the outcome variable, one at a time. The predictors (independent variables) in the model were: ATH status (no/yes), age, sex, BMI, smoking (pack years), physical activity index [START_REF] Telama | Physical activity from childhood to adulthood: a 21-year tracking study[END_REF], use of alcohol (portions per week), glasses of milk per day, HDL cholesterol, LDL cholesterol (due to alternatively apoB or total cholesterol), triglycerides, glucose, systolic blood pressure, and high sensitive CRP. The linear regression analysis for the above-mentioned outcome variables was performed by applying three different models: (model 1) using ATH status as a predictor, (model 2) Owing to skewed distributions, the values for triglycerides, insulin and CRP were log-transformed prior to the analyses. For glucose and the consumption of milk and cheese, the distribution remained too skewed after log transformation; consequently, the Kruskal Wallis test was employed.

The distributions of categorical variables among ATH groups were compared with the χ2test. A p-value less than 0.05 was considered significant.

RESULTS

The prevalence of LCT C/T -13910 genotypes C/C, C/T and T/T in our population were as follows:

17.6% (401/2,281), 48.9% (1,116/2,281) and 33.5% (764/2,281), respectively.

The association of ATH status with CHD risk factors

The relation of ATH status, as defined by C/T-13910 polymorphism (rs4988235), with CHD risk factors, dietary factors as well as indices of early atherosclerosis is shown in Table 1. The consumption of alcohol was highest among subjects with the C/C -13910 genotype (p=0.001) when compared to subjects without ATH (TT+CT). The overall consumption of milk products (milk product index on Table 1) was lower among subjects with ATH (p<0.001). The difference is explained by the differential consumption of the milk (p<0.001) as the consumption of the other dairy products was alike in the two groups (p=0.953). However, neither the total fat intake (g/day) nor the total intake of saturated fat differ in the groups studied (p=0.80 and p=0.99, respectively).

The association of ATH status and LCT C/T-13910 genotype with markers of early atherosclerosis

In univariate analysis, no significant association between LCT C/T-13910 genotype-defined ATH status and CIMT, CAC or brachial artery FMD was found among the entire study population (Table 1), or among females or males (p=NS for all groups).

The differently adjusted regression analyses-i.e., the unadjusted model 1, model 2 adjusted with age and lifestyle factors, and the fully adjusted model 3 also adding sex and CAD risk factors as predictors-gave essentially the same results for all major vascular end-points, namely CIMT, CAC and FMD. Therefore, the numerical results from only the fully adjusted regression model are shown in Table 2. In multiple linear regression analyses, after adjustment for all major covariates, there were no statistically significant associations between ATH status and CIMT, CAC or FMD among all subjects (Table 2) or when males and females were analysed in their own groups

DISCUSSION

This is the first general-population-based study suggesting that the genetic locus which causes ATH has no clinically significant effects on the early functional or structural markers of asymptomatic atherosclerosis in young Finns.

There is little previously published data on the effect of ATH on serum lipid concentrations [16,[START_REF] Russo | Hypolactasia and metabolic changes in post-menopausal women[END_REF]. In one earlier study, serum levels of insulin and glucose were found to be lower and HDL cholesterol levels higher in subjects with ATH compared to others [START_REF] Russo | Hypolactasia and metabolic changes in post-menopausal women[END_REF]. In our population-based study, we were not able to find any differences in serum levels of insulin and glucose between subject with and without ATH. Instead, the LCT C/C-13910 genotype was associated with HDL cholesterol in that it tended to be higher in subjects carrying the C/C-13910 genotype-when the population was stratified according to ATH status, however, the difference in HDL cholesterol remained statistically insignificant. Although the overall consumption of the dairy products is lower in subjects with the ATH, serum total or LDL cholesterol, insulin, glucose, sensitive CRP or homocysteine concentrations did not differ between subject with or without ATH. Therefore, our findings do not support the suggestion that differences in the dietary intakes between subjects with and without ATH are translated into differences in early atherosclerotic changes through an association with serum lipid levels.

The lower consumption of dairy products among the ATH subjects is in line with earlier results from Finnish, Estonian, and Austrian populations [START_REF] Lehtimäki | The effects of adult-type hypolactasia on body height growth and dietary calcium intake from childhood into young adulthood: a 21-year follow-up study--the Cardiovascular Risk in Young Finns Study[END_REF][START_REF] Anthoni | Molecularly defined adult-type hypolactasia among working age people with reference to milk consumption and gastrointestinal symptoms[END_REF]15,[START_REF] Obermayer-Pietsch | Adult-type hypolactasia and calcium availability: decreased calcium intake or impaired calcium absorption?[END_REF]. In addition, ATH was associated with higher consumption of alcoholic beverages among men, a new finding requiring confirmation in later investigations.

In epidemiological studies, the dietary consumption of milk [START_REF] Elwood | Milk, coronary disease and mortality[END_REF][START_REF] Strain | Milk consumption, lactose and copper in the aetiology of ischaemic heart disease[END_REF] and calcium [START_REF] Leskinen | The prevalence of peripheral arterial disease and medial arterial calcification in patients with chronic renal failure: requirements for diagnostics[END_REF][START_REF] Goldsmith | Vascular calcification: a stiff challenge for the nephrologist: does preventing bone disease cause arterial disease?[END_REF][START_REF] Seely | Possible connection between milk and coronary heart disease: the calcium hypothesis[END_REF], both of which are altered in connection with ATH, have been implicated as risk factors for CHD.

On the other hand, as a balancing view to these earlier epidemiological data, several recent studies have also indicated that milk and dairy products may yield some beneficial effect in relation to CHD risk factors, such as blood pressure and body weight control [START_REF] Pfeuffer | Milk and the metabolic syndrome[END_REF][START_REF] Alvarez-Leon | Dairy products and health: a review of the epidemiological evidence[END_REF][START_REF] Al-Delaimy | A prospective study of calcium intake from diet and supplements and risk of ischemic heart disease among men[END_REF][START_REF] Hajjar | Dietary calcium lowers the age-related rise in blood pressure in the United States: the NHANES III survey[END_REF][START_REF] Pereira | Dairy consumption, obesity, and the insulin resistance syndrome in young adults: the CARDIA Study[END_REF]. Therefore, our study which shows that ATH, a quite common condition, is not related to CIMT, carotid elasticity or the indicator of endothelial function, i.e., brachial artery FMD, makes an important contribution to the literature in this field. Our findings do not support either the suggestion that differences in the dietary intakes between subjects with and without ATH are translated into differences in early atherosclerotic changes directly without the effect on serum lipid levels. However, it is possible that the LCT loci affect CHD risk through mechanisms not reflected in CIMT or that the relatively young age range of the present cohort precludes the ability to detect an effect which may become discernible in older individuals. These possibilities require further investigations, and our results do not exclude the possibility that LCT C/T-13910 genotype or ATH status could be associated with more advanced, clinically symptomatic atherosclerotic changesi.e., coronary artery disease or MI-developing 20-30 years later.

The major limitation of our study traces back to our dietary questionnaire which only counts the number of milk and dairy product portions and does not account for the actual portion size. It therefore does not measure the true quantity of dairy product consumption. Although the sample size is seemingly large, the numbers and thus the statistical power may still be insufficient to formally exclude the association with blood pressure or vascular risk markers.

In conclusion, our results do not provide further evidence for the involvement of ATH, as defined by the LCT gene locus rs4988235, in determining early atherosclerosis risk in a large cohort of young Finnish individuals.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  adding age and lifestyle factors to model 1, and (model 3) adding sex and the remaining major CAD S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  risk factors as predictors to model 2. Due to high intercorrelations between apoB, LDL cholesterol and total cholesterol, these were not used simultaneously as predictors in the same statistical model.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society (data not shown). No significant association between LCT C/T-13910 genotypes and carotid IMT, CAC or brachial artery FMD was found among the entire study population or in females and males separately (data not shown).

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  distribution, the data on dairy products, triglycerides, CRP and insulin were log-transformed before analysis. ‡Distribution remained too skewed after log transformation; as a consequence, the Mann Whitney U-test was used here. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 1 .

 1 The association of adult-type hypolactasia status defined by the LCT C/T -13910 genotype (rs4988235) with the risk and dietary factors as well as preclinical indicators of atherosclerosis.Abbreviations: N, number of subjects; (g/week), grams/week; LDL, low density lipoprotein, HDL, high density lipoprotein; BP, blood pressure; CAC, carotid artery compliance. Note: *A physical activity index was calculated on the basis of a questionnaire. †Dairy products (index) represent subjects' habitual consumption of milk and dairy products as portions per day and has been assessed with a dietary questionnaire on habitual eating behaviour and food choices. Statistics: § 2 χ test for categorical variables and independent t-test for continuous variables. Due to skewed

	Values presented as mean ± standard deviation.
			Adult type hypolactasia status
	Variable	N (T/T+C/T, C/C) NO (T/T+CT)	YES (C/C)	P §
	Age (years)	1880, 401	31.7±5.0	31.9±4.9	0.512
	Sex (% males)	1880, 401	44.6	46.4	0.543
	Height (cm)	1867, 398	172.1±9.2	172.1±8.9	0.780
	Weight (kg)	1866, 398	74.5±15.9	74.8±16.0	0.739
	Body mass index (kg/m 2 )	1865, 401	25.1±4.4	25.1±4.61	0.763
	Smoking (pack years)	1748, 369	2.96±5.67	3.30±6.13	0.314
	Physical activity index*	1755, 368	9.96±2.36	9.75±2.36	0.120
	Dairy product index (p/day) †	1754, 369	3.03±1.86	2.39±1.54	<0.001
	Milk (glasses/day) ‡	1818, 386	1.79±1.84	1.12±1.50	<0.001
	Dairy products, without milk, (p/day) 1754, 369	1.25±0.60	1.25±0.56	0.953
	Alcohol consumption (g/week)	1852, 396	67.4±96.1	85.9±120.5	0.001
	Systolic BP (mmHg)	1862, 395	116.6±13.1	117.2±13.1	0.374
	Diastolic BP (mmHg)	1862, 395	70.7±10.9	70.9±10.4	0.768
	Total cholesterol (mmol/l)	1880, 401	5.16±0.98	5.17±0.99	0.803
	LDL cholesterol (mmol/l)	1855, 394	3.28±0.85	3.27±0.83	0.923
	HDL cholesterol (mmol/l)	1878, 401	1.29±0.31	1.31±0.35	0.120
	Triglycerides (mmol/l)	1880, 401	1.34±0.86	1.35±0.84	0.769
	C-reactive protein (mg/l)	1880, 401	1.94±3.99	1.86±3.73	0.707
	Glucose (mmol/l) ‡	1880, 401	5.06±0.89	5.02±0.48	0.374
	Intima-media thickness (mm)	1860, 395	0.581±0.092	0.583±0.096 0.657
	CAC (%/10 mmHg)	1852, 393	2.18±0.74	2.16±0.75	0.752
	Flow mediated dilatation (%)	1723, 376	8.00±4.41	7.98±4.4 6	0.944

© 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This study was financially supported by the Emil Aaltonen Foundation (T.L), the Academy of Finland (grant no. 77841, 210283, 117941), the Social Insurance Institution of Finland, the Turku University Foundation, the Juho Vainio Foundation, the Finnish Foundation of Cardiovascular Research, the Finnish Cultural Foundation as well as the Tampere and Turku University Central Hospital Medical Funds. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 14 Jan 2008 as manuscript CS20070360 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070360 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society


MD Arschang Valipour
email: arschang.valipour@wienkav.at

MD Martin Schreder

MD Michael Wolzt

MD Sleman Saliba

Sonja Kapiotis

Philipp Eickhoff

MD Otto Chris Burghuber

Sonja Di

Kapiotis

Circulating vascular

Keywords: chronic obstructive pulmonary disease, cardiovascular disease, vascular endothelial growth factor, systemic inflammation, c-reactive protein

Introduction

Vascular endothelial growth factor (VEGF), a soluble heparin binding glycoprotein, is a cytokine with potent angiogenic properties, enhancing vascular permeability and modulating thrombogenicity [START_REF] Ferrara | Molecular and biological properties of vascular endothelial growth factor[END_REF]. VEGF further serves as an endothelial cell survival factor, protecting endothelial cells against apoptosis and delaying endothelial cell senescence [START_REF] Gerber | Vascular endothelial growth factor induces expression of the antiapoptotic proteins BCL-2 and A1 in vascular endothelial cells[END_REF]. The biological properties of VEGF have led to interest in its role within the lung in both health [START_REF] Kaner | Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung[END_REF] and disease [START_REF] Thicket | Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome[END_REF]. In fact recent studies have suggested that the expression of VEGF in lung tissue obtained from patients with chronic obstructive pulmonary disease (COPD) may play a role in the pathogenesis of the disease [START_REF] Kasahara | Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema[END_REF][START_REF] Kranenburg | Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease[END_REF]. Kasahara and colleagues [START_REF] Kasahara | Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema[END_REF] have shown that VEGF protein expression is reduced in lung specimens obtained from patients with emphysematous type of COPD. The authors have suggested that the absence of VEGF, which is a trophic factor for endothelial cells, contributes to the development of emphysema. In consistency with these findings, Kanazawa et al. [START_REF] Kanazawa | Possible effects of vascular endothelial growth factor in the pathogenesis of chronic obstructive pulmonary disease[END_REF] observed a positive relationship between VEGF concentrations in induced sputum samples and FEV1 in patients with stable COPD.

Over the last few years VEGF levels have also been measured in the peripheral circulation as a biomarker of neovascularization and/or vascular remodeling. Several studies reported increased circulating VEGF levels in conditions in which tissue hypoxia and/or inflammation are critical, such as coronary heart disease [START_REF] Soeki | Serial changes in serum VEGF and HGF in patients with acute myocardial infarction[END_REF], sepsis [START_REF] Van Der Flier | Plasma vascular endothelial growth factor in severe sepsis[END_REF], cancer [START_REF] Li | Correlation of serum VEGF levels with clinical stage, therapy efficacy, tumor metastasis and patient survival in ovarian cancer[END_REF], and rheumatoid diseases [11]. Furthermore, there is cumulating evidence that circulating VEGF levels have prognostic significance particularly in cardiovascular disease [START_REF] Heeschen | Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes[END_REF], which could provide a mechanism to link the association between inflammation and promotion of cardiovascular disease. Both hypoxia and inflammation are also relevant to COPD. Thus the aims of the present study were threefold: 1. to determine circulating levels of VEGF and systemic inflammation in patients with COPD and appropriate controls 2. to assess the effects of acute deteriorations of respiratory health in COPD on circulating VEGF and systemic inflammatory markers during and after recovery from an acute exacerbation of COPD and 3.

to assess the relationship between markers of systemic inflammation, hypoxemia, and lung function parameters with circulating VEGF levels in both stable and exacerbed COPD.

Patient characteristics

The characteristics of patients and controls are summarized in Table 1. The groups were wellmatched with respect to age, gender, and BMI. By definition lung function parameters were normal in controls and significantly reduced in patients with COPD. There was no significant difference in the proportion of patients with oxygen therapy, hypertension, inhaled bronchodilator or corticosteroid use between patients with exacerbated and those with stable COPD. Mean (SD) duration of hospitalisation for patients with exacerbated COPD was 10.6 (3.7) days.

VEGF measurements

Coefficients of variation of the duplicate analyses of ELISA were generally less than 5%. The (median [IQR]) serum concentration of VEGF (359 [201-639] pg/ml) was significantly higher (p < 0.001) than the corresponding concentration of VEGFprp (178 [90-302] pg/ml), despite a higher platelet count in the latter (432 [350-591] platelets × 10 3 /μL of PRP versus 251 [202-309] platelets × 10 3 /μL of serum; p < 0.01). There was a significant correlation between VEGFser and VEGFprp (r = 0.681; p < 0.001), and between VEGFser and VEGFppp (r = 0.474; p < 0.01). Correlations did not change significantly when the data from the control subjects was subtracted.

Circulating VEGF and systemic inflammatory markers in the study groups

The groups were well-matched with respect to hemoglobin concentrations and platelet count (Table 2). Overall, VEGF levels were significantly higher (p < 0.01) in patients with acute exacerbated than in patients with stable COPD or controls with normal lung function. These findings were paralleled by an elevation in CRP, peripheral blood neutrophil cell count (PBNC), and circulating IL-6 levels in patients with exacerbated COPD. Fibrinogen levels were significantly higher in patients with COPD compared to controls (p < 0.01 for both), with no significant difference between stable and exacerbated patients. There were no statistically significant differences in TNF-α concentrations between the groups.

Circulating VEGF and systemic inflammation at clinical recovery

There was a significant reduction in VEGFser levels from 602 [457-883] during acute exacerbation to 490 [378-699] pg/ml (p = 0.032) six weeks after hospital discharge (Figure 1). There was a similar decrease in VEGFprp and VEGFppp levels (data not shown). The

Discussion

This study has investigated circulating levels of VEGF and markers of systemic inflammation in carefully selected patients with stable COPD, patients with acute exacerbated COPD, and age, weight and gender matched controls with normal lung function.

The origin of circulating VEGF:

Platelets have been proposed as the major source for circulating VEGF [START_REF] Gunsilius | Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood[END_REF]. VEGF is released from platelets during blood clotting [START_REF] Webb | Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease[END_REF], hemostatic plug formation [START_REF] Weltermann | Large amounts of vascular endothelial growth factor at the site of hemostatic plug formation in vivo[END_REF], and in response to inflammatory stimuli [START_REF] Caine | Platelet activation, coagulation and angiogenesis in breast and prostate carcinoma[END_REF]. VEGFser concentrations in the present report, however, were significantly higher than VEGFprp levels, despite a higher platelet count in the latter. These findings, suggest that in addition to platelets, a further VEGF pool exists in serum that cannot be explained by platelet activation alone. In a previous report from our laboratory, Mittermayer et al. [START_REF] Mittermayer | Marked increase in vascular endothelial growth factor concentrations during Escherichia coli endotoxin-induced acute inflammation in humans[END_REF] observed a significant increase in circulating VEGF levels in healthy volunteers after experimentally induced systemic inflammation. VEGF levels in that report were related to markers of neutrophil activation rather than clot formation, suggesting a mechanism of VEGF release different from that of the coagulation cascade. There is cumulating evidence that neutrophils contribute to circulating VEGF [START_REF] Gaudry | Intracellular pool of vascular endothelial growth factor in human neutrophils[END_REF]22]. According to Kusumanto and co-workers [22] approximately 60% of total circulating VEGF resides in neutrophils, whereas only 34% resides in platelets. Although we have not assessed the relative contribution of neutrophils to VEGF liberation into circulation, we observed a strong relationship between VEGFser levels and PBNC, which may support a role for neutrophils in blood-borne VEGF. In disease conditions, the amount of neutrophil-derived VEGF may further increase. Neutrophils may secrete VEGF in response to acute inflammatory stimuli associated with viral [START_REF] Ghildyal | Rhinovirus infects primary human airway fibroblasts and induces a neutrophil chemokine and a permeability factor[END_REF] and bacterial infections [START_REF] Van Der Flier | Streptococcus pneumoniae induces secretion of vascular endothelial growth factor by human neutrophils[END_REF], which are frequently associated with exacerbations of COPD.

Vascular endothelial growth factor in patients with COPD VEGF is a potent mediator of angiogenesis, which has multiple effects in lung development and physiology. An increasing number of reports suggest an important role for VEGF in the pathogenesis of COPD [START_REF] Kasahara | Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema[END_REF][START_REF] Kranenburg | Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Kanazawa | Possible effects of vascular endothelial growth factor in the pathogenesis of chronic obstructive pulmonary disease[END_REF]. Kasahara and co-workers [START_REF] Kasahara | Inhibition of VEGF receptors causes lung cell apoptosis and emphysema[END_REF] previously demonstrated that blockade of the VEGF receptor in rats resulted in emphysema via induction of alveolar septal cell apoptosis. Kanazawa and co-workers [START_REF] Kanazawa | Possible effects of vascular endothelial growth factor in the pathogenesis of chronic obstructive pulmonary disease[END_REF] Few recent studies also investigated circulating VEGF levels in patients with COPD [START_REF] Hurst | Use of plasma biomarkers at exacerbation of chronic obstructive pulmonary disease[END_REF][START_REF] Cheng | Increased expression of placenta growth factor in chronic obstructive pulmonary disease[END_REF][START_REF] Aldonyte | Analysis of systemic biomarkers in COPD patients[END_REF][START_REF] Kierszniewska-Stepien | Serum vascular endothelial growth factor and its receptor level in patients with chronic obstructive pulmonary disease[END_REF]. The range of VEGFser concentrations observed in our study population are comparable to those of previously published reports [START_REF] Cheng | Increased expression of placenta growth factor in chronic obstructive pulmonary disease[END_REF][START_REF] Aldonyte | Analysis of systemic biomarkers in COPD patients[END_REF]. Consistent with our findings, Cheng et al [START_REF] Cheng | Increased expression of placenta growth factor in chronic obstructive pulmonary disease[END_REF] have recently shown no significant differences in circulating VEGF concentrations between patients with stable moderate to severe COPD (FEV1% pred 47 ± 16%; VEGFser 282 ± 13pg/ml) and matched controls (VEGFser 318 ± 19pg/ml) without airflow obstruction. Kierszniewska-Stepien and co-workers [START_REF] Kierszniewska-Stepien | Serum vascular endothelial growth factor and its receptor level in patients with chronic obstructive pulmonary disease[END_REF] similarly reported no significant differences in VEGFser between controls and stable patients with severe COPD, however, the authors observed overall higher VEGFser levels in patients with mild COPD.

The latter findings suggest differential VEGF expression at different levels of disease severity. This hypothesis is further supported by the significant positive relationship between VEGFser levels and FEV1 observed in our report. Although it has yet to be determined whether VEGF concentrations in the blood mirror expressions of the cytokine in induced sputum samples, our findings may suggest a similar association between VEGF concentrations and airflow obstruction in patients with emphysematous type of COPD as reported previously [START_REF] Kanazawa | Possible effects of vascular endothelial growth factor in the pathogenesis of chronic obstructive pulmonary disease[END_REF].

Compared to stable COPD, patients with acute exacerbated COPD in the present report demonstrated higher circulating concentrations of VEGF and increased systemic inflammation. We furthermore observed a significant relationship between markers of systemic inflammation, such as c-reactive protein, interleukin-6 and fibrinogen, with VEGFser levels in acute exacerbated COPD.

Low grade systemic inflammation is common in patients with COPD [START_REF] Gan | Association between chronic obstructive pulmonary disease and systemic inflammation: a systemic review and a meta-analysis[END_REF]. The degree of systemic inflammation associated with COPD tends to increase over time [START_REF] Donaldson | Airway and systemic inflammation and decline in lung function in patients with COPD[END_REF] and is furthermore upregulated during acute exacerbations [START_REF] Hurst | Use of plasma biomarkers at exacerbation of chronic obstructive pulmonary disease[END_REF][START_REF] Wedzicha | Acute exacerbations of chronic obstructive pulmonary disease are accompanied by elevations of plasma fibrinogen and serum IL-6 levels[END_REF]. Systemic inflammation in COPD has been associated with an increased risk of developing CVD [33]. One mechanism for this may be an increase in procoagulatory factors such as IL-6 and fibrinogen, both which have also been linked to VEGF [START_REF] Caine | Platelet activation, coagulation and angiogenesis in breast and prostate carcinoma[END_REF]34,35]. Increased VEGF expression, in turn, may support prothrombotic effects via expression of the plasminogen activator inhibitor, von Willebrand factor, and tissue factor [36,37]. In fact, there is evidence that circulating VEGF levels are elevated in patients with acute coronary syndrome [START_REF] Soeki | Serial changes in serum VEGF and HGF in patients with acute myocardial infarction[END_REF][START_REF] Heeschen | Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes[END_REF]. In a report by Heeschen [START_REF] Heeschen | Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes[END_REF], patients with acute myocardial ischemia and serum VEGF levels above 500 pg/ml had the lowest 6-month survival rates. Similarly, Slevin and co-workers A recent report [START_REF] Hurst | Use of plasma biomarkers at exacerbation of chronic obstructive pulmonary disease[END_REF] similarly assessed several circulating biomarkers including VEGF in patients with COPD during a stable state as well during an exacerbation. In contrast to our findings, however, the authors did not observe a significant difference in median plasma VEGF between baseline (0.01 pg/ml) and exacerbation levels (0.01 pg/ml). Several methodological reasons may account for the discrepancy between our findings: 1. The authors have measured only free plasma levels of VEGF, and may therefore have missed to assess the contribution of platelets and neutrophils to circulating VEGF levels. 2. Hurst and co-workers used an antibody microarray to assess 36 different biomarkers in their patients. Compared to conventional ELISA, the former is basically a qualitative exploratory technique with overall lower sensitivity particularly in the low picogramm range, whereas the latter is a more accurate method to specifically quantify protein levels [39,40]. Moreover, the ELISA results in our report were verified by duplicates in all cases. 3. All patients in the present protocol were admitted to hospital because of acute exacerbated COPD, whereas only 3% of the patients studied by Hurst et al. [START_REF] Hurst | Use of plasma biomarkers at exacerbation of chronic obstructive pulmonary disease[END_REF] required hospitalization, indicating differences in exacerbation severity and therefore potential differences in the stimulus for VEGF release between the studies. Finally, we observed that recovery from an exacerbation resulted in a reduction of circulating VEGF levels and systemic inflammatory markers in patients with COPD. Similar results were obtained by McColley et al. [41], who have shown that antibiotic therapy decreased VEGF levels and inflammatory markers in patients with cystic fibrosis. Since all of our patients received systemic steroids and most received antibiotic therapy, we have to acknowledge that we cannot identify the effects of either on VEGF or systemic inflammation. Furthermore the impact of the observed changes in VEGFser levels on patient outcomes has yet to be determined in logitudinal studies.

Potential limitations of the study

On a molecular level hypoxia is an important stimulus for VEGF production [START_REF] Ferrara | Molecular and biological properties of vascular endothelial growth factor[END_REF], however, we did not observe a relationship between circulating VEGF levels and arterial oxygenation in Circulating VEGF in COPD p. 11 our report. In this context it has to be acknowledged that the hypoxic stimulus for VEGF production was diminished, as practically half of the COPD patients were on LTOT. On the other hand, there is experimental evidence that both platelets and neutrophils, the predominant sources of circulating VEGF, do not release VEGF in response to hypoxia [42]. In addition, it has previously been shown that the transcription of VEGF may be inhibited in patients with chronic hypoxia [43]. Thus, we suggest that hypoxemia associated with COPD seems to not be an independent stimulus for circulating VEGF.

We furthermore have to acknowledge that we did not perform echocardiography to rule out pulmonary hypertension, a potential confounder of circulating VEGF levels in patients with COPD. Tanaseanu and colleagues [44] recently reported elevated VEGF plasma concentrations in COPD patients with pulmonary hypertension compared to those without.

Nevertheless, given the close matching of the patient groups and the strong association between changes in systemic inflammation and circulating VEGF levels, it is unlikely that the presence of pulmonary hypertension may have significantly biased our findings.

Conclusions

Our findings suggest up-regulation of systemic inflammation and circulating VEGF levels in patients with acute exacerbated COPD. Given the compelling evidence implicating vascular factors in the pathogenesis of both chronic lung and cardiovascular disease processes, VEGF may be an important factor linking COPD to cardiovascular pathology.

  extended these observations, as they have shown differential VEGF expression in induced sputum samples from patients with COPD of different phenotypes. While the authors reported significantly lower VEGF sputum concentrations in emphysematous type of COPD, they observed significantly higher VEGF levels in patients with the bronchitis phenotype. Likewise there was either a positive or a S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 29 Feb 2008 as manuscript CS20070382 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070382 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical SocietyCirculating VEGF in COPD p. 9 negative relationship between FEV1 and VEGF sputum concentrations in emphysema or chronic bronchitis patients, respectively, suggesting a different role of angiogenesis in each phenotype.

  [38] demonstrated elevated Circulating VEGF in COPD p. 10 concentrations of VEGFser (588 ± 121pg/ml) in patients with acute ischemic stroke. Expression of VEGF in that report correlated with both infarct volume and clinical disability. Levels of VEGF detected in acute exacerbated COPD are comparable to those associated with individuals at high risk for cardiovascular morbidity and mortality. The significant relationship in our study between CRP -a marker strongly predictive for the development and prognosis of CVD -and VEGFser further supports this relationship.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 29 Feb 2008 as manuscript CS20070382 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070382 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 29 Feb 2008 as manuscript CS20070382 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070382 Circulating VEGF in COPD p. 16 33. Sin, D.D. and Man. S.F. (2003) Why are patients with chronic obstructive pulmonary disease at increased risk of cardiovascular disease? The potential role of systemic inflammation in chronic obstructive pulmonary disease. Circulation 107, 1514-1519 34. Cohen, T., Nahari, D., Cerem, L.W., Neufeld, G. and Levi, B.Z. (1996) Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 271, 736-741 35. Valipour, A., Litschauer, B., Mittermayer, F., Rauscher, H., Burghuber, O.C. and Wolzt, M. (2004) Circulating plasma levels of vascular endothelial growth factor in patients with sleep disordered breathing. Respir Med 98, 1180-1186 36. Nemerson Y. (1988) Tissue factor and hemostasis. Blood 71,1-8 37. Brock, T.A., Dvorak, H.F. and Senger, D.R. (1991) Tumor-secreted vascular permeability factor increases cytosolic Ca 2+ and von Willebrand factor release in human endothelial cells. Am J Pathol 138,213-221 38. Slevin, M., Krupinski, J., Slowik, A., Kumar, P., Szczudlik, A. and Gaffney, J. (2000) Serial measurment of vascular endothelial growth factor and transforming growth factor-beta 1 in serum of patients with acute ischemic stroke. Stroke 31, 1863-1870 39. Angenendt, P. (2005) Progress in protein and antibody microarray technology. Drug Discov Today 10, 503-11 40. Kusnezow, W. and Hoheisel, J.D. (2002) Antibody microarrays: promises and problems. Biotechniques Suppl, 14-23 41. McColley, S.A., Stellmach, V., Boas, S.R., Jain, M. and Crawford, S.E. (2000) Serum vascular endothelial growth factor is elevated in cystic fibrosis and decreases with treatment of acute pulmonary exacerbation. Am J Respir Crit Care Med 161,1877-1880 42. Levy AP. (1999) A cellular paradigm for the failure to increase vascular endothelial growth factor in chronically hypoxic states. Coron Artery Dis 10, 427-430 43. Koehne, P., Willam, C., Strauss, E., Schindler, R., Eckardt, K.U. and Buhrer, C. (2000) Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets. Am J Physiol Heart Circ Physiol 279, H 817-824 44. Tanseanu, C., Tudor, S., Tamsulea, I., Marta, D., Manea, G., Moldoveanu, E. (2007) Vascular endothelial growth factor, lipoprotein-associated phospholipase A2, sPselectin and antiphospholipid antibodies, biological markers with prognostic value in pulmonary hypertension associated with chronic obstructive pulmonary disease and systemic lupus erithematosus. Eur J Med Res 12, 145-151 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 29 Feb 2008 as manuscript CS20070382 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070382

Figure 1 -

 1 Figure 1 -VEGFser concentrations in patients with acute exacerbated COPD at admission and clinical recovery

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2008 The Authors Journal compilation © 2008 Biochemical SocietyCirculating VEGF in COPD p.[START_REF] Heeschen | Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes[END_REF]

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 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 SocietyCirculating VEGF in COPD p.[START_REF] Weltermann | Large amounts of vascular endothelial growth factor at the site of hemostatic plug formation in vivo[END_REF]

Acknowledgements

We thank Afrus Valipour for her help in the preparation and editing of the manuscript. We are grateful to Brigitte Litschauer for her help in laboratory analysis. This work was supported by a grant from the "Stiftung zur Förderung der Bekämpfung der Tuberkulose und anderer Lungenerkrankungen" (Foundation to support the fight against tuberculosis and other lung diseases).

List of abbreviations