Forte Amalia
email: amalia.forte@unina2.it

Finicelli Mauro

De Luca Pasquale

Nordström Ina

Onorati Francesco

Quarto Cesare

Santè Pasquale

Renzulli Attilio

Galderisi Umberto

Berrino Liberato

De Feo Marisa

Hellstrand Per

Rossi Francesco

Cotrufo Maurizio

Cascino Antonino

Cipollaro Marilena

Amalia Forte

INJURY TO RAT CAROTIDS CAUSES TIME-DEPENDENT CHANGES IN GENE EXPRESSION IN CONTRALATERAL UNINJURED ARTERIES

Keywords: vascular injury, remodelling, microarrays, inflammation, blood flow, Renin-Angiotensin System

Vascular surgery aimed at stenosis removal induces local reactions often leading to restenosis. While extensive analysis has been focused on pathways activated in injured arteries, little attention has been devoted to associated systemic vascular reactions. Aim of this study was to analyse changes occurring in contralateral uninjured rat carotids in the acute phase following unilateral injury.

WKY rats were submitted to unilateral carotid arteriotomy. Contralateral uniunjured carotids were harvested from 4 hrs to 7 days after injury. Carotids were also harvested from sham-operated rats and from uninjured rats. Carotid morphology and morphometry were examined. Affymetrix microarrays were used for differential analysis of gene expression. A subset of data was validated by Real-Time RT-PCR and verified at protein level by Western Blot.

1011 genes were differentially regulated in contralateral uninjured carotids from 4 hrs to 7 days after arteriotomy (p<0.0001, fold-change ≥2) and were classified in 19 gene ontology functional categories. To a lesser extent, mRNA variations occurred also in carotids of sham-operated rats.

Among the changes, up-regulation of members of the Renin-Angiotensin System was detected, with possible implications for vasocompensative mechanisms induced by arteriotomy. In particular, a selective increase of the N-domain Angiotensin Converting Enzyme 69-kDa isoform, and not the classical somatic 195 kDa isoform, was highlighted in contralateral uninjured carotids, suggesting that this 69-kDa isoenzyme could influence the local Angiotensin II production.

In conclusion, systemic reactions to injury occur in the vasculature, with potential clinical relevance and suggesting caution in the choice of control during experimental design in vivo.

INTRODUCTION

Surgical procedures to correct atherosclerotic arterial narrowing are always associated with some degree of injury to the vessel wall, which carries the risk of reocclusion of the vessel because of neointima and remodeling stimulated by the injury. This remains a serious problem despite recent advances in the use of drug-eluting stents [START_REF] Camenzind | Treatment of in-stent restenosis--back to the future?[END_REF]. Accordingly, the restenosis problem has been addressed by several approaches, including large-scale transcriptome analysis, to characterize patterns in the vascular response to injury with the aim to identify possible targets for therapy.

Animal studies and clinical observations indicate that change in carotid blood flow subsequent to unilateral carotid stenosis and/or endarterectomy or angioplasty have effects on flow in contralateral carotids [START_REF] Van Everdingen | MRA flow quantification in patients with a symptomatic internal carotid artery occlusion. The Dutch EC-IC Bypass Study Group[END_REF]. This serves to compensate for the disturbed blood flow, but may also affect the structure and function of the uninjured artery, which may have significant implications for the comparison of injured vs. contralateral uninjured arteries, as is often done. In addition, it is well known that vascular injury triggers the release in the plasma of a number of molecular mediators, including cytokines and growth factors, that can trigger a systemic pathophysiological effect which may be important for the clinical outcome. Observations in contralateral uninjured arteries following unilateral carotid surgery would be expected to elucidate such systemic effects. In previous studies [START_REF] Forte | Stenosis progression after surgical injury in Milan hypertensive rat carotid arteries[END_REF] we set up a model of rat carotid arteriotomy that mimics the injury affecting the arteries of patients submitted to endarterectomy rather than the trauma induced by standard balloon angioplasty. Our investigations, together with other studies based on a deep vascular injury [START_REF] Andersen | Remodeling rather than neointimal formation explains luminal narrowing after deep vessel wall injury: insights from a porcine coronary (re)stenosis model[END_REF][START_REF] Okamoto | Perivascular inflammation after balloon angioplasty of porcine coronary arteries[END_REF], support the key role of adventitia and of perivascular tissues in arterial stenosis. Recently, we reported time-resolved expression profiles from carotid arteries subjected to arteriotomy [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. Here we have used microarrays to analyse the time-dependent transcriptome variations in contralateral uninjured rat carotids after arteriotomy.

The results reveal that substantial changes in gene expression occur in these vessels and in shamcontrol carotids, thus implying a systemic reaction of the vasculature to surgery.

Microarray hybridization and analysis:

15 µg of each biotinylated cRNA were hybridized to Genechips 230 2.0 (Affymetrix). Microarray hybridization and analysis were performed as previously published [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. Results were filtered for flag (presence call), then for fold-change (FC) ≥2, obtaining a total of 15600 probe sets differentially expressed in the different conditions.

Statistical analysis was performed by the two-way ANOVA using a parametric test with variances assumed equal, p-value cut-off 0.0001. The Bonferroni multiple testing correction was applied. The false discovery rate under these conditions was proximal to zero (Supplemental file 1). Final data comply with the Minimum Information About a Microarray Experiment (MIAME) requirements and have been loaded in the EMBL-European Bioinformatics Institute (EBI) public database with the accession number E-MEXP-1278 (www.ebi.ac.uk). Differentially expressed genes were grouped for similar biological processes according to gene ontology (GO) definitions (www.geneontology.org) (supplemental file 2). A gene was associated with a gene ontology term if it was annotated by this term or by its child. Hierarchical clustering was performed on the gene lists by the Gene Tree algorithm using as similarity measure the Pearson correlation and the clustering algorithm average linkage.

Significant biological pathways implicated in time-dependent carotid reaction to arteriotomy and gene product association networks have been identified through DAVID (Database for Annotation, Visualization and Integrated Discovery) at http://david.abcc.ncifcrf.gov and by BioRag (Bioresource for array genes) at http://www.biorag.org (Supplemental files 3, 4).

Quantitative real-time RT-PCR

Quantitative real-time PCR (MJ Opticon II, Bio-Rad) was used to determine the copy number of mRNA for 8 genes (c-myc, AT1, ACE, Clcf1, Agt, Id2, Mcp-1, IL-1β) in contralateral uninjured carotids in comparison to carotids from uninjured rats. Real Time RT-PCR was performed as previously published [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. Validation of the microarray data was considered to be a significant (p<0.05) change in normalized PCR copy number in the same direction found in the microarray data. determine the fold difference for genes. The PCR efficiency was determined for each primer pair and was calculated using a dilution series and MJ Opticon II analysis software.

Western blot

Contralateral uninjured carotid arteries were harvested at 4 hrs (n=3), 48 hrs (n=3= and 7 days (n=4) after arteriotomy. Carotids from uninjured rats (n=3) and from sham-control rats (n=3) were also harvested. The vessels were immediately frozen in liquid nitrogen and then stored at -80°C. The frozen vessels were pulverized in liquid nitrogen and dissolved in Laemmli SDS-sample buffer. Protein concentrations were determined using EZQ Protein Quantitation assay (Molecular Probes).

Proteins were separated by SDS polyacrylamide gel electrophoresis using the Criterion precast gel system (Biorad). The same amount of protein (20 µg) was loaded in each lane. The amounts of Arginase I and Angiotensin Converting Enzyme (ACE) were determined by Western blot (WB) using antibodies from BD Bioscience, Pharmingen (Arginase I) and Santa Cruz Biotechnology (ACE H-170). Bands were visualized using enhanced chemiluminescence (Pierce) and analyzed with Quantity One software (Bio-Rad).

Statistical analysis

All statistical analysis was performed using GraphPad software (Prism 4.0). Statistical significance was determined using two-way ANOVA followed by Bonferroni's multiple comparison test.

RESULTS

Histological analysis of arteriotomy-injured carotids and of contralateral uninjured carotids:

Morphological analysis of cross-sections of carotids harvested from 4 hrs up to 30 days after arteriotomy showed changes mainly related to remodeling phenomena, while limited focal neointima was evident only in a few cases. Morphometric analysis revealed a progressive lumen reduction, with a maximal 60±9% narrowing 30 days after arteriotomy. Conversely, no morphological or morphometric differences among contralateral uninjured carotids harvested at different time points after arteriotomy were evident (data not shown).

Microarray transcriptome analysis in contralateral uninjured carotids at different time points after unilateral arteriotomy:

The microarray data showed consistency in gene expression amongst the replicates within each experimental group, but evidence of substantial transcriptional differences in contralateral uninjured carotids at different time points after unilateral arteriotomy was found. In Supplementary file 1 are listed a total of 1011 mRNA from uninjured carotids that were significantly affected at least at one time point after arteriotomy. Gene names, gene descriptions, biological function, Affymetrix Ids, accession numbers and expression levels are provided.

In uninjured carotids harvested at 4 hrs, 48 hrs and 7 days after arteriotomy, 728, 480 and 455 mRNAs, respectively, were significantly altered. Among the 1011 arteriotomy-modulated genes, the biological functions of 522 genes are known (Supplementary File 2). The remaining 489 genes were expressed sequence tags (ESTs), also listed in Supplementary file 2. In general, mRNA changes occurring in contralateral uninjured carotids after arteriotomy overlapped with changes detected in injured carotids relative to basal mRNA levels in uninjured rat carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF], but with a lower FC magnitude. In a few cases, mRNA expression trends showed an opposite behaviour in injured and contralateral uninjured carotids.

Heat map in Fig. 1 highlights, through a reference colour scale (green = 0; red = 5), the different temporal trends exhibited by GO functional categories. For example, genes included in cytoskeleton and contractile apparatus GO category clearly showed a biphasic up-regulation at 4 hrs and 7 days after arteriotomy, while other groups of genes (e.g. nucleic acid metabolism and protein metabolism) exhibited a marked up-regulation only at 4 hrs after arteriotomy and then gradually decreased to basal levels. Fig. 2 highlights the number of modulated genes for each GO functional category at different time points after arteriotomy in contralateral uninjured carotids. Genes included in each GO functional category are listed and described in Supplementary file 2. show similar data for the functional GO categories immune response, cytoskeleton and contractile apparatus, signal transduction and neurophysiological process, respectively. Graphs in Fig. 3F-L report the number of up-and down-regulated genes within the corresponding data sets. These graphs reveal a generally greater FC in arteriotomy-injured carotids, together with a greater number of activated genes and a more persistent activation in injured carotids in comparison with contralateral uninjured carotids. The graphs we selected for Fig. 3 reflect a general behavior observed for all the 19 GO functional categories we identified (Fig. 2).

Graphs in

Microarray data have been further analyzed to identify significant functional pathways activated after unilateral arteriotomy. A total of 15 most significant KEGG pathways (p<0.1) have been identified by conjunct application of DAVID and BioRag (Table 1, Supplementary Fig. 3). A modified Fisher Exact p-value (0<p<1) is provided for each functional KEGG pathway. The smaller the p-value, the more over-represented (enriched) will this pathway be in the gene list, relative to random selection. Supplementary file 4 includes the full list of genes included in each most significant pathway. The gene product association networks extracted from the 15 significant KEGG pathways are shown in Supplementary Fig. 4A and4B. The core of the network in Supplementary Fig. 4A is constituted by genes involved in MAPK signaling (green line), focal adhesion (yellow line) and cytokine-cytokine receptor interaction (red line).

Of interest, we identified a subgroup of 21 mRNAs that were significantly modified exclusively in contralateral uninjured and not in arteriotomy-injured carotids (Supplementary file 5). 12 out of the 21 mRNAs had a well-described biological function in rat, and of these 7 were up-regulated and 5 down-regulated after arteriotomy. ACE was selectively up-regulated in uninjured contralateral carotids at 48 hrs and 7 days after arteriotomy (FC 4.57 and 5.61, respectively). The marked upregulation of ACE in uninjured carotids could be responsible for an increase of local Angiotensin (Ang) II production. We also detected a marked 8.63-fold increase of Angiotensinogen (Agt) mRNA in contralateral carotids harvested 4 hrs after arteriotomy vs. a slight 2.12-fold increase measured in injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. Finally, the mRNA coding for the AT1 receptor for Ang II showed a comparable 3.39 and 4.37-fold increase in uninjured and injured carotids harvested 4 hrs after arteriotomy, respectively. Despite the subset of 21 genes selectively varied in contralateral uninjured carotids, we did not detect substantial differences in KEGG pathways identified in contralateral carotids in comparison to arteriotomy-injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. This is because the 12 selectively modified genes with described biological functions were found to belong to different functional pathways. The trend of Real Time RT-PCR results overlapped with the microarray data, although the magnitude of the FC was not identical (Table 2). For some genes, the Real Time RT-PCR revealed additional significant changes that were not included among microarray data. This could be related to the greater sensitivity of Real Time PCR compared to microarrays, or to filtering out of some results during statistical analysis due to the standard deviation in microarray replicates.

Western Blot verification of microarray data:

We verified by WB the expression levels of ACE and Arginase I on lysates from contralateral uninjured carotids at 4 hrs, 48 hrs and 7 days after arteriotomy, as well as from sham-control carotids and from carotids of uninjured rats. Results were normalised with respect to GAPDH house keeping gene level. N-domain ACE (69-kDa) showed a 2.24-fold increase in sham-control carotids and a 2.1-and 1.85fold increase in contralateral uninjured carotids 4 hrs and 48 hrs after arteriotomy. The expression of N-domain ACE then turned back to basal levels 7 days after unilateral arteriotomy (Fig. 4A). We did not detect the 195-kDa somatic isoform of ACE in carotid lysates, which was however present in control rat kidney lysate, together with the N-domain 69-and 136-kDa ACE isoforms (data not shown). The expression trend of the protein partially overlapped with that of mRNA, showing a significant 4.54-and a 5.61-fold increase in contralateral carotids harvested 48 hrs and 7 days after unilateral arteriotomy, respectively. In sham-control carotids and in contralateral uninjured carotids harvested 4 hrs after arteriotomy, ACE mRNA increased but showed a variability that caused this to be filtered out during statistical analysis. WB analysis revealed a 2.53-fold increase of Arginase I in contralateral uninjured carotids harvested 4 hrs after arteriotomy and a 4.28-fold increase in sham-control carotids (Fig. 4B). The expression of Arginase I was then back to basal levels at 48 hrs and 7 days after arteriotomy. The expression trend of the protein partially overlapped with that of mRNA, showing a significant 2.73-fold increase 48 hrs after arteriotomy, and a significant 1.7-fold increase in sham control carotids (not shown in microarray results as we selected a FC ≥ 2).

Microarray transcriptome analysis of sham-control carotids:

Only 149 mRNAs were significantly altered in sham-control carotids harvested at 30 min after surgery (Supplementary File 1). In this case, arteries were not manipulated and the vagus nerve not dissected. The majority of them were assigned to the inflammatory response and to the cytoskeleton and contractile apparatus (Supplementary file 2). Pathway analysis revealed a significant activation of fatty acid synthesis, ECM-receptor interaction and cytokine-cytokine receptor interaction in shamcontrol carotids (p<0.1).

Interestingly, a subset of mRNAs from sham-control carotids showed an expression trend opposite to that observed in injured carotids and/or in contralateral uninjured carotids. In particular, this was the case for mRNAs involved in inflammatory response (e.g. the macrophage inflammatory protein-1 alpha receptor, IL-6, the serine peptidase inhibitors Spin2c and Serpinb2, RT1-Ba, the chemokine ligand 9) and of other heterogeneous factors (e.g. c-fos, Cox6a2, Scn9a, Phosphoglycerate mutase 2).

These differences in mRNA expression between sham-control carotids and contralateral uninjured carotids may be related to feed-back regulative mechanisms and to the fact that sham-control carotids were harvested at 30 min after skin injury and carotid exposure, while contralateral carotids were harvested not earlier than 4 hrs after arteriotomy. High-density microarrays identified a large number of mRNA variations in contralateral uninjured carotids and sham-control carotids in a model of rat arteriotomy, allowing the dissection of patterns that could not have been discovered through analysis of individual genes. Microarrays were used in association with a method developed for mRNA linear amplification [START_REF] Eberwine | Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)-T7 primed cDNA[END_REF] that allowed the analysis of low amounts of RNA without affecting the fidelity of detection of differential gene expression and the false positive rate [START_REF] Patel | Widespread delta-like-1 expression in normal adult mouse tissue and injured endothelium is reflected by expression of the Dll1LacZ locus[END_REF]. Sample pooling before RNA extraction was necessary because of the low yield of RNA obtained from single samples, as we extracted RNA only from small carotid segments corresponding to those submitted to arteriotomy (less then 2 mm in length). Carotid pooling allowed the recovery of a sufficient amount of RNA for check of sample integrity and DNA absence, microarray hybridisation and Real Time RT-PCR for data validation. We used a large base of biological samples (n=15 rats for each time point) and stringent parameters to filter the data to avoid false gene identification and assign a level of significance to the overall results. Histological analysis did not reveal any significant morphological or morphometric change in contralateral uninjured carotids harvested at different time points after unilateral arteriotomy. This is in agreement with findings recently reported in a model of mouse carotid artery ligation, in which authors did not detect any proliferative activity in contralateral unligated vessels, but a prominently increased expression of Delta-like 1 gene [10].

Among the most significant KEGG pathways activated in contralateral uninjured carotids are those related to inflammation (e.g. antigen processing and presentation, B cell receptor signalling pathway, cytokine-cytokine receptor interaction), to cell-ECM adhesion and to the MAPK signalling pathway (Supplementary Fig. 3A). Of note, these pathways are not substantially different from those identified in arteriotomy-injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF].

Comparative analysis of GO categories in injured and contralateral uninjured carotids shown in Fig. 3 highlights differences in relative magnitude of FC and timing of variations of gene expression. In particular, the overall results show a lower mean FC value at all time points after arteriotomy in contralateral uninjured carotids than in injured carotids (Fig. 3A andF), together with differences in the number of regulated genes, most evident at 7 days after injury, when the majority of transcriptional activity in the contralateral artery, but not in injured carotids, has settled down. This indicates that variations of gene expression in contralateral uninjured carotids are transient. The overall results shown in Fig. 3A and F are similar to those obtained in the single GO categories (Fig. 3B-E), with the exception of the GO category "cytoskeleton and contractile apparatus", (Fig. 3D), in which the mean FC value rises again at 7 days after arteriotomy in contralateral uninjured carotids.

The genes and the pathways activated in contralateral uninjured carotids are presumably strictly related to changes occurring in arteriotomy-injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. Cytokines secreted by arteriotomyinjured vessels and by perivascular tissues may produce systemic effects detectable at molecular level at distal sites, as in our study, and could be predictable of future events of clinical relevance. In this context, Raman et al. [START_REF] Raman | Disease progression in contralateral carotid artery is common after endarterectomy[END_REF] showed that contralateral uninjured carotids of a cohort of patients submitted to unilateral endarterectomy had an annual rate of stenosis progression double that of ipsilateral carotids after endarterectomy. These authors did not identify any clinical or demographic risk factors associated with contralateral disease progression.

Other studies demonstrated that vascular injury causes changes in tissues of normal appearance located at a distance from the injury site. In particular, it has been demonstrated that SMC proliferation occurs at sites distant from vascular injury in aorta [START_REF] Reidy | Proliferation of smooth muscle cells at sites distant from vascular injury[END_REF]. Moreover, patients with acute myocardial infarction showed a severe vasodilator abnormality involving not only resistance vessels in infarcted myocardium, but also those in myocardium perfused by normal coronary vessels [START_REF] Uren | Reduced coronary vasodilator function in infarcted and normal myocardium after myocardial infarction[END_REF].

Finally, it has been demonstrated that renal artery manipulation causes a centripetal spread of endothelial cell replication along the periureteric collateral artery [START_REF] Hollenberg | Centripetal spread of arterial collateral endothelial cell hyperplasia after renal artery stenosis in the rat[END_REF]. The mediators of the above mentioned distal effects could be humoral vasoactive factors released immediately after the injury, able to play autocrine, paracrine and systemic effects. We have previously demonstrated that carotid arteriotomy induces synthesis of a number of mRNAs coding for cytokines, chemokines, hormones and growth factors, including IL-6 and Monocyte chemotactic protein (MCP-1/Ccl2) [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. These two factors appear of particular interest for their effect on contralateral uninjured carotids, as they could play a regulative role through a feedback mechanism. In fact, IL-6 mRNA showed a 135-fold increase in rat carotids 4 hrs after arteriotomy [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF] but a parallel marked decrease in contralateral uninjured and in sham-control carotids. These opposite expression trends could be related to a negative feedback mechanism of regulation of IL-6 mRNA in the cytokine cascade to inhibit excessive acute inflammation [START_REF] Saito | Expression and self-regulatory function of cardiac interleukin-6 during endotoxemia[END_REF]. Conversely, the increase of MCP-1 mRNA both in injured and in contralateral carotids was presumably due to a positive loop induced by circulating MCP-1 released at the arteriotomy site, as suggested by data obtained in human CD14-positive monocytes [START_REF] Sakai | MCP-1/CCR2-dependent loop for fibrogenesis in human peripheral CD14-positive monocytes[END_REF].

The activation of mRNAs involved in the inflammatory reaction in contralateral uninjured carotids could be presumably related not only to the release of systemic mediators from the injury site, but also to altered blood flow, that has been recently demonstrated to be able to trigger an inflammatory response in mouse mesenteric arteries [START_REF] Bakker | Blood flow-dependent arterial remodeling is facilitated by inflammation but directed by vascular tone[END_REF].

Finally, it can be also hypothesized that the vascular injury induced a marked inflammatory reaction triggering the priming of circulating leukocytes, which in turn can act as proatherogenic agents at local inflammatory sites. Similar mechanisms have been hypothesized in different vascular diseases [START_REF] Kolb | Presensitization accelerates allograft arteriosclerosis[END_REF][START_REF] Jacobi | Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury[END_REF].

Among other pathways and processes activated in contralateral uninjured carotids, we detected the activation of a large number of genes involved in neurophysiological processes (e.g. Dbh, Nefl, Pcp4, Nef3, Syt1). In particular, Dbh and Nef3 mRNAs at 4 hrs after arteriotomy had increased much more than in injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF], probably as a part of a neurocompensatory reaction to vascular injury.

Concomitantly, there was a massive decrease of the expression of genes involved in muscle contraction (e.g. troponin T2, myosin heavy chain polypeptides 3 and 8). In agreement with our data, Milner et al. [START_REF] Milner | Neurocompensatory responses to balloon-catheter-induced injury of the rat carotid artery[END_REF] detected neurocompensatory responses to balloon injury in rat carotids, comprising damage to the perivascular nerves and a transient increase in the density of sensory neuropeptidecontaining nerves innervating the contralateral, uninjured side.

Microarray data indicated also an up-regulation of mRNAs involved in oxidative stress, in particular of NAD(P)H oxidase subunits 22phox, gp91phox and Ncf4 48 hrs and 7 days after arteriotomy. Moreover, we measured a 4-fold increase of the small GTPase Rac2 in contralateral uninjured carotids 7 days after arteriotomy. Sod2, which exerts an antioxidant activity, showed a transient slight increase 4 hrs and 48 hrs after arteriotomy, followed by a marked decrease 7 days after injury.

In an attempt to distinguish between variations of gene expression specific for contralateral carotids and early systemic reactions of the vasculature to surgical procedures, we analysed gene expression variations also in sham-control carotids harvested 30' after skin incision and carotid exposure. The mRNA variations in sham-control carotids indicated that the carotid exposure, even in the absence of carotid manipulation and vagus nerve dissection, was sufficient to trigger a rapid reaction in the vasculature, both at mRNA and protein level (Fig. 4; Supplementary file 1). The two major GO categories activated in sham-control carotids signalled an early systemic inflammatory response and activation of the contractile apparatus. The slight changes of mRNAs we observed in sham-operated controls are in contrast with some studies [START_REF] Milner | Neurocompensatory responses to balloon-catheter-induced injury of the rat carotid artery[END_REF][START_REF] Fukada | Perivascular injury leads to a reduction in vascular reactivity of the collared and to an enhancement on contralateral carotid artery of rats[END_REF], but are in agreement with others [START_REF] Connolly | Cyclooxygenase isozyme expression and intimal hyperplasia in a rat model of balloon angioplasty[END_REF][START_REF] Li | Temporal evolution of gene expression in rat carotid artery following balloon angioplasty[END_REF].

Among the most significant KEGG pathways activated in contralateral uninjured carotids is the arginine and proline metabolism (Table 1). Arginase I plays a main role in this pathway, as it acts as a nitric oxide-synthase-alternative pathway for L-Arginine breakdown, leading to biosynthesis of urea and L-ornithine. Arginase I plays a role in the regulation of vascular tone and in vascular dysfunctions and is expressed by numerous cell types; moreover, Arginase I activity is increased by inflammatory molecules [START_REF] Wei | IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells[END_REF]. The increase of Arginase I mRNA and protein in contralateral uninjured and in sham-control carotids overlaps with the increase of Arginase I in arteriotomyinjured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. The greater increase of Arginase I protein in sham-control carotids harvested 30 min after surgery (Fig. 4B) than in contralateral carotids at 4 hrs may indicate that this protein is increased rapidly by a systemic inflammatory reaction, as suggested by Wei et al. [24]. This could also involve a prompt regulation of vascular tone after injury.

Our microarray and Real Time RT-PCR data highlight a preferential up-regulation of mRNAs coding for elements of local Renin-Angiotenin System (RAS) (Agt, AT1 receptor, ACE) in contralateral uninjured carotids relative to arteriotomy-injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. In particular, ACE mRNA showed a marked increase in contralateral uninjured carotids, while it was substantially unchanged in arteriotomy-injured carotids [START_REF] Forte | Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation[END_REF]. The broad spectrum of substrates for ACE and its wide distribution indicates that this enzyme may be involved not only in cardiovascular homeostasis but also in pathophysiological processes. WB integrated these data revealing an increase of the N-domain ACE isoform (69-kDa) in rat carotids and not of the classical somatic 195 kDa ACE isoform (Fig. 4A).

The N-domain 69-kDa and the 136-kDa N-domain ACE isoforms have been described in a variety of tissues, including aorta and heart [START_REF] Ronchi | N-domain angiotensin-converting enzyme isoform expression in tissues of Wistar and spontaneously hypertensive rats[END_REF], suggesting that these isoenzymes could influence local Ang II production, contributing to organ-specific regulation. In particular, the N-domain ACE isoforms are involved in the inactivation of vasodilator Ang1-7, a specific substrate for these isoforms, in addition to the conversion of Ang I to Ang II.

Despite the parallel increase of mRNAs, we cannot exclude that the Arginase I and ACE proteins detected in contralateral carotids derived from plasma residues infiltrating the intact vascular wall with a temporal expression profile related to changes in permeability of the vasculature.

Nevertheless, we detected only the 69-kDa N-domain ACE isoform in carotids, distinct from the ACE soluble isoform released in the plasma, with a molecular size similar to that of human 195-kDa somatic membrane-bound ACE [START_REF] Baudin | Purification and analysis of lung and plasma angiotensin I-converting enzyme by high-performance liquid chromatography[END_REF].

The importance of local vascular RAS is well established. Its activation in contralateral carotids after arteriotomy is in agreement with other studies hypothesizing that a RAS-mediated compensatory response may be caused by an increase in Ang II receptor density [START_REF] Fukada | Perivascular injury leads to a reduction in vascular reactivity of the collared and to an enhancement on contralateral carotid artery of rats[END_REF] and demonstrating that chemical sympathectomy in rats submitted to angioplasty induced an increase of DNA synthesis mediated by Ang II receptors exclusively in contralateral carotids [START_REF] Bruijns | Effects of chemical sympathectomy on angiotensin II-induced neointimal growth in the balloon-injured rat carotid artery[END_REF].

CONCLUSIONS AND PERSPECTIVES

The main findings of this study are that: i) carotid arteriotomy induces an extensive change of transcriptome in contralateral uninjured carotids; ii) local RAS is specifically up-regulated in contralateral uninjured carotids and in sham-control carotids, suggesting a role in the prompt regulation of vascular tone; iii) slight changes at mRNA and protein level have been detected also in sham control-carotids, indicating early systemic effects unrelated to altered carotid blood flow following arteriotomy; iv) contralateral uninjured carotids should be used with caution as reference control in vivo, since considerable changes occur also in these vessels. provided for each functional KEGG pathway is a modified Fisher Exact p-value. The smaller is the pvalue, the more enriched will be the pathway and consequently a gene list will be specifically associated (enriched) in a certain pathway than random chance. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Total RNA integrity was verified by electrophoresis on denaturing 1% agarose gel containing highly sensitive 2x GelStar Nucleic Acid Stain (Lonza) to ensure the absence of sample degradation. Absence of residual DNA was verified by PCR on total RNA without reverse transcription. 200 ng of each RNA sample were amplified and biotin-labeled with two rounds of amplification following the Affymetrix small sample labeling protocol.

Clinical

  Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T

  Fig. 3 highlight representative time-dependent differences between contralateral uninjured carotids and injured carotids. Graphs in Fig. 3A compare mean FC values for overall genechip data in Clinical Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T injured carotids (data from ref. 6) and in contralateral uninjured carotids (present study). Panels B-E

  Clinical Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T Real Time RT-PCR validation of microarray data: Real Time RT-PCR was done on a subset of mRNAs, either up-(c-myc, AT1, ACE, Agt, Mcp-1, IL-1β) or down-regulated (Id2, Clcf1) after arteriotomy. These mRNAs were chosen as representative of pathophysiological processes of interest in our injury model (e,g. immune reaction, nucleic acid metabolism, vascular remodeling), displaying a FC ranging from a high magnitude to near the 2-fold cut-off. Gene expression data were normalized with respect to mRNAs from carotids of uninjured rats.

Clinical

  Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION The analysis of systemic inflammatory and compensatory effects triggered by vascular injury procedures can have clinical relevance and be helpful for a better comprehension of the pathophysiological events occurring during and after surgery.

Clinical

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Clinical

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Clinical

  Science Immediate Publication. Published on 03 Jun 2008 as manuscript CS20080080 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080080 S t a g e 2 ( a ) P O S T -P R I N T

ClinicalFig. 1 :

 1 Fig. 1: Time-dependent expression of genes clustered in functional groups. Each row corresponds to a single gene; each column corresponds to the mean of three different experiments (reference colour scale: green = 0; red = 5).

Fig. 2 :

 2 Fig. 2: Functional GO classification of genes up-regulated (A) and down-regulated (B) at 4 hrs, 48 hrs and 7 days after arteriotomy in contralateral uninjured carotids.

Fig. 3 :

 3 Fig. 3: Mean fold-change (FG) values (A-E) and number of up-and down-regulated genes (L-H) at different time points after arteriotomy in injured and contralateral uninjured carotids for overall genechip results (A, L) and for the GO categories signal transduction, cytoskeleton and contractile apparatus, neurophysiological process and immune response (B-E, G-H). Data for injured carotids are from Ref. 6 and are shown here for comparison.

Fig. 4 :

 4 Fig. 4: Western Blot analysis of ACE and Arginase I expression in carotids from uninjured rats, sham-control rats and in contralateral uninjured carotids harvested at 4 hrs, 48 hrs and 7 days after arteriotomy. Original blots (A) and summarized data normalised with respect to GAPDH housekeeping gene (B) (mean values, n=3) are shown.

ClinicalFigure 1 ClinicalFigure 2 Clinical

 12 Figure 1

Figure 3 Clinical

 3 Figure 3

Figure 4 Clinical

 4 Figure 4

Number of genes Significance KEGG Pathways Time points after arteriotomy Time points after arteriotomy Cellular and regulatory pathways

		4h	48h	7 days	4h	48h	7 days
	Focal adhesion	21	16	12	9.2E-4	3.8E-3	1.4E-2
	ECM-receptor interaction	10		6	7.1E-3		4.2E-2
	Antigen processing and	9	8		6.1E-2	3.8E-2
	presentation
	Cell adhesion molecules (CAMS)	14	10		3.1E-2	9.7E-2
	Hematopoietic cell lineage		8	6		1.5E-2	4.4E-2
	B cell receptor signalling pathway	8		6	4.0E-2		3.1E-2
	Cytokine-cytokine receptor	12	12		9.0E-2	1.4E-2
	interaction
	Toll-like receptors signaling		7	6		3.8E-2	3.7E-2
	pathway
	MAPK signaling pathway	18			4.4E-2
	Complement and coagulation			5			5.7E-2
	cascades
	Cell communication	10			7.4E-2
	Type I diabetes mellitus		7			3.6E-2
	Tgf-beta signaling pathway	8			4.0E-2
	Axon guidance			2			5.7E-2
	Metabolic pathways
	Arginine and proline metabolism			4			7.8E-2

Table 1 :

 1 Most significant KEGG pathways identified by DAVID software in contralateral uninjured carotids harvested at different time points after arteriotomy (p<0.1). The number of genes included in each pathway at different points after arteriotomy and the P values are reported. The p-value

S t a g e 2 ( a ) P O S T -P R I N T 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 Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

To the memory of Prof. Antonino Cascino, deceased 6 April 2008. This work has been partially supported by Swedish Research Council (71X-28), The Swedish Heart Lung Foundation, the Torsten and Ragnar Söderberg Foundations and the Vascular Wall Programme, Lund University, to P.H. and by Ministero della Sanità, PF Sanità 2003 "Patologie infettive e insulto chirurgico: studi di genomica e proteomica nel remodeling vascolare" to A.C.

We are grateful to Ms. Maria Rosaria Cipollaro for technical assistance.

France). All animals were handled

The absence of evident morphological variations in contralateral uninjured arteries despite variations at mRNA and protein levels was probably related to their intact vascular wall, which inhibited cell proliferation and remodelling phenomena.

This study used healthy WKY rats as experimental animal model. Rodent models have the limitation that atherosclerosis-related pathological changes do not occur unless the animals are given special diets and/or have genetic dyslipidemias, as in several genetically modified mouse strains. The current analysis of gene expression following injury to healthy vessels thus represents changes related to the injury by itself without the complication of dyslipidemia or plaque formation. An analysis of differential expression in a model of stenosis induced in hypercholesterolemic animals would be of interest to simulate processes taking place in human atherosclerotic arteries. The vascular wall affected by an atherosclerotic plaque can show basal inflammation, endothelial dysfunction and proliferative activity that may influence not only its reactions to injury, but also the reactions of contralateral uninjured carotids exposed to the same blood and tissue environment.

To our knowledge, this is the first microarray-based analysis of time-dependent gene expression profiling in contralateral uninjured arteries in a model of vascular injury. The results raise questions with regard to potential adverse systemic effects of vascular injury, of possible pathophysiological and clinical significance.

Conflict of interest: none declared

Legends of supplementary files:

Supplementary file 1: Differentially expressed mRNAs in contralateral uninjured rat carotids harvested at 4 hrs, 48 hrs and 7 days after arteriotomy and in sham-control carotids harvested 30' after injury (these data are also available at www.ebi.ac.uk).

Supplementary file 2: Microarray data classification according to their GO biological functions. Supplementary file 3: Networks among genes included in significant (A) cellular and regulatory process and (B) metabolic KEGG pathways activated in contralateral uninjured carotids at 4 hrs, 48 hrs and 7 days after arteriotomy. The core of the network in A is constituted by genes involved in MAPK signalling (green line), cytokine-cytokine receptor interaction (red line) and focal adhesion (yellow line). The most significant pathway in B is arginine and proline metabolism. Faded nodes belong to pathways with a p>0.1. The thickness of the edge is proportional to the number of pathways to which are associated two genes.

Supplementary file 4: Gene classification in KEGG pathways.

Supplementary file 5: mRNAs modified exclusively in contralateral uninjured carotids.


MD Marlies Bosselaar
email: m.bosselaar@aig.umcn.nl

P Smits

C J Tack

Ischemia and insulin, but not ischemia and contraction, act synergistic in stimulating muscle glucose uptake in vivo in humans

Keywords: human in vivo, ischemia, euglycemic hyperinsulinemic clamp, forearm glucose uptake, forearm blood flow

Ischemia and insulin, but not ischemia and contraction, act synergistic in stimulating muscle glucose uptake in vivo in humans

Marlies Bosselaar, Paul Smits,

INTRODUCTION

Ischemia [START_REF] Cartee | Stimulation of glucose transport in skeletal muscle by hypoxia[END_REF], like muscle contraction [START_REF] Kraniou | Effects of exercise on GLUT-4 and glycogenin gene expression in human skeletal muscle[END_REF], induces translocation of intracellular glucose transporters (GLUT4) to the plasma membrane and subsequently stimulates glucose uptake in skeletal muscle tissue. Both stimuli appear independent of the insulin signalling cascade [1;3-7]. The mechanism behind ischemia and contraction-induced translocation of GLUT4 in skeletal muscle are only partly unresolved, but may involve several kinases that sense and transduce signals relating to changes in the intracellular environment during contraction/ ischemia (i.e., higher Ca 2+ , AMP concentrations) to other undefined proteins involved in GLUT4 movement [START_REF] Rose | Skeletal muscle glucose uptake during exercise: how is it regulated?[END_REF]. Intracellular Ca 2+ levels are related to the activity of the motor nerves, so proteins influenced by Ca 2+ , such as protein kinase C (PKC), calmodulin (CaM) and nitric oxide synthase (NOS) can be considered as feed forward regulation. For example, Ihleman et al [START_REF] Ihlemann | Calphostin C is an inhibitor of contraction, but not insulin-stimulated glucose transport, in skeletal muscle[END_REF] demonstrated that inhibition of CaM resulted in a reduction of contraction-induced glucose uptake. Also other proteins are linked to glucose uptake, such as AMP-activated protein kinase (AMPK) [START_REF] Bergeron | Effect of AMPK activation on muscle glucose metabolism in conscious rats[END_REF][START_REF] Hayashi | Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport[END_REF][START_REF] Kurth-Kraczek | 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle[END_REF]. AMPK is present in most mammalian cells and appears to act as a fuel gauge [START_REF] Hardie | Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status[END_REF]. Low energy cellular status such as ischemia and contraction, but also hyperosmolarity and heat shock, increase the AMP: ATP ratio, thereby activating AMPK [14;15]. A recent human in vivo study [START_REF] Frosig | Effects of endurance exercise training on insulin signaling in human skeletal muscle: interactions at the level of phosphatidylinositol 3-kinase, Akt, and AS160[END_REF], investigating the effects of endurance training, showed an increase in phosphorylation of AS160, which may be caused by changes in activity of AMPK acting upstream of AS160. As AMPK forms a potential pharmacological target for the treatment of type 2 diabetes [17;18], to elucidate the pathways regulating glucose uptake in skeletal muscle tissue is of great clinical relevance to the development of interventional strategies to improve glycaemic control in type 2 diabetes. Several in vitro studies have shown that the maximal effects of hypoxia and insulin on glucose transport are additive, whereas the effects of muscle contraction and hypoxia are not [1;14;19]. The latter implies that hypoxia and muscle contraction stimulate glucose transport via the same, insulin independent, mechanism. However, recent in vitro studies suggest that the mechanisms, responsible for ischemia and contraction induced glucose uptake do not share common pathways and are, at least partly, additive [6;20;21]. However the latter has not been confirmed in an in vivo human model. The present study aims to test the hypothesis that, ischemia-and insulin-induced glucose uptake are additive in vivo in healthy humans. Furthermore, we hypothesized that the combined effects of ischemia and muscle contraction on glucose uptake are not different from the impact of either ischemia or muscle contraction only. Therefore, we conducted two experiments. In protocol 1, we investigated the effects of ischemia in the presence and absence of a euglycemic hyperinsulinemic condition on forearm glucose uptake (FGU) and forearm blood flow (FBF). In protocol 2, we assessed the impact of ischemia, muscle contraction, and a combination of both on FGU and FBF.

METHODS

Subjects

A total of 18 healthy subjects were studied (2 groups of 9 subjects, consisting of 5 men and 13 females, age: 21.1 0.6 yr, BMI: 21.8 0.5 kg/m 2 , means±SD). All subjects underwent a clinical screening and a resting ECG. All subjects were healthy, i.e. not suffering from any acute or chronic illness and were not on any medications (except for oral contraceptives). The research was performed in accordance with the Declaration of Helsinki (2000) of the World Medical Association. The local ethics committee approved the study and all subjects gave written informed consent before participation.

Protocol 1: Impact of ischemia, insulin, and ischemia plus insulin on glucose uptake

We studied the physiological response to ischemia under normal physiological and euglycemic hyperinsulinemic conditions (clamp). First, the impact of 10 min of arterial occlusion on FGU and FBF was determined by inflating upper arm cuffs 100 mm Hg above systolic blood pressure. After 40 min of rest (pilot studies revealed that after 40 min the effects of ischemia (and contraction, see protocol 2) had totally disappeared, data not shown), a euglycemic hyperinsulinemic clamp was started. After 95 min, the impact of 10 min arterial occlusion was studied during euglycemic, hyperinsulinemic condition. We define "ischemia" as oxygen shortage of the muscle forearm, without reactive hyperaemia or muscle necrosis. After 10 min of ischemia, vasodilation will occur as a compensatory mechanism, and an increase in glucose uptake can be a consequence. We based this definition on a previous study of our colleagues Rongen et al [START_REF] Rongen | Annexin A5 scintigraphy of forearm as a novel in vivo model of skeletal muscle preconditioning in humans[END_REF]. They also used the same experimental procedure (ischemic exercise) as we did and have extended this with the Annexin A5 scan.

Protocol 2: Impact of ischemia, contraction, and ischemia plus contraction on glucose uptake

The effect of isometric contraction on FGU and FBF was determined during two 10 minperiods, during which muscle contraction and relaxation were alternated (5 sec at 30% of maximal hand grip power followed by 5 sec intermittent rest). After 40 min of subsequent rest, arterial occlusion was applied for 10 min by inflating upper arm cuffs 100 mm Hg above systolic blood pressure. After 40 min of rest, isometric contraction was repeated. After 10 min of contraction, contraction was combined with 10 min of arterial occlusion (or until exhaustion).

Experimental procedure

The experiments started at 8.00 AM after an overnight fast in a quiet, temperature controlled room (23-24 C). The subjects abstained from caffeine-containing foods and alcohol consumption for at least 24 h before the experiment. A catheter (Angiocath: 20-gauge, 48 mm, Becton Dickinson and Co., Sandy, Utah, USA) was inserted into the brachial artery of the non-dominant arm (experimental arm; the contralateral arm is the control arm) to obtain arterial blood samples and allow continuous blood pressure monitoring (Hewlett Packard GmbH, Böblingen Germany). In both arms a catheter (Venflon: 20-gauge, 32 mm, Becton Dickinson, Helsingborg, Sweden) was inserted retrograde into a deep forearm vein for deep venous blood sampling, enabling measurement of forearm arterio-venous glucose differences ( Gluc A-V ) in both arms. Blood samples were taken with an inflated wrist cuff for at least 5 min to exclude hand and skin flow to enter the deep venous system at the wrist. Bilateral FBF was measured by venous occlusion plethysmography [23;24]. In both protocols, FBF was measured immediately after the different stimuli (ischemia and contraction) were applied. In protocol 1 (euglycemic hyperinsulinemic clamp) an additional catheter (Venflon: 20-gauge, 32 mm, Becton Dickinson, Helsingborg, Sweden) was inserted into a vein on the dorsal side of a foot for infusion of insulin and glucose (20% glucose). After complete instrumentation, at least 30 min of rest was included to obtain a steady state before baseline data (FBF, Gluc A- V, plasma insulin level and hematocrit) were collected. During the euglycemic hyperinsulinemic clamp (protocol 1), 50 U insulin (Actrapid , Novo Nordisk, Farma BV, Alphen a/d Rijn, The Netherlands) was diluted in 47.5 ml 0.9% NaCl with the addition of 2 ml blood (to avoid adherence of insulin to tubing) to a concentration of 1 U/ml. Insulin was infused intravenously in a dose of 430 pmol•m -2 •min -1 (60 mU•m -2 •min -1 ). Subjects were clamped at fasting arterial glucose levels for 125 min. Euglycemia was maintained by a variable infusion of 20% glucose solution adjusted by arterial glucose measurements obtained at 5-min intervals.

Laboratory measurements and analysis

Plasma glucose levels were measured in duplicate using the glucose oxidation method (Beckman Glucose Analyzer II; Beckman, Fullerton, CA). Plasma insulin was assessed by inhouse radioimmunoassay (RIA).

Calculations and statistical analysis FBF, immediately obtained after 2 min of each intervention, was averaged to one value. FGU was calculated from Gluc A-V and FBF, assuming that:

Whole blood glucose = (1-[0.3 Hematocrit]) plasma blood glucose [25] FGU = Gluc A-V (1-[0.
3 Hematocrit]) FBF For calculation of whole body glucose disposal (M-value), we used the glucose infusion rate (GIR) over the last 30 min of the euglycemic hyperinsulinemic clamp before ischemia was applied, divided by body weight (µmol/min/kg). Differences in means were tested using paired Student's t-test. P<0.05 was considered statistically significant. Results and figures are expressed as means ± SEM, unless otherwise indicated.

RESULTS

Baseline characteristics

Baseline characteristics of the participants of both experiments did not differ. Provide details of the subjects here per experiment. In protocol 1, one person was excluded from the study because she fainted during the experiment (see below).

Protocol 1: Impact of ischemia, insulin, and ischemia plus insulin on glucose uptake 1: Metabolic response to ischemia: Gluc A-V did not change after 10 min of ischemia, see figure 1, middle. FGU increased from 0.6 0.1 to 5.5 1.9 mol/min per dl of forearm tissue, P= 0.03, versus baseline, see figure 1, bottom. 2: Metabolic response to insulin: After 95 min of systemic hyperinsulinemia, Gluc A-V increased in both arms (P<0.05 versus baseline for both and P= NS between 2 sides). FGU increased in the experimental and control arm (to 1.6 0.3 and 1.8 0.4 mol/min/dl respectively, P= 0.01 versus baseline, for both) with no differences between arms. During systemic insulin infusion, plasma insulin concentrations increased from 55 10 to 600 34 and to 551 27 pmol/l after 95 and 125 min respectively. The GIR was 37.2 2.8 mol/min/kg.

3: Metabolic response to ischemia plus insulin: Compared to insulin only,

Gluc A-V decreased significantly to 1.2 0.3 mmol/l after 10 min of ischemia combined with insulin. After ischemia, in the experiment arm, Gluc A-V returned to baseline values (1.8 0.3 mmol/l). FGU further increased to 15.6 2.2 mol/min/dl after 10 min of ischemia combined with insulin and was even synergistic when compared with the metabolic response to insulin and ischemia separately (P= 0.0003 and P= 0.0007 respectively). In the control arm, FGU increased to 2.2 0.4 mol/min/dl after 125 min of hyperinsulinemia (P= 0.03).

Protocol 1: Impact of ischemia, insulin, and ischemia plus insulin on blood flow 1: Hemodynamic response to ischemia: After 10 min of ischemia, FBF increased in the experimental arm to 14.8 1.9 ml/min per dl of forearm tissue, see figure 1, top (P= 0.0001). Heart rate (HR) did not change (P= 0.7), but systolic blood pressure decreased after 10 min of ischemia (from 121 2 to 109 3 mm Hg, P= 0.0005). See table 1 for heart rate and blood pressure values. 2: Hemodynamic response to insulin: After 95 min of hyperinsulinemia, there was no significant increase in FBF when compared with baseline values (from 1.4 0.3 to 1.6 0.4 ml/min/dl, P= 0.3). HR increased from 58 2 to 65 2 bpm (P= 0.01) and systolic blood pressure increased from 123 2 to 129 1 mm Hg (P= 0.001). MAP did not change significantly (P= 0.3). 3: Hemodynamic response to ischemia plus insulin: After 10 min of ischemia combined with insulin, FBF increased to 16.2 2.1 ml/min/dl (P= 0.0001 versus baseline), which did not differ from the vasodilator response to ischemia only. During the study, there was no significant increase in FBF in the control arm. HR was not affected by ischemia under hyperinsulinemic conditions (65 2 to 68 3 bpm, P= 0.3), but systolic blood pressure decreased during ischemia plus hyperinsulinemia (129 1 to 113 3 mm Hg, P= 0.00007).

Protocol 2: Impact of ischemia, contraction, and ischemia plus contraction on glucose uptake 1: Metabolic response to muscle contraction: Isometric contraction increased Gluc A-V in the experiment arm from 0.3 0.1 to 0.6 0.1 mmol/l (see figure 2, middle, P<0.05) and increased FGU from 0.3 0.1 to 2.7 0.8 mol/min/dl (P= 0.01 versus baseline, see figure 2, bottom.

2: Metabolic response to ischemia: After 10 min of ischemia,

Gluc A-V remained unchanged which is consistent with the findings in protocol 1. FGU increased from 0.5 0.1 to 4.4 0.8 mol/min/dl (P= 0.0008 versus baseline). FGU after ischemia was not significantly different compared to contraction only (P= 0.1).

3: Metabolic response to ischemia plus muscle contraction:

The combination of contraction and ischemia did not significantly change Gluc A-V (0.4 0.1 to 0.2 0.1 mmol/l, P= NS). There was no additive effect on FGU (from 0.6 0.2 to 5.3 1.6 mol/min/dl, P= 0.4, en P= 0.1 respectively versus ischemia and contraction separately). The arterial glucose (5.1 0.1 to 5.2 0.1 mmol/l) and the insulin levels (36±3 to 39±3 pmol/l) did not differ significantly (T=0 and 155 min respectively).

Protocol 2: Impact of ischemia, contraction, and ischemia plus contraction on blood flow 1: Hemodynamic response to muscle contraction: Isometric contraction increased FBF in the experimental arm from 1.5 0.2 to 5.1 0.8 ml/min/dl (P= 0.0004 versus baseline), see figure 2, top. HR and systolic blood pressure did not change. See table 2 for heart rate and blood pressure values. 2: Hemodynamic response to ischemia: Ischemia increased FBF from 2.2 0.3 to 14.8 2.2 ml/min/dl (P= 0.0003 versus baseline). HR increased from 60 2 to 63 2 bpm (P<0.05 versus baseline). The systolic blood pressure decreased from 128 4 to 116 3 mm Hg (P= 0.0001), which is comparable to the observed changes in protocol 1 after 10 min of ischemia.

3: Hemodynamic response to ischemia plus muscle contraction:

After the combination of contraction and ischemia, there was a larger increase in FBF (maximal increase from 2.1 0.3 to 25.3 3.4 ml/min/dl, P= 0.0003 and P= 0.0007 respectively versus contraction or ischemia alone). FBF in the control arm did not significantly change over the course of the experiment. HR increased from 63 2 to 75 5 bpm and MAP increased from 92 3 to 109 4 mm Hg, P<0.05 versus baseline for both.

Side effects

During the experiments with ischemia and contraction (protocol 2), subjects reported no sideeffects, except for cramps in the forearm muscle during the combination of exercise and contraction. All subjects found the combination of ischemia and contraction hard to carry out. Half of the participants were able to perform exercise during ischemia for 8 to 10 min (men found this easier than women), the others found this more difficult, but could combine this in most of the times for at least 5-6 min. During the last 4-5 min the subject also contract there muscles on a less regular basis. Thus, although the exercise stimulus may have been somewhat less intense, the participants still performed substantial muscle contraction during ischemia and that indeed the exercise stimulus during ischemia does not really differ from exercise only. In protocol 1, one person fainted during the euglycemic hyperinsulinemic clamp as a result of pain at the site of the catheter in the experimental arm. This person was excluded from the study. Other subjects in protocol 1 reported no side-effects at all.

DISCUSSION

The present study shows that ischemia and insulin are synergistic in stimulating effects on forearm glucose uptake in vivo in human skeletal muscle, while ischemia and muscle contraction are not. The observed differential effects of these stimuli on glucose uptake seem to be unrelated to changes in muscle blood flow.

These findings suggest that ischemia and muscle contraction stimulate glucose uptake in muscle tissue through the same metabolic pathways and, as such, imply that the pathways responsible for ischemia-induced glucose uptake are at least partly different from the mechanism responsible for insulin stimulated glucose uptake. The latter is supported by our observation that ischemia-induced glucose uptake is of a similar magnitude compared with ischemia plus contraction induced glucose uptake, and that the combined impact of insulin plus ischemia on glucose uptake is even greater than the sums of both separately. These findings seem consistent with earlier in vitro studies, which exposed isolated rat and human skeletal muscle to hypoxia. Cartee et al [START_REF] Cartee | Stimulation of glucose transport in skeletal muscle by hypoxia[END_REF] have shown that in perfused rat hind limb muscle, hypoxia increases the translocation of glucose transporters to the plasma membrane. Furthermore, they showed that the maximal effect of hypoxia and insulin on glucose transport was additive, whereas the effect of exercise and hypoxia was not. The latter provides evidence that hypoxia and exercise stimulate glucose transport via the same mechanism. Azevedo et al [START_REF] Azevedo | Hypoxia stimulates glucose transport in insulin-resistant human skeletal muscle[END_REF] examined muscle tissue derived from lean, obese and obese type 2 diabetic subjects and reported similar results. The authors showed that hypoxia in combination with insulin significantly stimulates glucose transport in all muscle tissue samples that were examined, suggesting that the glucose transport pathway is intact in muscle tissue in obese as well as in obese, type 2 diabetic patients when stimulated by hypoxia. More recent studies [START_REF] Hayashi | Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism[END_REF] have examined the effect of metabolic stressors on glucose transport and AMPK activation in rodent muscle. The glucose transport rates in the isolated muscle increased 5-fold above basal values during contraction and 8-fold during hypoxia. The latter was accompanied by a substantial activation of AMPK in skeletal muscle. However, insulin did not affect AMPK activity, but stimulated glucose transport 6.5-fold above baseline, again indicating that ischemia and muscle contraction stimulated glucose uptake are insulin-independent. In a previous human in vivo study [START_REF] Niklasson | Muscle glucose uptake is effectively activated by ischemia in type 2 diabetic subjects[END_REF], the effects of ischemia on glucose uptake was measured by using muscle micro dialysis in patients with type 2 diabetes and healthy subjects. The authors showed that glucose uptake is efficiently activated by ischemia in both healthy and type 2 diabetic subjects, and suggest that the activation of a pathway alternative to the insulin signalling cascade is not impaired [START_REF] Niklasson | Muscle glucose uptake is effectively activated by ischemia in type 2 diabetic subjects[END_REF]. The present findings are to our knowledge the first to show the differential effects of ischemia, muscle contraction and/ or hyperinsulinemia on skeletal muscle in vivo in humans. Recent in vitro studies suggest that part of the mechanism responsible for muscle contraction induced glucose uptake are different from ischemia stimulated glucose uptake and involve pathways independent of AMPK. The latter would imply that the impact of ischemia and muscle contraction on glucose uptake may be, at least partly, additive [6;20;21]. Nielsen et al [START_REF] Nielsen | A possible role for AMPactivated protein kinase in exercise-induced glucose utilization: insights from humans and transgenic animals[END_REF] concluded that a role for AMPK in exerciseinduced glucose uptake cannot be excluded, but that one or more additional pathways are involved in mediating glucose uptake in skeletal muscle tissue during exercise. So far, these speculations have not been further studied in an in vivo human model. The molecular mechanism responsible for hypoxia-(and exercise-) stimulated glucose uptake in human skeletal muscles remains to be fully elucidated, but it seems evident that AMPK plays a central role in this process. A limitation of the present study is that we were unable to obtain muscle biopsies of the forearm, because of ethical and safety reasons. So we cannot prove whether ischemia and/ or contraction did or did not phosphorylate AMPK in healthy subjects. As AMPK activation has important stimulating effects on both fat and carbohydrate metabolism, it represent an interesting target for intervention strategies in the prevention and/ or treatment of obesity and/ or type 2 diabetes [17;18]. Unravelling the exact physiological pathways responsible for the impact of ischemia, contraction and/ or insulin on fat and carbohydrate metabolism in liver, adipose tissue as well as muscle tissue in humans will proof to be of great clinical relevance in the treatment of chronic metabolic diseases. Another (possible) limitation of our study is that over activity of the sympathetic activation can influence glucose uptake negatively. The subjects experienced protocol 1 not as painful at all. Only the subjects in protocol 2 experienced some muscle cramps in their forearm during the combination of ischemia and contraction (see Side effects). We also showed that heart rate en blood pressure increased during this period. Thus, with the exception of the combined exercise ischemia protocol, pain-induced activation of the sympathetic nervous system is unlikely to have affected our results. Although less likely, it cannot be excluded that the pain associated with the combined exercise ischemia protocol might induce sympathetic activation, which in itself than may damp subsequent glucose uptake stimulation.

Although our study was mainly designed to investigate the metabolic effects of ischemia to contraction/ insulin in vivo in humans, our secondary goal was to explore the effects of ischemia on forearm blood flow and its potential contribution to glucose uptake. In the present study, we show that ischemia is a potent vasodilator, which is in accordance with previous findings [START_REF] Abbink | Vascular effects of metabolic inhibition by 2-deoxy-D-glucose in humans[END_REF][START_REF] Riksen | Augmented hyperaemia and reduced tissue injury in response to ischaemia in subjects with the 34C > T variant of the AMPD1 gene[END_REF][START_REF] Beckerath | Hypoxic vasodilatation in isolated, perfused guinea-pig heart: an analysis of the underlying mechanisms[END_REF]. Furthermore, exercise stimulates insulin-induced capillary recruitment in rodent skeletal muscle and, as such, augments glucose uptake [START_REF] Rattigan | Exercise training improves insulin-mediated capillary recruitment in association with glucose uptake in rat hindlimb[END_REF]. The stimulatory effect of muscle contraction on blood flow has also been described in vivo in humans. A prolonged exercise bout has been shown to increase FBF in healthy men under hyper- [START_REF] Hallsten | Insulin-and exercisestimulated skeletal muscle blood flow and glucose uptake in obese men[END_REF][START_REF] Bisquolo | Previous exercise attenuates muscle sympathetic activity and increases blood flow during acute euglycemic hyperinsulinemia[END_REF][START_REF] Dela | Effect of training on interaction between insulin and exercise in human muscle[END_REF] as well as under normoinsulinemic conditions [START_REF] Hunter | Active hyperemia and vascular conductance differ between men and women for an isometric fatiguing contraction[END_REF][START_REF] Thompson | Forearm blood flow responses to fatiguing isometric contractions in women and men[END_REF][START_REF] Gonzales | Forearm blood flow follows work rate during submaximal dynamic forearm exercise independent of sex[END_REF]. Based on the findings that especially in protocol 1, the increase in FGU is not preceded by an increase of FBF, which is also less visible in protocol 2 (see figure 1 and 2), it seems that the increase in FGU is not clearly correlated by an increase in FBF. This suggests that the differential stimulating effects on glucose uptake are unrelated to the changes in blood flow (measured with plethysmography). The latter is consistent with the idea that increased capillary recruitment is of a greater importance than an increase in total blood flow to augment (insulin/ contraction-) mediated glucose uptake [30;37;38]. However, in the present study, capillary recruitment was not assessed. Hyperinsulinemia was not associated with a significant increase in vasodilation. The latter is likely to be attributed to the relative short period of insulin infusion, because insulin-induced vasodilation is slow in onset, takes at least 3 hours to obtain its maximal effects and shows a high inter-individual variability [39;40]. We observed an increase in heart rate and systolic blood pressure (not in MAP) during insulin infusion, which is also well-described, although conflicting data consist on this issue [41;42]. Strikingly, we observed a decrease of systolic blood pressure after 10 min of ischemia. This is probably the result of a false low intra-arterial blood pressure in combination with an extreme high blood flow in the brachial artery after hypoxia.

We conclude that ischemia and insulin independently stimulate glucose uptake in human skeletal muscle and that their stimulating effects on glucose uptake are synergistic. In contrast, contraction and ischemia stimulate forearm glucose uptake to a similar extend, so their impact on glucose uptake is not additive. Therefore, these data suggest that ischemia and contraction use similar physiological pathways to stimulate glucose uptake, and these are different from the stimulating effects of insulin. The differential effects on glucose uptake are not clearly related to the concomitant changes in blood flow. It will be of important clinical benefit to further unravel the mechanisms responsible for ischemia, contraction and insulin

LEGENDS TO FIGURES

Figure 1 :

 1 Figure 1: Upper panel: Forearm blood flow. Middle panel: Arterio-venous glucose difference. Lower panel: Forearm glucose uptake, all before and after 10 minutes of ischemia, before and after euglycemic hyperinsulinemic clamp and after the combination of ischemia and hyperinsulinemia (protocol 1). Open square = experimental arm. Closed square = control arm. I = ischemia, Clamp = euglycemic hyperinsulinemic clamp. * indicates P<0.05. Significant levels for other time points not indicated. Please notice that the time schedule is not correct.

Figure 2 :

 2 Figure 2:Upper panel: Forearm blood flow. Middle panel: Arterio-venous glucose difference. Lower panel: Forearm glucose uptake, all before and after 2 times of contraction (10 minutes each), before and after ischemia and after the combination of ischemia and contraction (protocol 2). Open square = experimental arm. Closed square = control arm. I = ischemia, C = contraction. * indicates P<0.05. Significant levels for other time points not indicated. Please notice that the time schedule is not correct.

Table 1 : Effect of ischemia, hyperinsulinemia and the combination of ischemia plus hyperinsulinemia on heart rate, systolic and diastolic BP

 1 Syst means systolic blood pressure (BP), diast means diastolic BP. * P< 0.05 versus baseline, ¶ P< 0.05 versus before insulin, # P< 0.05 versus after 95 min of insulin.

		Baseline	After	Before	After 95 min	After ischemia+
			ischemia	insulin	insulin	insulin
	Heart rate (bpm)	59±5	60±3	58±2	65±2 ¶	68±3
	Syst BP (mm Hg)	121±2	109±3*	123±2	129±1 ¶	113±3#
	Diast BP (mm Hg)	66±2	66±2	67±2	68±2	67±2

Table 2 : Effect of ischemia, contraction and the combination of ischemia plus contraction on heart rate, systolic and diastolic BP

 2

		Baseline	After	Before	After	Before isch	After isch and
			contraction	ischemia	ischemia	and contr.	contr.
	Heart rate (bpm)	60±2	61±2	60±2	63±2*	63±2	75±5#
	Syst BP (mm Hg)	126±3	127±3	128±4	116±3*	132±4	137±6
	Diast BP (mm Hg)	68±2	69±2	68±2	68±2	71±3	85±3#
	Isch means ischemia, contr means contraction, syst means systolic blood pressure (BP), diast
	means diastolic BP.

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Clinical Science Immediate Publication. Published on 20 Jun 2008 as manuscript CS20080087 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080087 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

We thank Luc van Loon and Aarnout Jansen van Roosendaal for their contribution to this work. This work was supported by a grant of the Dutch Diabetes Foundation. Cees J. Tack is supported by NIDDM DK-0691881.