Mª Arantxa Ortega

Mª Teresa Pérez De Prada

Petra J Mateos-Cáceres

Priscila Ramos Mozo

Juan J González-Armengol

Juan M González

Del Castillo

Javier Martín Sánchez

Pedro Villarroel

José L Santiago

Ricardo J Bosch

Teresa Pérez

Juan M González Del Castillo

Carlos Macaya

Pedro Esbrit

Antonio J López-Farré

Effect of parathyroid-hormone-related protein on human platelet activation

Keywords: PTHrP, human platelets, platelet activation, acute myocardial infarction, mitogen-activated protein kinase

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INTRODUCTION

The acute coronary syndrome is caused by rupture of the atherosclerotic plaque and subsequent occlusive or subocclusive thrombus formation. Activated platelets then release a variety of vasoactive substances that promote platelet aggregation. Increased platelet activation occurs in the setting of cardiovascular disease related to acute coronary syndrome. However, this process is complex and influenced by a myriad of cellular and plasma-derived mediators. In fact, the normal response of platelets can be altered by either an increase or decrease in pro-aggregating or anti-aggregating stimuli, respectively.

Parathyroid hormone (PTH)-related protein (PTHrP), the causative factor of humoral hypercalcemia of malignancy, is now known to be widespread in normal fetal and adult tissues [START_REF] Philbrick | Defining the roles of parathyroid hormone-related protein in normal physiology[END_REF]. Due to N-terminal sequence homology between PTH and PTHrP, both peptides interact with a common receptor, the G protein-coupled PTH type 1 receptor (PTH1R), and thus stimulate cAMP formation, protein kinase (PK) C, and mitogen-activated protein kinase (MAPK) activities in different cell types [START_REF] Carpio | Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways[END_REF][START_REF] Clemens | Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets[END_REF][START_REF] Fortino | ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts[END_REF]. In contrast to PTH, which acts as a classical endocrine hormone, PTHrP normally exerts its effects locally in an autocrine, paracrine, and even intracrine fashion [START_REF] Philbrick | Defining the roles of parathyroid hormone-related protein in normal physiology[END_REF][START_REF] Clemens | Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets[END_REF]. Recent evidence strongly suggests that PTHrP may function as a pro-inflammatory mediator in various pathophysiological settings [START_REF] Funk | A role for parathyroid hormone-related protein in the pathogenesis of inflammatory/autoimmune diseases[END_REF]. Thus, PTHrP may contribute to the lethal effects of several pro-inflammatory cytokines during multi-organ failure in endotoxemia [START_REF] Funk | A role for parathyroid hormone-related protein in the pathogenesis of inflammatory/autoimmune diseases[END_REF][START_REF] Funk | Endotoxin increases parathyroid hormone-related protein mRNA levels in mouse spleen. Mediation by tumor necrosis factor[END_REF][START_REF] Funk | Parathyroid hormone-related protein is induced in the adult liver during endotoxemia and stimulates the hepatic acute phase response[END_REF]. There are also certain evidences supporting the notion that PTHrP, through its pro-inflammatory properties, could be involved in the mechanisms of renal injury [START_REF] Ortega | Role of parathyroid hormonerelated protein in tubulointerstitial apoptosis and fibrosis after folic acid-induced nephrotoxicity[END_REF][START_REF] Ortega | Role of the renin-angiotensin system on the parathyroid hormone-related protein overexpression induced by nephrotoxic acute renal failure in the rat[END_REF]. Moreover, recent data suggest that PTHrP may also have complex roles in cardiovascular pathology [START_REF] Clemens | Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets[END_REF]. PTHrP is present in smooth muscle cells and macrophages within human atherosclerotic lesions, where it might act as both proinflammatory and pro-atherogenic factor [START_REF] Ishikawa | Expression of parathyroid hormone-related protein in human and experimental atherosclerotic lesions: functional role in arterial intimal thickening[END_REF][START_REF] Martín-Ventura | Possible role of parathyroid hormone-related protein as a proinflammatory cytokine in atherosclerosis[END_REF].

As mentioned above, platelet activation is a key process related to both plaque instability and the compromised blood flow after coronary ischemia [START_REF] Gawaz | Role of platelet in coronary thrombosis and reperfusion of ischemic myocardium[END_REF][START_REF] Hughes | In vivo platelet activation following myocardial infarction and acute coronary ischaemia[END_REF]. To our knowledge, there are no current data on a possible effect of PTHrP on platelet activation, but some investigators have reported some conflicting effects of PTH in platelets. One of these studies has shown that acute administration of physiological concentrations of PTH induced an increase in platelet [Ca 2+ ] i associated with an increased arterial blood pressure in humans [START_REF] Fliser | Subacute infusion of physiological doses of parathyroid hormone raises blood pressure in humans[END_REF]. Moreover, a significant correlation between serum PTH and platelet [Ca 2+ ] i levels has been found in normotensive patients with primary hyperparathyroidism; although other investigators failed to observe such relationship in human subjects with either essential hypertension or secondary hyperparathyroidism [START_REF] Fardella | Intracellular calcium and blood pressure: comparison between primary hyperparathyroidism and essential hypertension[END_REF][START_REF] Poch | Parathyroid hormone and platelet cytosolic calcium concentration in essential hypertension[END_REF]. Furthermore, PTH has shown to display either pro-or anti-aggregating effects in human platelets [START_REF] Benigni | Inhibition of human platelet aggregation by parathyroid hormone. Is cyclic AMP implicated?[END_REF][START_REF] Kirichuk | Role of parathyroid hormone in regulating the functional activity of platelets[END_REF]. Taken together, however, these results support the notion that PTH might directly interact with platelets; although no attempts have been made to characterize the putative PTH receptor -e.g., the PTH1R or the PTH-specific PTH2 receptor [START_REF] Usdin | Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor[END_REF]-in platelets. Moreover no studies have yet been done to assess whether PTHrP may also be involved in the regulation of platelet activation. Therefore, in the present study, we investigated: 1) the effect of PTHrP on platelet aggregation, as well as the putative intracellular mechanism involved in this PTHrP action, and 2) the presence of the PTH1R in human platelets, and possible changes in its expression in platelets from patients during an acute coronary syndrome.

MATERIALS AND METHODS

Reagents

Platelet-rich plasma (PRP) preparation

PRP was obtained from 31± 3 years old healthy volunteers, who were not taken aspirin or any other anti-platelet or anti-inflammatory drugs from at least 15 days before blood extraction. Healthy volunteers signed informed consent. PRP isolation was carried out as previously reported [START_REF] López-Farré | Effects of aspirin on platelet-neutrophil interactions. Role of nitric oxide and endothelin-1[END_REF]. Briefly, whole blood was obtained in 10%

(vol/vol) acid-citrate-dextrose, and centrifuged at 800xg for 15 min, at 20°C. PRP was collected, and platelet number was counted by a Coulter counter. Platelet number was adjusted to 2.5x10 8 cells ml -1 with platelet-poor plasma obtained from the same subject.

Platelet aggregation

Platelet activation was registered in a lumiaggregometer (Aggrecorder, four channels; Chrono-Log Corporation, Harvertown, Pennsylvania, USA) by the change in light transmission. A platelet-poor plasma sample was used as control for precalibration at 100% light transmission. PRP (0.5 ml) was incubated at 37ºC for 10 min with continuous stirring in the aggregometer, and was then stimulated with: ADP (10 -6 mol L -1 ), arachidonic acid (5 x 10 -4 mol L -1 ), thrombin (0.02 U ml -1 ), or collagen (2 g mL -1 ), in the presence or absence of different PTHrP peptides. In some experiments, PRP samples were pre-incubated for 10 min with different inhibitors before PTHrP and ADP were added. For calculation, only turbidimetry values at 8 min were used; a time period corresponding to the maximal or near-maximal value of the first wave of platelet aggregation [START_REF] López-Farré | Effects of aspirin on platelet-neutrophil interactions. Role of nitric oxide and endothelin-1[END_REF].

Western blot analysis

Platelets were obtained from PRP by centrifugation at 600xg for 10 min, at room temperature. Isolated platelets were then homogenized in 8 mol L -1 urea, 2% CHAPS w/v, 40 mmol L -1 dithiothreitol, and 0.01% (w/v) bromophenol blue. After centrifugation at 900g, for 15 min at 4°C, the pellet was discarded. Protein (40 g/lane estimated by bicinchonimic acid reagent (Pierce, Rockford, IL), using bovine serum albumin (BSA) as standard), were separated on 10% polyacrylamide-SDS gels under reducing conditions. Proteins were then blotted onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes were then blocked with 5% BSA in phosphate-buffered saline (PBS) with 0.05% Tween-20, by overnight incubation at 4°C. Membranes were then incubated with the anti-PTH1R antibody (sc-12722, Santa Cruz Biotechnology) at 1:1000 dilution, or rabbit polyclonal antibodies against extracellular signal-regulated kinase ERK 1/2 and phosphor pERK 1/2 (Cell Signaling Technology, Beverly, MA, USA) each at 1:1000 dilution, for 1 h at room

temperature. An anti. -actin (Sigma Aldrich) at 1:5000 dilution, was used as a loading control. After extensive washing, the membranes were incubated with peroxidaseconjugated goat anti-mouse IgG and anti-rabbit IgG respectively for 1 h at room temperature and developed by enhanced chemiluminescence (ECL, Amersham). The bands were quantitated by densitometric scanning (ImageQuant, Molecular Dynamics; Sunnyvale, CA). Pre-stained protein markers were used for molecular mass determinations.

Flow cytometry

For flow cytometry studies, blood samples (0.2 ml) were immediately fixed with 1% p-formaldehyde overnight, at 4°C. The blood cells were then washed two times with PBS, and incubated for 30 min at 25°C with the anti-PTH1R antibody (sc-12722 FITC, Santa Cruz Biotechnology). After extensive washing with PBS, the samples were analyzed in a flow cytometer (Beckton Dickinson, San José, CA, USA) as described elsewhere [START_REF] De Miguel | A 4-trifluoromethyl derivative of salicylate, triflusal, stimulates nitric oxide production by human neutrophils: role in platelet function[END_REF]. The number of platelets expressing PTH1R was determined by analysis of a constant number of platelets (5x10 3 ) for each sample, using forward and side-light

Dot-blot in plasma

For dot-blot analysis, 10 g of total plasma proteins were loaded onto the nitrocellulose membrane in a dot-blot filtration mani-fold apparatus. The amount of proteins was determined by bicinchonimic acid reagent. As described above for Western blot analysis, the membranes were blocked with 5% BSA in PBS with 0,05% Tween-20, and then incubated with the monoclonal antibody against PTH1R (sc-12722, Santa Cruz Biotechnology) at 1:1000 dilution, for 1 h at room temperature. Following incubation with peroxidase-conjugated goat anti-mouse IgG, membranes were developed by ECL.

As loading control, a parallel blot containing the same amount of samples was performed and stained with Coomassie.

Statistical analysis

Results are expressed as mean SEM. Statistically significance was assessed by Mann-Whitney test. P<0.05 was considered significant.

RESULTS

Effect of PTHrP (1-36) on platelet activation

Spontaneous platelet activation (% light transmission: < 8 %) was not significantly modified by PRP incubation with increasing concentrations of PTHrP (1-36) (% light transmission: 14 3, corresponding to this peptide at 10 -7 mol L -1 ).

However, PTHrP (1-36) dose-dependently enhanced ADP-induced platelet activation (Figure 1A). Figure 1B PTHrP: 60 6, pNS). A similar pro-aggregating effect than with PTHrP was also observed with 10 -7 mol L -1 PTH (1-34) (Figure 1C). In contrast, PTHrP (107-139), at 10 - 7 mol L -1 , which does not seem to signal through the PTH1R [START_REF] Valín | C-terminal Parathyroid hormonerelated protein (PTHrP) (107-139) stimulates intracellular Ca 2+ through a receptor different from the type 1 PTH/PTHrP receptor in osteoblastic osteosarcoma UMR 106 cells[END_REF], failed to modify ADP-induced platelet activation (Figure 1C). Platelet incubation with 10 -6 mol L -1 PTHrP (7-34), a PTH1R antagonist [START_REF] Nutt | Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH 2 by substitution of PTH amino acids at positions 10 and 11[END_REF], did not by itself modify ADP-induced platelet activation, but blocked the enhancer ability of 10 -7 mol L -1 PTHrP (1-36) on this activation (% inhibition: 89 3). We next tested whether PTHrP (1-36) would also interact with the effect of other known platelet agonists. We found that this peptide PTHrP (1-36), at 10 -7 mol L -1 , also increased the ability of thrombin, collagen and arachidonic acid to induce platelet activation (Figure 2).

PTH1R expression in human platelets

Western blot analysis of human platelet extracts revealed a single band of apparent molecular weight 90-kDa [START_REF] Izquierdo | The parathyroid hormone-related protein system and diabetic nephropathy outcome in mice with steptozotocin-induced diabetes[END_REF], consistent with the presence of the PTH1R in these cells (Figure 3). This was further confirmed by flow cytometry analysis, showing that PTH1R immunoreactivity was present on the platelet surface (% platelets expressing the PTH1R: 19.7 2.2%).

Intracellular pathways associated with the effect of PTHrP (1-36) on human platelet activation

Additional studies were carried out to perform an initial characterization of the mechanism involved in platelet activation by PTHrP . We found that H89 (10 -6 mol L -1 ) and Rp-cAMPS (25 x 10 -6 mol L -1 ), two PKA inhibitors [START_REF] Chijiwa | Inhibition of forskolininduced neurite outgrowth and protein phosphorylation by a newly sinthetized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-((pbromocinnamyl)-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), of PC12D pheocromocytoma cells[END_REF][START_REF] Wang | Regulation of kainate receptors by cAMP-dependent protein kinase and phosphatase[END_REF], and also BIM (25 x 10 -9 mol L -1 ), a PKC inhibitor [START_REF] Alessi | PD098059 is a specific inhibitor of the activation of mitogen-activated kinase kinase in vitro and in vivo[END_REF], partially decreased the promoting effect of PTHrP on ADP-induced platelet activation (Table 1). None of these inhibitors significantly modified ADP induced platelet activation (Table 1). PD098059 (10 -5 mol L -1 ), a specific inhibitor of the mitogen-activated kinase (MAPK) upstream of ERK 1/2 [START_REF] Toullec | The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase[END_REF], abolished the effect of PTHrP (1-36) on ADPinduced platelet activation (Figure 4). In the absence of PTHrP, PD098059 (10 -5 mol L - 1 ) failed to significantly modify ADP-induced platelet activation (Figure 4). Moreover, PTHrP (1-36) (10 -7 mol L -1 ) rapidly ( 15 min) increased (2-fold vs control) ERK 1/2 phosphorylation in human platelets (Figure 5). No changes in total ERK½ expression in platelets was observed under the different experimental conditions (data not shown).

ERK activation by G-protein-coupled receptors may occur via Ras [START_REF] Luttrell | Regulation of tyrosine kinase cascades by G-protein-coupled receptors[END_REF]. We found here that the FTase inhibitor III (10 -8 mol L -1 ), which inhibits protein prenylation and thus Ras activation [START_REF] Nagasu | Inhibition of human tumor xenograft growth by treatment with the farnesyl transferase inhibitor B956[END_REF][START_REF] Qian | Design and structural requirements of potent peptidomimetic inhibitors of p21ras farnesyltransferase[END_REF], abrogated the effect of PTHrP (1-36) on ERK 1/2 phosphorylation in human platelets (Figure 5), as well as on ADP-induced platelet aggregation. Thus, this aggregation in the presence of this PTHrP peptide (at 10 -7 mol L - 1 ) plus the FTase inhibitor III was (% light transmission): 62 4, not different than the corresponding value with ADP alone (% light transmission: 60 3).

PTH1R expression in platelets from patients during an AMI

Table 2 shows the clinical features of both AMI patients and patients with similar cardiovascular risk factors without AMI (control group). The percentage of platelets from patients during AMI expressing PTH1R in their surface was: 17.2 2.8 % which was not different from that observed in platelets from the control group (19.4 2.6 % pNS). Figure 3 shows a representative flow cytometry analysis of PTH1R in platelets from an AMI patient. However, Western blot analysis demonstrated that platelet content of PTH1R were significantly higher in platelets from the control group compared with that observed in the platelets from AMI patients (Figure 6). Moreover, dot-blot analysis demonstrated that plasma obtained from patients during AMI contained higher amounts of circulating PTH1R levels than those in plasma from patients in the control group (Figure 7).

DISCUSSION

PTHrP was initially isolated from hypercalcaemia-associated tumours. However, in the absence of malignancy, PTHrP rather acts as a local growth factor or cytokine in a broad variety of tissues [START_REF] Philbrick | Defining the roles of parathyroid hormone-related protein in normal physiology[END_REF][START_REF] Clemens | Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets[END_REF]. A growing body of evidence suggests that PTHrP might be an important mediator of the inflammatory host response. In this setting, PTHrP would participate in a network of redundant pathways involving various cytokines and growth factors, e.g., interleukin-6 and angiotensin II, to promote inflammation and fibrosis (Reviewed in 6).

Inflammation is a key process associated with instability of atherosclerotic lesions [START_REF] Ross | Atherosclerosis: an inflammatory disease[END_REF]. Following atheroma rupture, the formation of an occlusive thrombus is the major event related to myocardial infarction and unstable angina. This is accompanied by an inflammatory response which is responsible for most acute coronary syndromes.

In fact, increased plasma levels of inflammatory markers have been detected in this scenario, and the degree of inflammation has been associated with these patients' outcome [START_REF] Gawaz | Role of platelet in coronary thrombosis and reperfusion of ischemic myocardium[END_REF][START_REF] Hughes | In vivo platelet activation following myocardial infarction and acute coronary ischaemia[END_REF][START_REF] Eikelboom | Antiplatelet agents in acute coronary syndromes[END_REF][START_REF] Mateos-Cáceres | Proteomic analysis of plasma from patients during an acute coronary syndrome[END_REF].

Studies in human atherosclerotic lesions following atherectomy have shown an intense PTHrP staining in both macrophages and smooth muscle cells which seems to correlate with the severity of the lesion [START_REF] Ishikawa | Expression of parathyroid hormone-related protein in human and experimental atherosclerotic lesions: functional role in arterial intimal thickening[END_REF][START_REF] Martín-Ventura | Possible role of parathyroid hormone-related protein as a proinflammatory cytokine in atherosclerosis[END_REF][START_REF] Nakayama | Coronary atherosclerotic smooth muscle cells overexpress human parathyroid hormone-related peptides[END_REF]. Moreover, we recently demonstrated that PTHrP and monocyte chemoattractant factor-1 colocalize in these cells in the atherosclerotic plaque [START_REF] Martín-Ventura | Possible role of parathyroid hormone-related protein as a proinflammatory cytokine in atherosclerosis[END_REF]. These findings suggest that PTHrP may acts as a proinflammatory factor contributing to plaque breakdown and thrombus formation. The factors responsible for PTHrP overexpression in atherosclerosis are unknown. However, vasoactive factors such as thrombin and norepinephrine, two upregulated agents in this condition and well known platelet activators, could induce PTHrP in the plaque neointima [START_REF] Hongo | Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation[END_REF][START_REF] Pirola | Angiotensin II regulates parathyroid hormone-related protein expression in cultured rat aortic smooth muscle cells through transcriptional and post-transcriptional mechanisms[END_REF].

In the present study, we examine whether PTHrP might directly interact with platelet activation. PTHrP (1-36) was found to increase significantly the effect of various known platelet aggregation inducers in human platelets. Our findings also indicate that this effect is likely to occur through the PTH1R, based on the following evidences: First, this receptor was detected -for the first time to our knowledge-in human platelets using Western blot and flow cytometry; second, this effect of PTHrP was similar to that of PTH , which bind to the PTH1R with the same affinity [START_REF] Clemens | Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets[END_REF]; third, it was abolished by PTHrP , a PTH1R antagonist [START_REF] Nutt | Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH 2 by substitution of PTH amino acids at positions 10 and 11[END_REF].

Moreover, our results suggest that the N-terminal region of PTHrP is essential to enhance platelet activation, since PTHrP (107-139), which does not interact with the PTH1R [START_REF] Valín | C-terminal Parathyroid hormonerelated protein (PTHrP) (107-139) stimulates intracellular Ca 2+ through a receptor different from the type 1 PTH/PTHrP receptor in osteoblastic osteosarcoma UMR 106 cells[END_REF], was inefficient in this regard.

The finding that PTHrP (1-36) not only enhanced the effect of ADP but also that of other inducers of platelet activation suggests that this peptide might interact with an ADP-postreceptor mechanism in human platelets. PTH1R activation usually stimulates both cAMP/PKA and [Ca 2+ ] i /PKC pathways through G s and G q , respectively, in many target cells [START_REF] Clemens | Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets[END_REF][START_REF] Mannstadt | Receptors for PTH and PTHrP: their biological importance and functional properties[END_REF]. In the present study, the promoting effect of PTHrP (1-36) on platelet activation was partially inhibited by either PKA or PKC inhibitors. This suggests that both signal transduction pathways can act independently as mediators of this effect of PTHrP (1-36) in human platelets. Interaction with the PTH1R may also activate MAPK signaling in a Ras-dependent or -independent manner in different cell types [START_REF] Carpio | Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways[END_REF][START_REF] Fortino | ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts[END_REF][START_REF] Swarthout | Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent[END_REF]. Our present findings suggest the existence of a cross-talk between PTHrP (1-36) activation of PKA and PKC and MAPK pathways in human platelets, since the enhancing effect of this peptide on ADP-inducing platelet activation was abrogated by a MAPK inhibitor. It is noteworthy that in the absence of PTHrP (1-36), this MAPK inhibitor did not affect ADP-induced platelet activation, in agreement with previous findings using platelet activators [START_REF] Borsch-Haubold | Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets[END_REF]. Accordingly with the MAPK pathways involvements of PTHrP-dependent platelet activation, PTHrP (1-36) rapidly activated ERK ½ phosphorylation in human platelets. Present data also support the notion that Ras-dependent ERK activation is a key distal signaling pathway integrating the control of platelet activation by PTHrP . It is of interest in this regard, that this PTHrP domain has shown to activate several G-protein-coupled pathways apparently converging in ERK activation to modulate osteoblast cell differentiation [START_REF] Carpio | Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways[END_REF]. The true role of ERK activation on platelet activation is currently controversial [START_REF] Borsch-Haubold | Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets[END_REF][START_REF] Rosado | Role of the ERK pathway in the activation of store-mediated calcium entry in human platelets[END_REF].

However, this activation might induce Ca 2+ influx [START_REF] Rosado | Role of the ERK pathway in the activation of store-mediated calcium entry in human platelets[END_REF], and thus be responsible, at least in part, for the pro-aggregating effect of PTHrP in human platelets. This hypothesis awaits further studies.

As mentioned above, PTHrP is abundant in human atherosclerotic plaques [START_REF] Ishikawa | Expression of parathyroid hormone-related protein in human and experimental atherosclerotic lesions: functional role in arterial intimal thickening[END_REF][START_REF] Martín-Ventura | Possible role of parathyroid hormone-related protein as a proinflammatory cytokine in atherosclerosis[END_REF]. Previous data and present results support the hypothesis that, after plaque rupture, human platelets would be exposed to PTHrP, and thus might promote both the inflammatory reaction and platelet activation. The presence of PTH1R in platelets, as demonstrated here, might provide a new significance to the previously reported increase in serum PTH early after acute myocardial ischemia [START_REF] Joborn | Platelet and plasma catecholamines in relation to plasma minerals and parathyroid hormone following acute myocardial infarction[END_REF]. It is unknown whether the recently identify PTH-specific receptor, the PTH2 receptor [START_REF] Usdin | Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor[END_REF], could also be present in platelets. However, present data suggest that, in this pathological setting, both PTH and PTHrP might act alone or in concert through the PTH1R to promote platelet aggregation. In the present study, we failed to find significant alterations in the number of platelets expressing the PTH1R in their surface in AMI patients, compared to that in another group of cardiovascular patients without AMI, matched by age, sex and cardiovascular risk factors, used as controls. However, despites the fact that AMI patients were administered anti-platelet therapy (aspirin) before platelet isolation, the total amount of PTH1R in platelets from these patients were markedly reduced compared with those from control. Therefore, taken together these findings suggest that PTH1R may be also contained into the platelets, probably stored in the platelet granules, and when platelets are activated, as occur during AMI, may rapidly been released into the circulation. In this regard, platelets contain other receptors, such as CD40 ligand and P-selectin, which localize to the platelet surface upon activation, and are rapidly shed into the circulation [START_REF] Chiu | Levels and value of soluble Pselectin following acute myocardial infarction: evaluating the link between soluble P-selectin levels and recruitment of circulating white blood cells and the marker for the rapid diagnosis of chest pain[END_REF][44][45]. Therefore, we assessed whether such mechanism might also apply to the PTH1R, which could thus be detected in plasma in AMI patients. Our results reveal, for the first time to our knowledge, the presence of this receptor in plasma. In addition, its plasma levels were significantly increased in plasma during AMI. The source of the circulating PTH1R is presently unknown, however, since it might originate not only from platelets but also from other cells of the ruptured plaque environment. In addition, it did not discard that platelets during AMI may interact with PTHrP in the ruptured atherosclerotic plaques which may favour the activation of the expressed PTH1R on the platelet surface enhancing platelet activation during the acute coronary syndrome. In any case, our finding is intriguing and raises the question of the putative functional role of the PTH1R in AMI as a marker of thrombosis or inflammation. In this regard, it would be interesting to explore in future studies the possible relation of the increase in PTH1R plasma levels with that of other soluble platelet receptor, namely sCD40 ligand and P-selectin [START_REF] Chiu | Levels and value of soluble Pselectin following acute myocardial infarction: evaluating the link between soluble P-selectin levels and recruitment of circulating white blood cells and the marker for the rapid diagnosis of chest pain[END_REF]46,47], in the setting of cardiovascular disease.

In conclusion, our findings demonstrate for the first time to our knowledge, that human platelets express the PTH1R. In addition, this receptor seems to mediate a pro-

Human PTHrP ( 1 -

 1 36) was kindly supplied by Drs. A.F. Stewart and A. García Ocaña (Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA). Human PTH (1-34) and human PTHrP (107-139) were obtained from Sigma (St. Louis, MO, USA), and (Asn 10 , Leu 11 , D-Trp 12 ) PTHrP (7-34) amide [PTHrP (7-34)] from Bachem (Bubendorf, Switzerland). N-[2-((pbromocinnamyl)amino)-ethyl]-5BIM), 2'-amino-3'-methoxyflavone (PD098059) and farnesyltransferase (FTase) inhibitor III were obtained from Calbiochem (San Diego, CA, USA). Affinity-purified mouse monoclonal anti-PTH1R antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against phosphorylated extracellular-regulated kinase (ERK) 1/2 (Thr 202 /Tyr 204 ) were from Cell Signaling Technology (Beverly, MA, USA). ADP, thrombin, collagen, and arachidonic acid were purchased from Chrono-par Reagents (Havertown, PA, USA).

  shows a representative trace of platelet aggregation triggered by ADP in the presence of different PTHrP concentrations. As mentioned, PTHrP potentiated the maximal platelet aggregation response to ADP but PTHrP did not significantly change the slope of the platelet curve (ADP: 56 4 min; ADP+10 -7 mol L -1

Clinical

  Science Immediate Publication. Published on 14 May 2007 as manuscript CS20070010 © 2007 The Authors Journal compilation © 2007 Biochemical Society Parathyroid hormone-related protein and platelet activation

Figure 1 .-

 1 Figure 1.-Bar graphs showing the effect of increasing concentrations of PTHrP (1-36) (A,B) and that of PTH (1-34) and PTHrP (107-139) (each at 10 -7 mol L -1 ) (C) on ADP (10 -6 mol L -1 )-induced platelet activation. (B) Representative platelet aggregation trace showing the effect of ADP alone and in the presence of increasing concentrations of PTHrP. (C) Bar graph showing the effect ADP+PTH(1-34) and ADP+PTHrp(107-139)(each at 10 -7 ml L -1 ) on ADP-induced platelet activation. For comparison the effect of ADP+PTHrP (10 -7 ml L -1 ) was also included. Results are mean ± SEM of four independent experiments. * p<0.05, with respect to ADP alone.

Figure 2 .

 2 Figure 2.-Bar graph showing the effect of PTHrP (1-36) (10 -7 mol L -1 ) on platelet activation induced by thrombin (0.02 U ml -1 ), collagen (2 g mL -1 ) and arachidonic acid (AA) (5 x 10 -4 mol L -1 ). Results are mean ± SEM of three independent experiments. *p<0.05, with respect to the corresponding value in the absence of PTHrP (1-36).

Figure 3 .-

 3 Figure 3.-Representative autoradiogram showing the PTH1R protein expression in human platelet extracts from two different normal subjects (V 1 and V 2 ) by Western blot (see text for details). At the bottom is shown the flow cytometry analysis to detect PTH1R expression on the platelet surface of platelets obtained from patients during AMI and patients with similar cardiovascular risk factors without AMI. In the middle of the figure is showed the autofluorescence of platelets used as negative control. which was substracted from the positive value of the samples. In addition, a non-specific mouse IgG 1 -isotype antibody was also used as negative control (in the figure first column on the left).

Figure 4 .-

 4 Figure 4.-Bar graph showing the abrogating effect of the MAPK inhibitor PD098059 (10 -5 mol L -1 ) on the pro-aggregating ability of PTHrP (1-36) (10 -7 mol L -1 ) in ADPstimulated human platelets. Results are mean ± SEM of three independent experiments. *p<0.05, with respect to ADP alone. †p<0.05, with respect to ADP+PTHrP (1-36).

Figure 5 .

 5 Figure 5.-Representative autoradiogram showing ERK 1/2 activation by PTHrP (1-36) (10 -7 mol L -1 ) and its blockade by the FTase inhibitor III (10 -8 mol L -1 ) by Western blot. Protein loading was similar in each well, as assessed by Ponceau S staining (not shown).

Figure 6 .

 6 Figure 6.-Representative Western blot showing the total content of PTH1R in platelets from patients during AMI and patients in the absence of AMI (control group). Densitometric values are mean SEM. * p<0.05 with respect to the control group.

Figure 7 .-

 7 Figure 7.-Representative dot-blot showing the presence of PTH1R protein in plasma of both groups of patients, during AMI and in the absence of AMI (control group). Protein loading was similar in each well as assessed by Coomassie staining (at the bottom). Corresponding densitometric values as mean SEM are shown. * p<0,05 with respect to the control group.

Clinical Science Immediate Publication. Published on 14 May 2007 as manuscript CS20070010 scatter

  bivariate plots. Non-specific mouse IgG 1 isotype antibody (sc-2855, Santa Cruz Biotechnology) was used as negative control.Both platelets and plasma were obtained from six consecutive patients, aged 75 4 years (range 69 to 83), during an acute myocardial infarction (AMI), admitted in the Emergency Unit of Hospital Clínico San Carlos. AMI was diagnosed on the basis of the classic criteria of prolonged chest pain accompanied by serial changes on the standard 12-lead electrocardiogram, as well as raised plasma creatine kinase (CK) (more than twice the upper limit of the normal value measured in the biochemistry laboratory and CK-MB fraction >10% total CK). Two patients presented electrocardiographic ST

	Patients

elevation whereas the other four patients had not ST elevation (non-Q-wave infarction).

A criterion for enrollment was admission within 24 hours after the onset of chest pain.

A group of 5 patients matched by age, sex and cardiovascular risk factors were enrolled as a control group. None of the patients was taking any anti-inflammatory and antithrombotic drug, except aspirin (250 mg) in the AMI group, administered 20-60 min before blood extraction. Patients with history of neoplastic or autoimmune diseases or infections, or any surgical procedure, in the preceding six months were not included.

Both control and AMI patients gave informed consent, and the study was approved by the Ethics Committee of Hospital Clínico San Carlos.

Clinical Science Immediate Publication. Published on 14 May 2007 as manuscript CS20070010

Table 1 .-Effect of PKC (BIM) and PKA (H89, Rp-cAMPS) inhibitors on the enhancer ability of PTHrP (1-36) on ADP-induced human platelet activation.

 1 Results are mean ± SEM of four different experiments. * p<0.05, with respect to the corresponding value in the presence of ADP. † p<0.05, with respect to ADP + PTHrP(1- 36).

	Clinical
		% Platelet aggregation
		ADP	ADP + PTHrP (1-36)
		59 ± 2	83 ± 3*
	BIM	53 ± 2	70 ± 2* †
	H89	48 ± 3	72 ± 3* †
	Rp-cAMPS	58 ± 2	68 ± 2* †

Science Immediate Publication. Published on 14 May 2007 as manuscript CS20070010

Clinical Science Immediate Publication. Published on 14 May 2007 as manuscript CS20070010 © 2007 The Authors Journal compilation © 2007 Biochemical Society Parathyroid hormone-related protein and platelet activation

© 2007 The Authors Journal compilation © 2007 Biochemical Society Parathyroid hormone-related protein and platelet activation

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGMENTS

This work has been supported by grants from Instituto de Salud Carlos III (Red HERACLES, RD06/0009 and PI050363), and Ministerio de Ciencia y Tecnología (SAF2002-04356-C03-03). A. Ortega was supported by Conchita Rábago Foundation, and is currently a recipient of a research contract from "Juan de la Cierva" Program from the Spanish Ministry of Education and Science. M.T. Pérez de Prada was supported by Red Heracles. Authors thank Begoña Larrea for secretarial assistance.


Bo Westman
email: bo.westman@sts.sll.se

Lars Weidenhielm

Olav Rooyackers

Katarina Fredriksson

Jan Wernerman

Folke Hammarqvist

Solveig Mrs

Mrs Eriksson

Liselotte

Knee replacement surgery as a human clinical model of the effects of ischemia/reperfusion upon skeletal muscle

Keywords: Ischemia-reperfusion, skeletal muscle, knee replacement surgery, glutathione, amino acids, energy rich compounds

come

Knee replacement surgery as a human clinical model of the effects of ischaemia/reperfusion upon skeletal muscle

INTRODUCTION:

The metabolic effects of ischemia and reperfusion on skeletal muscle tissue have been extensively described in animal studies. The relevance of these findings for man is not obvious.

Therefore we have investigated clinical situations involving ischemia followed by reperfusion.

Changes in muscle glutathione, energy and amino acid metabolism have been described using major vascular surgery in order to characterise a human model [1,2]. However the influence of abdominal aneurysm and aorto-bifemoral bypass surgery on lower limb muscle metabolism is attributable both to the surgical trauma and to the ischemia-reperfusion. The characteristic postoperative changes are dominating over the signs of major oxidative stress regarding muscle amino acid pattern as well as glutathione status. In addition, during the major vascular surgery procedures studied, no complete ischemia is at hand, related to the existence of collaterals and shunting. Therefore a clinical human ischemia-reperfusion model was explored using knee replacement surgery in tourniquet ischemia. This clinical model could possibly be more suitable for studies on ischemia and reperfusion in human skeletal muscle and the effects on glutathione metabolism, reflecting the scavenging status.

Application of a tourniquet and inflation up to double the systolic arterial blood pressure is described to induce ischemia with anaerobic metabolism and oxidative stress [START_REF] Mathru | Tourniquet-induced Exsanguination in Patients Requiring Lower Limb Surgery: An Ischemia-Reperfusion Model of Oxidant and Antioxidant Metabolism[END_REF][START_REF] Appell | Administration of tourniquet. II. Prevention of postischemic oxidative stress can reduce muscle edema[END_REF]. Tourniquet ischemia is frequently used in orthopaedic surgery and metabolic tissue alterations are seen both during ischemia and during the following reperfusion such as decreased levels of phosphocreatine, increased lactate, increased glycogenolysis, and decreased pH [START_REF] Haljamäe | Human skeletal muscle energy metabolism during and after complete tourniquet ischemia[END_REF][START_REF] Larsson | Electrolyte changes in muscle tissue and plasma in tourniquet-ischemia[END_REF][START_REF] Larsson | The effect of long-term arterial occlusion on energy metabolism of the human quadriceps muscle[END_REF][START_REF] Häggmark | Time course of muscle metabolic changes during tourniquet ischemia in man[END_REF][START_REF] Sjöholm | The effect of long-term circulatory occlusion on pH and energy metabolism of the quadriceps muscle in man[END_REF]. After the release of tourniquet in knee replacement surgery an increased production of reactive oxygen species (ROS) is seen [10]. Although not always clinically significant, several studies show that an increased oxidative stress occurs during knee replacement surgery using tourniquet ischemia with activation of polymorphonuclear granulocytes and increased levels of H 2 O 2 in femoral vein blood from the reperfused leg [START_REF] Mathru | Tourniquet-induced Exsanguination in Patients Requiring Lower Limb Surgery: An Ischemia-Reperfusion Model of Oxidant and Antioxidant Metabolism[END_REF][START_REF] Willy | Systemic effects of extremityischemia reperfusion surgical trauma. Assessment of tourniquet ischemia induced activation of polymorphonuclear netruophic granulocytes[END_REF]. Moreover, increased expressions of ICAM-1 (intercellular cell-adhesion molecule-1) in muscle and of MnSOD (manganese superoxide dismutase), GST-mu (glutathione transferase-mu) in granulocytes and increases of IL6 and IL8 in plasma suggest an association between inflammation, redox markers and ischemia and reperfusion [START_REF] Huda | Inflammatory and redox responses to ischeamia/reperfusion in human skeletal muscle[END_REF].

The aim of this study was to investigate the effects of total ischemia and reperfusion in a clinical model on muscle metabolism as reflected by glutathione status, amino acid pattern and Patients (n=15) without any history of vascular disease, undergoing knee replacement surgery due to gonarthrosis were included in the study. The patients were non-smokers and not on corticosteroid therapy, and they were all metabolically healthy. The study has been carried out in accordance with the Declaration of Helsinki and the protocol has been approved by the Ethics Committee of the Karolinska Institute. Informed consent was obtained from the patients after informing them about the purpose, the nature and risk of the procedures in the study.

All patients were given epidural anaesthesia with Xylocain R (Astra, Södertälje Sweden) during operation and during the following 24 hours. Ischemia was induced just prior to surgery by a tourniquet that was inflated to double the patients systolic arterial blood pressure. Muscle biopsies were obtained from the vastus lateralis portion approximately 15 cm above the knee on the leg to be operated on before ischemia, at maximal ischemia just before deflating the tourniquet and after 24 hours of reperfusion with a needle biopsy technique described by Bergström et al [14]. The biopsies after 24 hours of reperfusion were obtained under local anaesthesia down to the muscular fascia, but not into the muscular compartment.

The patients were randomised to receive either mannitol with an osmolality of 930 mosm/kg (Fresenius Kabi, Uppsala at a dose of 12.5 ml/kg/day; n=8) or glucose (Fresenius Kabi, Uppsala) at a concentration of 50 mg/ml, at a dose of 18.75 ml/kg/day; n=7). The infusions were given as a continuous infusion during 24 hours after the induction of tourniquet ischemia.

The patients were allowed to drink non-caloric fluid during the postoperative period and were given Ringer's acetate to cover the basal need for fluid.

Muscle samples

A percutaneous muscle biopsy of approximately 150 mg was obtained at each sampling occasion. The muscle biopsy was taken approximately 15-20 cm below the lower border of the tourniquet cuff. The muscle biopsy was divided into three smaller portions, weighed on an electro balance, then plunged into liquid nitrogen and stored at -80 C prior to analyse.

Biochemical analyses

In a subgroup of patients (n=7) muscle samples were obtained preoperatively, at maximal ischemia and after 24 hours of reperfusion for analyses of lactate, ATP, creatine (Cr) and Westman et al 6 phosphocreatine (PCr) as described in detail elsewhere [2,[START_REF] Harris | Glycogen, glycolytic intermediates and high energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values[END_REF]. Total creatine (tCr) was calculated as the sum of Cr and PCr. In addition the mitochondria were assembled and the activities of citrate synthase and complexes I and IV were determined as described in detail elsewhere [16].

Glutathione and cysteine were analysed as described in detail previously [START_REF] Luo | Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography[END_REF]. The oxidised form of glutathione (GSSG) was calculated by the difference between total (tGSH) and reduced glutathione (GSH) divided by two. The redox-status of glutathione was expressed as the ratio between GSH and tGSH (GSH/tGSH). The concentrations of thiols are expressed as mmol/kg ww of muscle.

For amino acid analyses HPLC employing postcolumn derivatisation with ophtaldialdehyde was used [18]. The concentrations of amino acids were expressed as mmol/kg ww of muscle.

Statistical analyses

The values are expressed as medians with lower and upper quartiles. All patients in the two groups were combined in this comparison. When comparing results during the reperfusion period with preoperative levels a nonparametric ANOVA (Freidman's ANOVA) comparing medians, was used followed by Wilcoxon test for post-hoc comparisons. Spearman rank test was used for correlations. A p-value < 0.05 was considered as statistically significant for comparisons. Westman et al 7

Clinical

RESULTS

The patients included in the study were 71.0 (63.0 -76. 0) years old (mannitol 68.5 (63.5 -74.3) and glucose group 73.0 (62.0 -79.0) respectively). Ischemia time was 83.0 (56.0 -92.0) minutes (mannitol 84.0 (75.0 -93.0) and glucose group (78.0 (50.0 -88.0). The tissue was not exposed to glucose or mannitol until the reperfusion started after removal of the tourniquet. Therefore all samples obtained preoperatively and at maximal ischemia were combined in the calculations reflecting the preoperative and maximal ischemia condition. During the reperfusion period the values are presented for the combined group as well as for the two treatment groups.

Energy rich compounds and mitochondrial enzymes (Figure 1)

Lactate increased during ischemia by 420 % as a sign of anaerobic metabolism (p<0.05). PCr decreased at maximal ischemia by 69 % (p < 0.05) with a reciprocal increase in Cr by 91 % (p<0.05). At 24 h of reperfusion both PCr and Cr recovered back to the preoperative levels. tCr and ATP remained unchanged during the whole study period as compared to the preoperative values. Ischemia time and changes in lactate and PCr at maximal ischemia were not statistically correlated. The mitochondrial enzymes citrate synthase and Complexes I and IV were not affected by ischemia and reperfusion.

Thiols (Figure 2, and electronic supplement table 1)

At maximal ischemia an increase was seen in GSH by 8% (p<0.05). At the same time the oxidised form of glutathione (GSSG) decreased by 21% (p<0.01), with a consequent increase in the redox-status of glutathione (GSH/tGSH) by 4% (p<0.01). At 24 h reperfusion decreases were seen in GSH and tGSH by 27% (p<0.01) and 22% (p<0.01) respectively, while an increase was seen in cystine (CySS), the oxidised form of cysteine by 23% (p<0.01).

At 24 h of reperfusion the effect of mannitol was studied. No changes in GSH and tGSH were seen in the patients randomised to mannitol, while decreases by 31% (p < 0.05) and by 26% (p< 0.05) respectively were seen in the control group as compared to the preoperative levels.

There were no statistically significant changes between the two groups. (Data are given in an electronic supplement, Table 1). After 24 hour of reperfusion the pattern was quite different with decreases seen in threonine by 23 % (p<0.05), serine by 18 % (p<0.05), glutamate 51 % (p<0.001), glutamine by 32 % (p<0.01), glycine by 15 % (p<0.01), ornithine by 50 % (p<0.01), lysine by 42 % (p<0.01), histidine by 27 % (p<0.01), arginine by 48 % (p<0.01), BAA by 41 % (p<0.01), EAA by 21 % (p<0.01), and in tAA by 28 % (p<0.01) in the combined group together with an increase in the sum of AAA by 15 % (p<0.01). (Data are given in an electronic supplement, Table 2). Between the two groups given mannitol or placebo no differences were seen in the muscle amino acid levels after the 24 h reperfusion period. (Data are given in an electronic supplement, Table 3).

No correlations were seen between changes in biochemical markers and length of ischemia.

DISCUSSION

.

In our search for a human clinical model of ischemia and reperfusion, here knee replacement surgery was elucidated. The ischemia part of the model showed alterations in muscle amino acid and glutathione metabolism, which have not been described before. The reperfusion part of the model on the other hand showed changes in muscle amino acid and glutathione metabolism, which to a large extent represent what is generally seen postoperatively.

In our earlier efforts to use a human clinical model to study ischemia/reperfusion in skeletal muscle we have used major vascular surgery [1, 2]. In terms of amino acid and glutathione metabolism in muscle, these models have not been very different from what is seen following major surgical procedures in general. In abdominal aortic aneurysm repair the possibility of a too short ischemia time was discussed [2]. When similar results were obtained also when the ischemia time was doubled in aorto-bifemoral graft surgery, the fact that this type of vascular surgery does not represent total ischemia related to the existent of collaterals, was considered [1]. In the present model, knee replacement surgery, a total ischemia in terms of halted blood flow was obtained. Another advantage with the present protocol was that combined circulatory isolation was combined with the use of an epidural block for anaesthesia. Hence no interference from circulating hormones or neural impulses was at hand in the muscle during ischemia. At reperfusion the advantages during the ischemia time were not longer present. In the literature metabolic changes of ischemia followed by reperfusion during cross ligament repair in tourniquet ischemia has been reported [START_REF] Larsson | The effect of long-term arterial occlusion on energy metabolism of the human quadriceps muscle[END_REF]. Today this mode of surgical approach is obsolete, as this type of surgery is performed during arthroscopy not employing tourniquet ischemia.

The ischemia during 60-90 minutes was total as reflected by the increases of lactate and creatine levels with a reduction of phosphocreatine. Muscle ATP concentration was unaltered, reflecting that cellular energy status and indirectly the membrane potential were preserved. This fact is important for the interpretation of the rest of the results. A decrease in cellular ATP level is associated with necrosis and cellular disintegration [START_REF] Labbe | The extent and distribution of skeletal muscle necrosis after graded periods of complete ischemia[END_REF], in which case the amino acid gradients across cell membranes may no longer be kept stable.

For glutathione a 10 % increase in reduced glutathione with an unaltered total glutathione pool in muscle was seen at maximal ischemia. This was also accompanied by a slight increase in the Westman et al 10 redox ratio of glutathione. The physiological significance of this increase is not totally clear to us, but it indicates that the appearance of radical oxygen species was very low. A possible interpretation is that the redox ratio at maximal ischemia actually represents more or less a "maximal" value of the equilibrium between reduced glutathione and total glutathione in muscle tissue. This occurred in a situation when no oxygen was available. The redox status of cystine on the other hand was not different between basal situation and maximal ischemia.

The free amino acid pattern at maximal ischemia was different from what is seen at "maximal ischemia" during major vascular surgery. In this study all free amino acid concentrations except for glutamate were elevated. In contrast, during vascular surgery, immediately before reperfusion, most free amino acid concentrations decrease except for alanine [1, 2]. The ischemia pattern of muscle free amino acids seen here may be interpreted as a mobilisation of free amino acids from protein degradation in excess of ongoing synthesis. The most pronounced increases were seen in the alanine and glutamine concentrations. This may reflect transaminations to the carbon skeleton of pyruvate/lactate to form alanine and alphaketoglutarate/glutamate to form glutamine. The decrease in glutamate may consequently reflect transaminations into both glutamine and alanine production. As oxygen depending processes involving the Krebs cycle or an export via venous blood were not possible, the result was an increase in most amino acid concentrations. As the intracellular concentrations of the individual different amino acids are not proportional to their occurrence in muscle proteins, the percentage increases given must be considered in relation to the actual concentrations. This indicated for example de novo production of glutamine, which may explain the decrease in glutamate. The constant uptake of glutamate in muscle tissue described during all physiological situations so far investigated [20], which was now interrupted during ischemia, is also a factor to consider in this explanation.

When major vascular surgery was used as an ischemia model, the disadvantage of the possible incomplete ischemia and also the possibility of an incomplete epidural block must be considered. Although the aorta was clamped, it is well known that a collateral circulation exists. As for the completeness of the epidural block, during vascular surgery the patients were subjected to general anaesthesia in combination with epidural anaesthesia. Hence the efficacy of the epidural block may have been sufficient for postoperative pain relief without totally blocking the neurogenic part of the impact of surgery. The pattern of muscle free amino acid seen in major vascular surgery just before reperfusion is more in line with the general Westman et al 11 postoperative pattern. This pattern develops characteristically over time [21,[START_REF] Hammarqvist | Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance[END_REF]. Initially there is an increase in alanine concentration together with a decrease in glutamate, while all other amino acids are unaltered. This pattern is also described during a combined stress hormone infusion [START_REF] Hammarqvist | Stress hormone and amino acid infusion in healthy volunteers; short term effects on protein synthesis and amino acid metabolism in skeletal muscle[END_REF]. At 6-12 hours postoperatively decreases in the free concentrations of a number of amino acids appear [21]. In particular the branched chain amino acid decrease at this time point. Later on, at 24 h after the traumatic insult, the characteristic postoperative pattern, with decreases in glutamate, glutamine and the basic amino acids together with increases of the aromatic and branched chain amino acids, is seen [21,[START_REF] Hammarqvist | Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance[END_REF]. With the result from the present study at hand, it is obvious that the amino acid pattern seen just before reperfusion during major vascular surgery was merely dominated by the changes in the amino acid patterns in response to trauma rather than to ischemia.

A secondary aim of this study was to use a scavenging substance, mannitol, to investigate whether the postoperative glutathione pattern may be modified. Administration of mannitol, a free radical scavenger diminishes oedema in the muscular compartment and prevents compartment syndrome, mostly by its hyperosmolar properties, and has been shown to reduce muscular necrosis and increase restitution of energy production by free radical scavenging. This has been shown in an animal model in which preparation of the hind-limb followed by clamping of surgically isolated vessels outruled the influence of circulation through collaterals [START_REF] Oredsson | The effect of Mannitol on reperfusion Injury and postischemic compartment pressure in skeletal muscle[END_REF]. In that model positive effects were seen both of mannitol and allopurinol leading to reduced signs of reperfusion injury. Moreover, mannitol has been shown to reduce the negative effects of reperfusion elicited by reactive oxygen species in acute vascular surgery [24,[START_REF] Adiseshiah | Reperfusion injury in skeletal muscle: a prospective study in patients with acute limb ischaemia and claudicants treated by revascularization[END_REF] and in models of ischemia reperfusion [26][27][START_REF] Aldman | The effect of mannitol on reperfusion after severe induced subtotal ischemia of the leg. A hemodynamic, biochemical and metabolic study in a pig hind limb[END_REF].

As stated above the reperfusion part of the knee replacement model was less convincing, as the traumatic insult elicited by the surgical procedure probably was the dominating influence upon the muscle glutathione and amino acid metabolism during this time period. Nevertheless, a slight different pattern was seen in the two groups. At 24 h of reperfusion the decrease in total and reduced glutathione concentrations in muscle attained statistical significance in the control group, but not in the mannitol group. Still the difference between the two groups was numerically minimal and it was not statistically significant. Considering the small numerical difference and the imperfection of the reperfusion part of the model, the significance of this finding is not clear. Evaluation of mannitol treatment as an adjunct during reperfusion in man

  Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070025 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical

  Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070025 © 2007 The Authors Journal compilation © 2007 Biochemical Society Westman et al 8 Amino acids (Figure 3 a and b, and electronic supplement table2 and 3)Muscle glutamate decreased by 29 % (p<0.001) at maximal ischemia. At maximal ischemia increases in the muscle concentrations of threonine by 5 % (p<0.05), glutamine by 4 % (p<0.05), alanine by 65 % (p<0.001), lysine by 6 % (p<0.05), histidine by 10 % (p<0.05), and arginine by 6 % (p<0.01) were seen. Increases were also seen in the total sum of all amino acids (tAA) by 8 % (p<0.05), in the basic amino acids (lysine, histidine and arginine; BAA) by 8 % (p<0.01), in the aromatic amino acids (AAA) by 7 % (p<0.05) and in the essential amino acids (EAA) by 21 % (p<0.01).

Clinical

  Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070025 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical

  Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070025 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1

 1 Figure 1 Concentrations of energy rich compounds and activities of mitochondrial enzymes in skeletal muscle preoperatively (pre), at maximal ischemia (max), and after 24 hour of reperfusion (reperf) in patients (n=15) undergoing knee replacement surgery employing tourniquet ischemia. The symbols are explained on the figure, left hand y-axis (atp = adenosinetriphosphate, pcr = phosphocreatine, ct = creatine, tcr = total creatine, lac = lactate, cs = citrate synthase), right hand y-axis (compl I = complex I, compl IV = complex IV). * denotes different from preoperative values, p < 0.05.

Figure 2

 2 Figure 2Concentrations of thiols (mmol/kg ww) and the redox ratio (%) of glutathione in skeletal muscle preoperatively (pre), at maximal ischemia (max), and after 24 hour of reperfusion (reperf) in patients (n=15) undergoing knee replacement surgery employing tourniquet ischemia. The symbols are explained on the figure, left hand y-axis (cysh = cysteine, tcys = total cysteine, cyss = cystine, gsh redox = the ratio gsh/tgsh), right hand y-axis (gsh = reduced glutathione, tgsh = total glutathione, gssg = reduced glutathione). *, ** denotes different from preoperative values, p < 0.05 and p < 0.01 respectively.

Figure 3

 3 Figure 3 Concentrations of selected amino acids in skeletal muscle preoperatively (pre), at maximal ischemia (max), and after 24 hour of reperfusion (reperf) in patients (n=15) undergoing knee replacement surgery employing tourniquet ischemia. The symbols are explained on the figure, panel a (glu = glutamate, gln = glutamine, gly = glycine, ala = alanine, lys = lysine), panel b (thr = threonine, ser = serine, orn = ornithine, his = histidine, arg = arginine). *, **, *** denotes different from preoperative values, p < 0.05, p < 0.01, p < 0.001 respectively.

Clinical Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070025 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Weinbroum, A. A., I. Shapira, A. R.B. and A. Szold (2003) Mannitol dose-dependently attenuates lung perfusion injury following liver ischemia reperfusion: a dose-response study in an isolated perfused double-organ model. Lung 180, 327-338

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Thunblad and Mrs Christina Hebert in performing the study. The study was supported by grants from Swedish Medical Research Council (projects 04210, 14244, 20319).


Klaus Stark

Katharina Neureuther

Kamil Sedlacek

Wibke Hengstenberg

Marcus Fischer

Andrea Baessler

Silke Wiedmann

Andreas Jeron

Stephan Holmer

Jeanette Erdmann

Heribert Schunkert

Dr Christian Hengstenberg
email: christian.hengstenberg@klinik.uni-regensburg.de

Common Y402H variant in complement factor H gene is not associated with susceptibility to myocardial infarction and its related risk factors

Keywords: Genetics, Association study, Polymorphism, Myocardial infarction, CFH

Recently, the genetic variant Y402H in complement factor H gene (CFH) was associated with increased risk for myocardial infarction (MI) in a prospective Caucasian cohort. In another nested case-control study, however, the CFH-Y402H variant did not carry susceptibility to MI.

The aim of the present study was to test for association between the CFH-Y402H variant and MI in a large case-control sample with familial background for coronary artery disease (CAD).

A total of 2,161 individuals from the German MI family study were studied by questionnaire, physical examination and biochemical analyses. MI patients (n=1,188; 51.4 ± 8.6 years at first MI) were recruited from families with at least two members affected by MI and/or severe CAD. Spouses, sisters-in-law, and brothers-in-law, respectively, without MI/CAD were included as unaffected controls (n=973; 56.9 ± 9.8 years). Genotyping was performed using a TaqMan assay. Common Y402H variant in the CFH gene was not associated with classical cardiovascular risk factors (diabetes, hypercholesterolemia, hypertension, obesity, smoking, and C-reactive protein serum levels). No association could be found between the CFH-Y402H variant and susceptibility to MI. Separate analyses in both men and women revealed no gender-specific influence of the gene variant on cardiovascular risk factors or MI. This investigation could not replicate the association between the common CFH-Y402H variant and susceptibility to MI in our large Caucasian population which is enriched for genetic factors. We conclude that the CFH-Y402H variant has no relevant risk modifying effect in our population.

Introduction

Coronary artery disease (CAD) and myocardial infarction (MI) are the leading causes of morbidity and mortality in the Western world [START_REF] Murray | Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Study[END_REF]. In epidemiological studies, factors like smoking, obesity, high blood pressure, elevated cholesterol levels, and diabetes have been identified to increase cardiovascular risk. Additionally, a strong genetic component was also documented in the etiology of CAD [START_REF] Marenberg | Genetic susceptibility to death from coronary heart disease in a study of twins[END_REF][START_REF] Zdravkovic | Heritability of death from coronary heart disease: a 36-year follow-up of 20 966 Swedish twins[END_REF]. To unravel the underlying genes, several genomewide analyses have been performed and revealed chromosomal loci with linkage to CAD or MI [START_REF] Broeckel | A comprehensive linkage analysis for myocardial infarction and its related risk factors[END_REF][START_REF] Farrall | Genome-wide mapping of susceptibility to coronary artery disease identifies a novel replicated locus on chromosome 17[END_REF][START_REF] Francke | A genome-wide scan for coronary heart disease suggests in Indo-Mauritians a susceptibility locus on chromosome 16p13 and replicates linkage with the metabolic syndrome on 3q27[END_REF][START_REF] Harrap | Genome-wide linkage analysis of the acute coronary syndrome suggests a locus on chromosome 2[END_REF][START_REF] Hauser | A genomewide scan for early-onset coronary artery disease in 438 families: the GENECARD Study[END_REF][START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF][START_REF] Pajukanta | Two loci on chromosomes 2 and X for premature coronary heart disease identified in early-and late-settlement populations of Finland[END_REF][START_REF] Samani | A genomewide linkage study of 1,933 families affected by premature coronary artery disease: The British Heart Foundation (BHF) Family Heart Study[END_REF][START_REF] Wang | Premature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis[END_REF]. However, until today only few genes and variants responsible for prevalence to MI in the general population are known [START_REF] Helgadottir | The gene encoding 5lipoxygenase activating protein confers risk of myocardial infarction and stroke[END_REF][START_REF] Watkins | Genetic susceptibility to coronary artery disease: from promise to progress[END_REF].

In the pathophysiology of atherosclerosis, inflammation is hypothesized to play an important role for plaque formation and its destabilization and therefore causing CAD and MI [START_REF] Hansson | Inflammation, atherosclerosis, and coronary artery disease[END_REF][START_REF] Ross | Atherosclerosis--an inflammatory disease[END_REF].

Complement factor H (CFH) as a part of innate immunity contributes to inflammation processes. CFH provides binding sites for C3b, heparin as well as sialic acids, and also interacts with C-reactive protein (CRP) [START_REF] Rodriguez De | The human complement factor H: functional roles, genetic variations and disease associations[END_REF], which has been linked in several studies to CAD, MI and stroke [START_REF] Wilson | The novel role of C-reactive protein in cardiovascular disease: risk marker or pathogen[END_REF].

Recently, an association between a common CFH gene variant and increased risk for MI was reported in a prospective cohort study: Single nucleotide polymorphism (SNP) rs1061170, representing a tyrosine-histidine change at amino acid position 402 in the CFH protein, showed hazard ratios up to 1.77 in 226 MI cases during a mean of 8.4 years of follow-up of 5,237 individuals from the Rotterdam Study [START_REF] Kardys | A common polymorphism in the complement factor H gene is associated with increased risk of myocardial infarction: the Rotterdam Study[END_REF]. However, another prospective study with 335 MI cases did not show an influence of Y402H variant on susceptibility to MI [START_REF] Zee | Complement factor H Y402H gene polymorphism, C-reactive protein, and risk of incident myocardial infarction, ischaemic stroke, and venous thromboembolism: A nested case-control study[END_REF].

The German MI family study [START_REF] Broeckel | A comprehensive linkage analysis for myocardial infarction and its related risk factors[END_REF][START_REF] Baessler | Long-term effects of in-hospital cardiac rehabilitation on the cardiac risk profile. A case-control study in pairs of siblings with myocardial infarction[END_REF][START_REF] Fischer | Distinct heritable patterns of angiographic coronary artery disease in families with myocardial infarction[END_REF][START_REF] Reinhard | Association of the metabolic syndrome with early coronary disease in families with frequent myocardial infarction[END_REF] provides well-characterized MI patients as well as unrelated, healthy controls for association studies. Here, we investigated the association between the CFH-Y402H genetic variant and susceptibility to MI and known cardiovascular risk factors, as well as CRP serum levels.

Methods

Study sample

The study sample consisted of individuals from the German MI family study. Selection criteria have been described previously [START_REF] Broeckel | A comprehensive linkage analysis for myocardial infarction and its related risk factors[END_REF][START_REF] Baessler | Long-term effects of in-hospital cardiac rehabilitation on the cardiac risk profile. A case-control study in pairs of siblings with myocardial infarction[END_REF]. All participants were studied by standardized questionnaire, physical examination and biochemical analyses at inclusion (n=2,161) and five-year follow-up (n=1,780).

Baseline characteristics of the 2,161 investigated participants in this study at the time point of inclusion are summarized in Table 1. The present analyses included independent MI cases (n=1,188) with a positive family history (at least one additional family member who had suffered from MI or severe CAD, defined as treatment with percutaneous coronary intervention or coronary artery bypass graft). Control individuals (n=973) consisted of married-in spouses, sisters-in-law, and brothers-in-law, respectively. Thereof, 617 controls were confirmed to be free of any cardiovascular symptoms and events during the follow-up period. Of the remaining 356 control individuals follow-up examination was not completed from the time of the present study. Consanguineous individuals were excluded. Research has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Written informed consent was obtained from all subjects, and the ethics committee of the University of Regensburg approved the study.

Definition of risk factors

Diabetes was defined as a history of diabetes mellitus or intake of antidiabetic medication.

Individuals with former or current smoking habit were classified as smokers. Obesity was defined as body mass index ≥ 30 kg/m². Study subjects receiving antihypertensive therapy or with a systolic blood pressure (BP) ≥ 140 mmHg or diastolic BP ≥ 90 mmHg were classified as hypertensive. Hypercholesterolemia was defined as low-density lipoprotein (LDL) cholesterol ≥ 160 mg/dL or use of a lipid lowering therapy.

Genetic analysis

Genomic DNA was isolated from whole blood samples using the PureGene DNA Purification System Blood Kit (Gentra, Minneapolis, MN, USA). DNA samples were genotyped using 5' exonuclease TaqMan ® technology (Applied Biosystems, Foster City, CA, USA) with differently fluorescence labeled probes including non-fluorescence quencher and minor groove binder (MGB) [START_REF] De Kok | Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes)[END_REF][START_REF] Livak | Allelic discrimination using fluorogenic probes and the 5' nuclease assay[END_REF]. For SNP rs1061170, a Custom TaqMan ® SNP Genotyping Assay (Applied Biosystems) was used with forward primer: For each genotyping experiment 10 ng DNA was used in a total volume of 5 µl containing 1x TaqMan ® Universal PCR Master Mix (Applied Biosystems). PCR reaction and post-PCR endpoint plate read was carried out according to the manufacturer's instructions using the Applied Biosystems 7900HT Real-Time PCR System. Sequence Detection System software version 2.2 (Applied Biosystems) was used to assign genotypes applying the allelic discrimination test [START_REF] Livak | Allelic discrimination using fluorogenic probes and the 5' nuclease assay[END_REF]. Case and control DNA was genotyped together on the same plates.

Duplicates of samples (15 %) were employed to assess intraplate and interplate genotype quality. No genotyping discrepancies were detected. Assignment of genotypes was performed by a person without knowledge of the affection status.

Statistical analysis

Genotype distribution within the groups of cases and controls, respectively, was compared with values predicted by Hardy-Weinberg equilibrium using the χ²-test. Differences in allele frequencies between dichotomous traits were calculated with the same method. Genotype distribution between cases and controls assuming dominant or recessive genetic models were performed using logistic regression analysis. Linear regression analysis was employed for comparison of genotype distributions with continuous variables, whereas ln(CRP) serum levels were used. Potential interaction between each traditional cardiovascular risk factor and genotype was tested in separate logistic regression analyses including cross-product term.

Prevalence odds ratios (OR) with their 95% confidence intervals (CI) were reported. A twosided P value ≤ 0.05 was considered statistically significant. All analyses were carried out using JMP IN 5.1 (SAS Institute, Cary, NC, USA). Power analysis was performed applying the G*Power program [START_REF] Erdfelder | GPOWER: A general power analysis program[END_REF].

Results

From 2,187 DNA specimens, a total of 2,161 individuals were genotyped successfully and therefore the overall call rate was 98.8 %. Baseline characteristics of this study sample are shown in Table 1. The proportion of women was lower in the patient group (n=375) than in the control group (n=636). As expected, the MI patients (n=1,188) had a higher prevalence of classical cardiovascular risk factors (hypercholesterolemia, hypertension, obesity, diabetes, and smoking habit) than did the control subjects (n=973). In MI patients, the mean age at first MI was 51.4 ± 8.4 years. Anthropometric and biochemical measurement were performed at time-point of inclusion at a mean age of 57.9 ± 9.2 years (58.7 ± 8.6 years for MI patients, 56.9 ± 9.8 years for controls).

Genotype distribution of CFH gene variant Y402H was analyzed in the whole population and in sub-groups separately. The Hardy-Weinberg equilibrium was always fulfilled. Hence, test for allele frequency difference and co-dominant model gave nearly the same P values (not shown). Therefore, additional to the allele frequency comparisons we reported results from dominant and recessive genetic models (Table 2). In our study with 2,161 different DNA samples, the frequency of the H allele was 36.7%. Genotype frequencies in the whole study group were 40.4%, 45.8%, and 13.8% for YY, YH, and HH, respectively.

CFH-Y402H variant and susceptibility to MI and risk factors

The observed allele frequency and genotype distributions of the CFH-Y402H variant were not significantly different between 1,188 MI cases and 973 unaffected controls. In addition, no association could be found between the Y402H variant and classical cardiovascular risk factors, namely diabetes, hypercholesterolemia, hypertension, obesity, and smoking habit (Table 2). We also performed adjusted analyses to exclude confounding effects of the differently distributed risk factors between MI cases and controls and found no significant association between CFH-Y402H variant and MI (data not shown).

Additionally, no association between CFH-Y402H variant and CRP serum levels at date of inclusion was observed (P = 0.16). Due to the limited sample size with CRP data points at inclusion date (497 of 2,161 individuals; 471 of them classified as MI patients) we used fiveyear follow-up values (mean age 62.9 ± 8.7 years), where 1,284 CRP values were measured. Again, no association between CFH-Y402H variant and CRP serum level could be found (P = 0.97). From the follow-up examination, the available CRP measurements were from 855 MI patients (mean age 63.2 ± 8.1 years, i. e. on the average of 11.8 years after first MI) and 429 controls (mean age 62.5 ± 9.1 years), respectively. No significant association between CRP serum levels at follow-up examination and MI was observed (P = 0.09).

To test for gender-specific influences of CFH-Y402H variant, we performed the analyses in

Discussion

Inflammation and components of the innate immunity -and thereby potentially complement activation -play a major role in the pathophysiology of atherosclerosis [START_REF] Oksjoki | Role of complement activation in atherosclerosis[END_REF]. The CFH gene, encoding a plasma protein essential for regulation of the alternative complement pathway, is a good candidate for genetic susceptibility to MI. Two recent studies showed inconsistent results on the association of CFH-Y402H variant with MI in prospective cohorts [START_REF] Kardys | A common polymorphism in the complement factor H gene is associated with increased risk of myocardial infarction: the Rotterdam Study[END_REF][START_REF] Zee | Complement factor H Y402H gene polymorphism, C-reactive protein, and risk of incident myocardial infarction, ischaemic stroke, and venous thromboembolism: A nested case-control study[END_REF]. Thus, additional data is needed to assess the impact of this variant to the genetic etiology of MI.

In the present case-control association study, the CFH-Y402H variant was neither associated with an increased risk for MI in patients with a strong familial background for cardiovascular disease nor with classical cardiovascular risk factors, such as diabetes mellitus, hypercholesterolemia, hypertension, obesity, and smoking (Table 2). Serum CRP as a nonspecific indicator of inflammation has gained great importance as a risk marker in cardiovascular disease, although the magnitude of its risk stratification potential has been debated [START_REF] Danesh | C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease[END_REF]. Since the CRP binding site of CFH protein is localized within the region of Y402H variant [START_REF] Discipio | Human polymorphonuclear leukocytes adhere to complement factor H through an interaction that involves alphaMbeta2 (CD11b/CD18)[END_REF] and its genotype potentially determine CRP levels [START_REF] Johnson | Individuals homozygous for the age-related macular degeneration risk-conferring variant of complement factor H have elevated levels of CRP in the choroid[END_REF], we analyzed a possible association of CFH-Y402H and CRP serum levels in our study group. At follow-up, CRP serum levels were available from 1,284 participants. No association between serum CRP and Y402H genotypes was detectable, indicating that CRP serum levels are not influenced by the CFH gene variant in a measurable fashion in our cohort. Additionally, CRP serum level at follow-up examination is not associated with susceptibility to MI in our study.

However, it has to be noted that in this study group baseline as well as follow-up values for serum CRP are measured after a mean of 7.3 and 11.8 years, respectively, after suffering from MI. It is thus questionable whether these values can be related to the pathophysiology of the MI event.

Within the CFH gene the amino acid position 402 is encoded in exon 9. This region is part of a strong linkage disequilibrium (LD) block that did not cover the whole gene with respect to a r² value of 0.8 by analyzing data from the HapMap project [START_REF]The International HapMap Consortium (2005) A haplotype map of the human genome[END_REF]. However, Y402H variant is the only one so far showing positive association with MI in a prospective cohort study [START_REF] Kardys | A common polymorphism in the complement factor H gene is associated with increased risk of myocardial infarction: the Rotterdam Study[END_REF]. In upcoming genome-wide association (GWA) studies on MI, e. g. using Affymetrix © GeneChip ® Human Mapping 500K Array Set, CFH-Y402H variant will not be analyzed directly due to absence of this SNP on the chips. Altogether, information of 18 SNPs within the 95.5-kb CFH gene will be available from these GWA studies. Recent studies on the CFH gene dealing with association between the Y402H variant and age-related macular degeneration showed that other polymorphisms within the CFH gene also contribute to disease susceptibility independent from Y402H genotype [START_REF] Li | CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration[END_REF][START_REF] Maller | Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration[END_REF]. This emphasises the relevance of analyzing the Our non-replication of the recently reported association between CFH-Y402H variant and MI is unlikely to be a result of insufficient power. The sample size with 1,188 MI cases and 973 controls has greater than 96% power to detect a weak to moderate gene effect with an effect size = 0.2 at a significance level of 0.05.

A possible limitation of the study is the recruitment of our MI patients: only patients who survived at least one MI and had an affected sibling with CAD or MI were included. Hence, there was a selection bias for both MI surviving patients and familial form of the disease. On the other hand, however, due to the family-based character of our patient selection, the genetic background for etiology of MI is high in our study group thus enabling us to investigate even small genetic effects. Using married-in spouses or their relatives as control subjects, the risk of population stratification between affected MI patients and unaffected individuals is minimized. Additionally, main part of control group was validated as being free of any cardiovascular symptoms and events during the five-year follow-up period.

Like other candidate genes, CFH should be included to analyses in upcoming association studies of CAD and MI to reveal more information about the contribution of this gene and its variants to the genetic etiology of the disease.

  5'-CTT TAT TTA TTT ATC ATT GTT ATG GTC CTT AGG AAA ATG TTA TTT -3', reverse primer: 5'-GGC AGG CAA CGT CTA TAG ATT TAC C -3', probe 1: VIC-5'-TTT CTT CCA TAA TTT TG -3'-MGB, probe 2: Clinical Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070028 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society FAM-5'-TTT CTT CCA TGA TTT TG -3'-MGB (fluorophore VIC ® and 6FAM™, respectively, and MGB are quoted; SNP position is underlined; probes were designed on the reverse chromosomal strand).

Clinical

  Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070028 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society both men and women separately. Neither susceptibility to MI nor cardiovascular risk factors (diabetes, hypercholesterolemia, hypertension, obesity, and smoking habit) showed genderspecific association with Y402H variant. Likewise, no gender-related association between CFH-Y402H genotypes and CRP serum levels from the follow-up examination could be found (data not shown). Additional analysis in a age-and sex-matched sub-sample (n=1,200) with 300 cases and 300 controls from both men and women also showed no association between risk factors, MI and CFH-Y402H genotypes (data not shown). Clinical Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070028 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Clinical

  Science Immediate Publication. Published on 2 May 2007 as manuscript CS20070028 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society Y402H variant separately. Additionally, in the background of GWA studies future focus should be given on remaining part of CFH gene as a potential candidate locus for MI susceptibility.

Table 2 .

 2 CFH-Y402H genotype distribution according to affection status

								Affected			Unaffected		Difference in	Dominant model	Recessive model
							Genotype (n)		Genotype (n)		allele frequency	(genotype HH+YH vs. YY)	(genotype HH vs. YH+YY)
							YY	YH	HH	MAF	YY	YH	HH	MAF	P value	OR (95% CI)	P value	OR (95% CI)	P value	OR (95% CI)
	Myocardial infarction		484	540	164	0.365	390	449	134	0.368	0.83	0.99 (0.87-1.12)	0.76	0.97 (0.82-1.16)	0.98	1.00 (0.78-1.28)
	Diabetes mellitus	*		104	115	33	0.359	770	874	265	0.368	0.71	0.96 (0.79-1.17)	0.78	0.96 (0.74-1.26)	0.73	0.93 (0.62-1.36)
	Hypercholesterolemia	*	558	637	176	0.361	316	349	122	0.377	0.29	0.93 (0.82-1.06)	0.80	0.98 (0.82-1.17)	0.08	0.80 (0.63-1.03)
	Hypertension	*			679	758	230	0.365	193	229	68	0.372	0.68	0.97 (0.84-1.12)	0.59	0.77 (0.86-1.16)	0.96	0.99 (0.75-1.34)
	Smoking	*				507	617	183	0.376	366	372	115	0.353	0.12	1.11 (0.97-1.26)	0.06	1.19 (1.00-1.41)	0.73	1.04 (0.81-1.35)
	Obesity	*					186	210	53	0.352	686	775	242	0.370	0.33	0.96 (0.79-1.08)	0.66	0.95 (0.77-1.18)	0.19	0.81 (0.58-1.10)
	MAF: minor allele frequency
	Unadjusted odds ratios (OR) are given

* Definition is given in Table

1

' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Acknowledgement

We gratefully acknowledge the excellent technical assistance of Martina Köhler, Josef Simon, and Michaela Vöstner. We appreciate support from the German MI family study team, and 15 German cardiac rehabilitation centers. This study was supported by the Deutsche Forschungsgemeinschaft (He1921/9-1, Ho1073/8-1, and Schu672/12-1), the National Genome Network (NGFN2; 01GS0417 and 01GS0418 to C.H. and H.S., respectively), the Ernst-und Berta-Grimmke-Stiftung (C.H. and H.S.), the Wilhelm-Vaillant-Stiftung (C.H., S.H., H.S.), and the Deutsche Stiftung für Herzforschung (C.H. and H.S.), Germany.

The authors declare no conflict of interest.

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